Reaction mass of 4,4'-methylenediphenyl diisocyanate and Amines, soya alkyl

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A number of genotoxicity studies were included as part of NONS registrations. Bacterial reverse mutation assays (Ames tests) have been conducted on 3,3'-dioctadecyl-1,1'-methylenebis(4,1-phenylene)diurea (PU12/A123; EC 406-690-3), 3,3'-dicyclohexyl-1,1'-methylenebis(4,1-phenylene)diurea (R95; EC 406-370-3), N,N''-(methylenedi-4,1-phenylene)bis[N'-octyl]urea (A124; EC 445-760-8), and a mixture of: 3,3'-dicyclohexyl- 1,1'-methylenebis(4,1-phenylene)diurea; 3-cyclohexyl-1-(4-(4-(3-octadecylureido)benzyl)phenyl)urea; 3,3'-dioctadecyl-1,1'-methylenebis(4,1-phenylene)diurea (PU10; A002; PU18;EC 406-530-2),. The results from all tests concluded that the substances were not mutagenic in the presence or absence of metabolic activation. Additionally, an in vitro mammalian chromosome aberration test was also performed on N,N''-(methylenedi-4,1-phenylene)bis [N'-octyl]urea (A124; EC 445-760-8). The study concluded that no chromosomal abnormalities were observed in either the presence or absence of metabolic activation during the two replicate tests, and therefore the conclusion is that this substance is not a clastogen.

 

As part of an updated testing program, in vitro bacterial reverse mutation assays (Ames tests) were conducted on:

- Reaction mass of 4,4'-methylenediphenyl diisocyanate and Amines, soya alkyl (A003; EC 905-837-3)

- Reaction product of MDI, Octadecylamine and Magnesium Hydroxide (PU05; EC 944-730-6)

- Reaction product of MDI and p-toluidine (PU07; EC 926-809-7)

- Reaction product of MDI, Octylamine and Cyclohexylamine (PU08; EC 926-119-6)

- Reaction product of MDI, Octylamine and Hexamethylenediamine (PU09; EC 924-670-7)

- Polyurea, produced by reacting diphenylmethane diisocyanate with octylamine and dodecyl amine (R03; EC 812-490-0)

- Polyurea, produced by reacting diphenylmethane diisocyanate with octyl amine and stearyl amine (R04; EC 812-491-6)

- Reaction product of 4,4'-methylenediphenyl diisocyanate, ethylenediamine and dodecylamine (R05; EC 924-043-8)

Negative results were observed for all substances tested, indicating the substances were not mutagenic in the presence or absence of metabolic activation.

 

Under the conditions of the tests, the substances within the MDI category did not demonstrate a mutagenic or clastogenic response, and thus are considered to be non-mutagenic.

 

 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22/10/2021 - 25/11/2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997, corrected 2020

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2008

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

1998

Principles of method if other than guideline:

Based on provisions in the guidelines:
The Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy, Trade and Industry (METI), and Ministry of the Environment (MOE) Guidelines of 31 March 2011
ICH S2(R1) guideline adopted June 2012 (ICH S2(R1) Federal Register. Adopted 2012; 77:33748-33749)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

- Salmonella: Histidine
- E. Coli: Tryptophan

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

- Type of metabolic activation system: Lyophilized phenobarbital/beta-naphthoflavone induced rat liver S9 and cofactors mix (MutazymeTM)
- Source of S9: Prepared in lab before use
- S9 mix: Final concentrations of S9-mix of approximately 10% (v/v) MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium phosphate buffer (100 mM, pH 7.4)
- Concentration: 0.5 mL of S9-mix was added to the molten, trace amino-acid supplemented media

Test concentrations with justification for top dose:

Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate
Pre-Incubation Method: 15, 50, 150, 500, 1500 and 5000 μg/plate.
The maximum concentration was 5000 μg/plate (the OECD TG 471 maximum recommended dose level).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: The test item was insoluble in sterile distilled water, dimethyl sulphoxide, dimethyl formamide and acetonitrile at 50 mg/mL, acetone at 100 mg/mL and tetrahydrofuran at 200 mg/mL in solubility checks performed in–house. The test item formed the best doseable suspension in acetone at 100 mg/mL, therefore, this solvent was selected.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments: Two - plate incorporation and pre-incubation methods

METHOD OF TREATMENT/ EXPOSURE:
- Test substance in agar (plate incorporation) and pre-incubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 20 minutes (pre-incubation method)
- Exposure duration/duration of treatment: 48 to 72 hours

Evaluation criteria:

Evaluation criteria -
There are several criteria for determining a positive result. Any one, or all, of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537.
A test item is considered non-mutagenic (negative) in the test system if the above criteria are not met.

Statistics:

Statistical significance was not included as part of the result evaluation.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Checks: Prior to use, the relevant strains were checked for characteristics (deep rough character, ampicillin resistance, UV light sensitivity and histidine or tryptophan auxotrophy), viability and spontaneous reversion rate and all checks were found to be satisfactory.
- Sterility: The amino acid supplemented top agar and the S9-mix used in both experiments were shown to be sterile. The test item formulation was also shown to be sterile.
- Precipitation: A precipitate of the test item (white and particulate in appearance) was noted at and above 500 g/plate in both the presence and absence of S9-mix. This precipitate did not prevent the scoring of revertant colonies.

STUDY RESULTS
- Concurrent vehicle control data: The solvent (acetone) control plates gave counts of revertant colonies within the normal range.
- Concurrent negative control data: Results for the untreated controls (spontaneous mutation rates) and viability were considered to be acceptable.
- Concurrent positive control data: All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation.
- Validity: As the controls were valid, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Remarks on result:

other: all strains/cell types tested

Table 1: Experiment 1 – Without Metabolic Activation (Plate Incorporation)

Test Period		From: 28 October 2021					To: 31 October 2021
S9-Mix (-)	Dose Level Per Plate	Number of revertants (rounded mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (Acetone)	105 116 120	(114) 7.8#	26 28 18	(24) 5.3	16 24 38		(26) 11.1	18 15 17	(17) 1.5	6 9 11	(9) 2.5
	1.5 µg	125 141 134	(133) 8.0	17 23 17	(19) 3.5	26 24 29		(26) 2.5	22 18 23	(21) 2.6	6 13 7	(9) 3.8
	5 µg	133 145 149	(142) 8.3	14 14 14	(14) 0.0	34 25 29		(29) 4.5	19 21 19	(20) 1.2	9 9 12	(10) 1.7
	15 µg	131 118 128	(126) 6.8	19 24 22	(22) 2.5	33 25 25		(28) 4.6	18 21 20	(20) 1.5	14 12 6	(11) 4.2
	50 µg	156 119 125	(133) 19.9	19 10 19	(16) 5.2	20 30 31		(27) 6.1	21 18 18	(19) 1.7	10 10 9	(10) 0.6
	150 µg	126 110 138	(125) 14.0	21 15 15	(17) 3.5	21 35 31		(29) 7.2	16 15 21	(17) 3.2	10 11 6	(9) 2.6
	500 µg	119 P 115 P 114 P	(116) 2.6	9 P 19 P 19 P	(16) 5.8	22 P 20 P 21 P		(21) 1.0	16 P 18 P 19 P	(18) 1.5	11 P 7 P 8 P	(9) 2.1
	1500 µg	117 P 138 P 128 P	(128) 10.5	17 P 20 P 19 P	(19) 1.5	25 P 32 P 24 P		(27) 4.4	19 P 18 P 15 P	(17) 2.1	10 P 6 P 12 P	(9) 3.1
	5000 µg	107 P 89 P 102 P	(99) 9.3	20 P 27 P 19 P	(22) 4.4	25 P 21 P 30 P		(25) 4.5	16 P 11 P 15 P	(14) 2.6	8 P 10 P 17 P	(12) 4.7
Positive controls S9-Mix (-)	Name	ENNG		ENNG		ENNG			4NQO		9AA
	Dose Level	3 µg		5 µg		2 µg			0.2 µg		80 µg
	No. of Revertants	469 472 487	(476) 9.6	681 593 772	(682) 89.5	427 439 398		(421) 21.1	103 104 91	(99) 7.2	435 322 380	(379) 56.5

ENNG        N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO         4-Nitroquinoline-1-oxide

9AA           9-Aminoacridine

P               Test item precipitate

#         Standard deviation

Table 2: Experiment 1 – With Metabolic Activation (Plate Incorporation)

Test Period		From: 28 October 2021					To: 31 October 2021
S9-Mix (+)	Dose Level Per Plate	Number of revertants (rounded mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (Acetone)	134 112 123	(123) 11.0#	17 29 19	(22) 6.4	32 39 31		(34) 4.4	23 17 15	(18) 4.2	16 8 14	(13) 4.2
	1.5 µg	139 129 119	(129) 10.0	21 21 22	(21) 0.6	23 22 21		(22) 1.0	21 15 20	(19) 3.2	13 9 12	(11) 2.1
	5 µg	104 129 114	(116) 12.6	12 26 20	(19) 7.0	30 27 24		(27) 3.0	21 24 17	(21) 3.5	23 12 11	(15) 6.7
	15 µg	129 105 130	(121) 14.2	19 20 21	(20) 1.0	28 26 27		(27) 1.0	15 20 18	(18) 2.5	15 15 14	(15) 0.6
	50 µg	140 127 126	(131) 7.8	18 16 21	(18) 2.5	33 26 23		(27) 5.1	24 17 17	(19) 4.0	7 13 8	(9) 3.2
	150 µg	116 101 112	(110) 7.8	13 19 18	(17) 3.2	37 31 22		(30) 7.5	19 23 14	(19) 4.5	9 9 20	(13) 6.4
	500 µg	112 P 130 P 130 P	(124) 10.4	25 P 20 P 21 P	(22) 2.6	27 P 24 P 29 P		(27) 2.5	19 P 17 P 14 P	(17) 2.5	15 P 12 P 16 P	(14) 2.1
	1500 µg	100 P 104 P 95 P	(100) 4.5	13 P 21 P 21 P	(18) 4.6	29 P 29 P 28 P		(29) 0.6	14 P 15 P 16 P	(15) 1.0	14 P 17 P 13 P	(15) 2.1
	5000 µg	104 P 105 P 114 P	(108) 5.5	19 P 23 P 20 P	(21) 2.1	21 P 34 P 24 P		(26) 6.8	17 P 14 P 21 P	(17) 3.5	13 P 9 P 16 P	(13) 3.5
Positive controls S9-Mix (+)	Name	2AA		2AA		2AA			BP		2AA
	Dose Level	1 µg		2 µg		10 µg			5 µg		2 µg
	No. of Revertants	865 722 662	(750) 104.3	176 164 181	(174) 8.7	213 202 192		(202) 10.5	231 249 207	(229) 21.1	141 132 119	(131) 11.1

BP          Benzo(a)pyrene

2AA        2-Aminoanthracene

P            Test item precipitate

#         Standard deviation

Table 3: Experiment 2 – Without Metabolic Activation (Pre-Incubation)

Test Period		From: 22 November 2021					To: 25 November 2021
S9-Mix (-)	Dose Level Per Plate	Number of revertants (rounded mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (Acetone)	109 82 106	(99) 14.8#	11 11 7	(10) 2.3	17 12 12		(14) 2.9	22 16 20	(19) 3.1	8 9 8	(8) 0.6
	15 µg	102 107 95	(101) 6.0	10 11 10	(10) 0.6	17 10 17		(15) 4.0	10 16 25	(17) 7.5	9 4 8	(7) 2.6
	50 µg	106 93 105	(101) 7.2	16 7 14	(12) 4.7	19 9 16		(15) 5.1	28 21 19	(23) 4.7	9 7 3	(6) 3.1
	150 µg	61 101 108	(90) 25.4	C 14 9	(12) 3.5	22 27 15		(21) 6.0	24 20 11	(18) 6.7	6 8 5	(6) 1.5
	500 µg	67 P 60 P 63 P	(63) 3.5	6 P 18 P 9 P	(11) 6.2	19 P 21 P 22 P		(21) 1.5	11 P 21 P 20 P	(17) 5.5	7 P 12 P 9 P	(9) 2.5
	1500 µg	86 P 85 P 88 P	(86) 1.5	9 P 6 P 9 P	(8) 1.7	19 P 11 P 21 P		(17) 5.3	16 P 22 P 26 P	(21) 5.0	7 P 5 P 11 P	(8) 3.1
	5000 µg	92 P 124 P 115 P	(110) 16.5	12 P 10 P 8 P	(10) 2.0	17 P 14 P 20 P		(17) 3.0	21 P 18 P 21 P	(20) 1.7	14 P 11 P 4 P	(10) 5.1
Positive controls S9-Mix (-)	Name	ENNG		ENNG		ENNG			4NQO		9AA
	Dose Level	3 µg		5 µg		2 µg			0.2 µg		80 µg
	No. of Revertants	1894 872 1099	(1288) 536.7	2793 2718 3243	(2918) 283.9	1034 1069 1049		(1051) 17.6	163 154 117	(145) 24.4	87 210 208	(168) 70.4

ENNG        N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO         4-Nitroquinoline-1-oxide

9AA           9-Aminoacridine

P               Test item precipitate

#         Standard deviation

Table 4: Experiment 2 – With Metabolic Activation (Pre-Incubation)

Test Period		From: 22 November 2021					To: 25 November 2021
S9-Mix (+)	Dose Level Per Plate	Number of revertants (rounded mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (Acetone)	130 147 134	(137) 8.9#	7 7 16	(10) 5.2	21 25 25		(24) 2.3	23 32 28	(28) 4.5	13 9 9	(10) 2.3
	15 µg	156 154 185	(165) 17.3	6 13 16	(12) 5.1	31 19 22		(24) 6.2	27 30 24	(27) 3.0	5 7 10	(7) 2.5
	50 µg	145 141 158	(148) 8.9	13 13 11	(12) 1.2	20 19 16		(18) 2.1	22 24 31	(26) 4.7	19 15 10	(15) 4.5
	150 µg	129 144 150	(141) 10.8	16 18 13	(16) 2.5	16 25 17		(19) 4.9	17 30 18	(22) 7.2	13 16 10	(13) 3.0
	500 µg	160 P 135 P 152 P	(149) 12.8	12 P 9 P 14 P	(12) 2.5	28 P 16 P 21 P		(22) 6.0	30 P 24 P 38 P	(31) 7.0	14 P 5 P 12 P	(10) 4.7
	1500 µg	151 P 152 P 129 P	(144) 13.0	17 P 15 P 16 P	(16) 1.0	20 P 17 P 30 P		(22) 6.8	38 P 33 P 24 P	(32) 7.1	11 P 12 P 15 P	(13) 2.1
	5000 µg	182 P 143 P 123 P	(149) 30.0	16 P 10 P 9 P	(12) 3.8	17 P 14 P 17 P		(16) 1.7	28 P 21 P 35 P	(28) 7.0	14 P 10 P 12 P	(12) 2.0
Positive controls S9-Mix (+)	Name	2AA		2AA		2AA			BP		2AA
	Dose Level	1 µg		2 µg		10 µg			5 µg		2 µg
	No. of Revertants	641 592 562	(598) 39.9	97 118 133	(116) 18.1	98 74 128		(100) 27.1	177 176 176	(176) 0.6	99 118 163	(127) 32.9

BP          Benzo(a)pyrene

2AA        2-Aminoanthracene

P            Test item precipitate

#         Standard deviation

Conclusions:

The in vitro gene mutation potential in bacteria of PU05 was determined to be negative.

Executive summary:

The in vitro gene mutation potential in bacteria of PU05 was tested in an Ames test (OECD 471) and was determined to be negative. Bacterial S. typhimurium and E. coli strains were treated with suspensions of PU05 at eight dose levels in triplicate, both with and without metabolic activation, via plate incorporation and pre-incubation assays. The vehicle, positive and negative controls confirmed the sensitivity of the assay and the efficacy of the S9-mix.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Remarks:

This study is included in a NONS registration and therefore has been evaluated by a relevant competent authority and is considered to be reliable.

Qualifier:

according to guideline

Guideline:

other: Annex V (Ames)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Remarks:

and TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S9

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 10 ... 5000 μg/plate
Concentration range in the main test (without metabolic activation): 10 ... 5000 μg/plate

Vehicle / solvent:

Solvent: Dimethylsulfoxide

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

other: 4-nitro-ortho-ohenylenediamine and 2-aminoanthracene

Details on test system and experimental conditions:

Concentration of the test substance resulting in precipitation: 333 μg/plate

Species / strain:

other: Strains as specified above, preliminary test

Metabolic activation:

with

Genotoxicity:

not specified

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>5000 µg/plate

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

primary culture, other: Strains as specified above, preliminary test

Metabolic activation:

without

Genotoxicity:

not specified

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

> 5000 µg/plate

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

other: Strains as specified above, main test

Metabolic activation:

with

Genotoxicity:

not specified

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

> 5000 µg/plate

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

other: Strains as specified above, main test

Metabolic activation:

without

Genotoxicity:

not specified

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>5000 µg/plate

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

Observations: The plates incubated with the test substance showed normal background growth at concentrations up to 5000 µg/plate. No increases in the numbers of revertant colonies were seen at any of the concentrations of the test substance with or without metabolic activation.

Positive control substances sodium azide, 4-nitro-ortho-ohenylenediamine) and 2-aminoanthracene led to increases in induce revertant colonies in the appropriate bacterial strains.

Conclusions:

The Ames test was negative with metabolic activation and without metabolic activation.

Executive summary:

The test item was investigated for in vitro genetic toxicity at the concentration in an Ames test with a concentration range of 10 to 5000 μg/plate, with and without metabolic activation, following a standard guideline. The study included TA98, TA100, TA1535, TA1537 and TA1538 strains. Positive control substances were also included. The Ames test was negative with metabolic activation and negative without metabolic activation as no increases in the numbers of revertant colonies were seen at any of the concentrations of the test substance, with or without metabolic activation.

The study is a GLP compliant, guideline study and is acceptable with restrictions for assessment of this endpoint.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Remarks:

This study is included in a NONS registration and therefore has been evaluated by a relevant competent authority and is considered to be reliable.

Qualifier:

according to guideline

Guideline:

other: Annex V (Ames)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S9

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 10 ... 5000 μg/plate
Concentration range in the main test (without metabolic activation): 10 ... 5000 μg/plate

Vehicle / solvent:

Solvent: Dimethylsulfoxide

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

not specified

Details on test system and experimental conditions:

Concentration of the test substance resulting in precipitation: 333 μg/plate

Species / strain:

other: Strains specified above

Remarks:

Preliminary test

Metabolic activation:

with

Genotoxicity:

not specified

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>5000 ug/plate

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

other: Strains specified above

Remarks:

Preliminary test

Metabolic activation:

without

Genotoxicity:

not specified

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

> 5000 ug/plate

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

other: Strains specified above

Remarks:

Main test

Metabolic activation:

with

Genotoxicity:

not specified

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

> 5000 ug/plate

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

other: Strains specified above

Remarks:

Main test

Metabolic activation:

without

Genotoxicity:

not specified

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>5000 ug/plate

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

Observations: No increases in the number of revertant colonies were seen in any of the bacterial strains treated with the test substance, either in the presence or absence of S9.
Positive control substance led to significant increases in the number of revertant colonies compared with controls.

Conclusions:

The Ames test was negative with metabolic activation and negative without metabolic activation.

Executive summary:

The test item was investigated in an in vitro genetic toxicity study (Ames test) at a concentration range of 10 to 5000 μg/plate, along with a positive control and solvent control. The strains TA98, TA100, TA1535 and TA1537 were included in the test. The test was conducted with and without metabolic activation. The Ames test was negative with metabolic activation and negative without metabolic activation.

The study is a GLP compliant, guideline study and is acceptable with restrictions for assessment of this endpoint.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Remarks:

This study is included in a NONS registration and therefore has been evaluated by a relevant competent authority and is considered to be reliable.

Qualifier:

according to guideline

Guideline:

other: Annex V

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S9

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 10 ... 5000 μg/plate
Concentration range in the main test (without metabolic activation): 10 ... 5000 μg/plate
Concentration of the test substance resulting in precipitation: 5000 μg/plate

Vehicle / solvent:

Solvent: DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxic at 5000 μg/plate, with and without metabolic activation (in one experiment only)

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: No increase in revertants was observed up to > 5000 μg/plate in the preliminary test. No toxicity observed up to 5000 μg/plate for TA98 and TA100, with and without metabolic activation.

Conclusions:

The Ames test concluded that the test item was negative with and without metabolic activation.

Executive summary:

The test item was investigated for in vitro genetic toxicity in Salmonella typhimurium bacteria strains TA98, TA100, TA1535, TA1537 and TA1538 following EU Annex V guidelines. Bacteria were exposed to the test item in the concentration range of 10 to 5000 μg/plate, with and without metabolic activation. The test item was observed to precipitate at the highest concentration of 5000 μg/plate. The test item was cytotoxic at 5000 μg/plate for TA1538, however this was observed in one experiment only. The test concluded that the test item was negative with and without metabolic activation.

The study is a GLP compliant, guideline experimental study and is acceptable with restrictions for assessment of this endpoint.

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Remarks:

This study is included in a NONS registration and therefore has been evaluated by a relevant competent authority and is considered to be reliable.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Remarks:

and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9 mix prepared from the microsomal fraction (S9 mix fractions) of rat liver induced by Aroclor 1254.

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 3.13 ... 50 μg/plate
Concentration range in the main test (without metabolic activation): 3.13 ... 50 μg/plate

Vehicle / solvent:

Solvent: Ethanol

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Details on test system and experimental conditions:

The product is insoluble in vehicles usually used in this test. A suspension of homogeneous appearance could not be obtained, therefore an extract produced in ethanol has been tested. The doses are therefore expressed in μL of ethanol extract per plate.
The highest dose tested is 50 μL per plate which corresponds to the highest volume used for this vehicle in this type of test in the laboratory

Species / strain:

other: main test

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

other: Main test

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Conclusions:

The Ames test concluded that the test item was negative with metabolic activation and negative without metabolic activation.

Executive summary:

The test item was investigated for in vitro genetic toxicity at the concentration range of 3.13 to 50 μg/plate in an Ames test. The study was conducted with and without metabolic activation following the OECD 471 and EU Method B13/14 guidelines. The Ames test concluded that the test item was negative with metabolic activation and negative without metabolic activation.

The study is a GLP compliant, guideline experimental study and is acceptable with restrictions for assessment of this endpoint.

Reference 6

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Remarks:

This study is included in a NONS registration and therefore has been evaluated by a relevant competent authority and is considered to be reliable.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

human lymphoblastoid cells (TK6)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix prepared from the microsomal fraction (S9 mix fractions) of rat liver induced by Aroclor 1254.

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 12 ... 15 μg/mL
Concentration range in the main test (without metabolic activation): 12 ... 15 μg/mL

Vehicle / solvent:

Solvent: Ethanol

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Details on test system and experimental conditions:

Exposure period (with metabolic activation): 3 hours
Exposure period (without metabolic activation): 44 hours
Expression time:
First test: 3 hours exposure with and without S9 mix.
Second test: exposure of 20 and 44 hours without S9 mix.
Exposure of 3 hours with S9 mix.
Selection time:
First test: collection 20 hours after the start of treatment (about 1 and a half normal cell cycle).
Second test: collection 20 and 44 hours after the start of treatment (about 1 and a half normal cell cycle, and 24 hours later).

Species / strain:

other: main test

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

other: Main test

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

No dose-dependent anomaly observed during the two trials

Conclusions:

The study concluded that the test was negative with metabolic activation and negative without metabolic activation.

Executive summary:

The test item was investigated for in vitro genetic toxicity at a concentration range of 12 to 15 μg/mL. The test was conducted with and without metabolic activation following the OECD 473 and EU Method B10 guidelines. The test item is insoluble in the vehicles usually used in this test and no suspension of homogeneous appearance could be obtained, therefore an extract of the product in ethanol was tested. The doses are therefore expressed in μL of ethanol extract per tube (containing 5.5 mL of final treatment volume). The highest dose tested is 15 μL per tube which corresponds to the largest volume used for this vehicle in this type of test in the laboratory.

No dose-dependent anomaly was observed during these two trials. The study concluded that the test item was negative with metabolic activation and negative without metabolic activation.

The study is a GLP compliant, guideline experimental study and is acceptable with restrictions for assessment of this endpoint.

Reference 7

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22/10/2021 - 19/11/2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997, updated 2020

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2008

Deviations:

no

Principles of method if other than guideline:

Based on provisions in the guidelines:
The Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy, Trade and Industry (METI), and Ministry of the Environment (MOE) Guidelines of 31 March 2011
ICH S2(R1) guideline adopted June 2012 (ICH S2(R1) Federal Register. Adopted 2012; 77:33748-33749)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

- Salmonella: Histidine
- E. Coli: Tryptophan

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

Bacteria

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Bacteria

Metabolic activation:

with and without

Metabolic activation system:

- Type of metabolic activation system: Lyophilized phenobarbital/beta-naphthoflavone induced rat liver S9 and cofactors mix (MutazymeTM)
- Source of S9: Prepared in lab before use
- S9 mix: Final concentrations of S9-mix of approximately 10% (v/v), MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium phosphate buffer (100 mM, pH 7.4)
- Concentration: 0.5 mL of S9-mix was added to the molten, trace amino-acid supplemented media

Test concentrations with justification for top dose:

Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate
Pre-Incubation Method: 15, 50, 150, 500, 1500 and 5000 μg/plate.
The maximum concentration was 5000 μg/plate (the OECD TG 471 maximum recommended dose level).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: The test item was insoluble in sterile distilled water, dimethyl sulphoxide (DMSO), dimethyl formamide and acetonitrile at 50 mg/mL, acetone at 100 mg/mL and tetrahydrofuran at 200 mg/mL in solubility checks performed in–house. The test item formed the best doseable suspension in DMSO at a maximum concentration of 25 mg/mL, therefore, this solvent was selected.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments: Two - plate incorporation and pre-incubation methods

METHOD OF TREATMENT/ EXPOSURE:
- Test substance in agar (plate incorporation) and pre-incubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 20 minutes (pre-incubation method)
- Exposure duration/duration of treatment: 48 to 72 hours

Evaluation criteria:

Evaluation criteria -
There are several criteria for determining a positive result. Any one, or all, of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537.
A test item is considered non-mutagenic (negative) in the test system if the above criteria are not met.

Statistics:

Statistical significance was not included as part of the result evaluation.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Checks: Prior to use, the relevant strains were checked for characteristics (deep rough character, ampicillin resistance, UV light sensitivity and histidine or tryptophan auxotrophy), viability and spontaneous reversion rate (all checks were found to be satisfactory).
- Sterility: The amino acid supplemented top agar and the S9-mix used in both experiments were shown to be sterile. The test item formulation was also shown to be sterile.
- Precipitation: A precipitate of the test item (off-white and particulate in appearance) was noted at and above 500 µg/plate (plate incorporation method) and at and above 1500 µg/plate (pre-incubation method) in both the presence and absence of S9-mix. This precipitate did not prevent the scoring of revertant colonies.

STUDY RESULTS
- Concurrent vehicle control data: The number of revertant counts for the solvent (dimethyl sulphoxide (DMSO)) control plates were within or close to the normal range. Three revertant colony counts (WP2uvrA untreated control dosed after the first mutation test and TA98 vehicle control dosed in the absence of S9-mix and TA1537 untreated control dosed after the second mutation test) were marginally below the minimum level of the in-house historical untreated/vehicle control minima for the tester strains. These counts were considered acceptable as the other vehicle and untreated control counts were within expected range and the tester strains responded very well with the respective positive controls in both the presence and absence of S9 mix.
- Concurrent negative control data: Results for the untreated controls (spontaneous mutation rates) and viability were considered to be acceptable.
- Concurrent positive control data: All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation.
- Validity: As the controls were valid, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Remarks on result:

other: all strains/cell types tested

Table 1: Experiment 1 – Without Metabolic Activation (Plate Incorporation)

Test Period		From: 26 October 2021					To: 29 October 2021
S9-Mix (-)	Dose Level Per Plate	Number of revertants (rounded mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (DMSO)	99 109 107	(105) 5.3#	18 14 17	(16) 2.1	16 13 15		(15) 1.5	19 13 20	(17) 3.8	12 10 10	(11) 1.2
	1.5 µg	122 134 132	(129) 6.4	20 16 8	(15) 6.1	17 13 22		(17) 4.5	15 12 21	(16) 4.6	4 9 4	(6) 2.9
	5 µg	100 141 114	(118) 20.8	16 15 9	(13) 3.8	10 13 10		(11) 1.7	21 18 18	(19) 1.7	5 10 15	(10) 5.0
	15 µg	112 125 114	(117) 7.0	11 11 13	(12) 1.2	12 23 13		(16) 6.1	13 19 12	(15) 3.8	12 15 10	(12) 2.5
	50 µg	137 135 122	(131) 8.1	13 9 9	(10) 2.3	15 12 15		(14) 1.7	10 26 19	(18) 8.0	11 11 17	(13) 3.5
	150 µg	135 108 106	(116) 16.2	21 9 14	(15) 6.0	13 14 10		(12) 2.1	21 20 23	(21) 1.5	15 4 14	(11) 6.1
	500 µg	111 P 117 P 92 P	(107) 13.1	12 P 17 P 21 P	(17) 4.5	21 P 26 P 24 P		(24) 2.5	17 P 15 P 12 P	(15) 2.5	5 P 5 P 11 P	(7) 3.5
	1500 µg	129 P 130 P 169 P	(143) 22.8	11 P 7 P 14 P	(11) 3.5	18 P 15 P 20 P		(18) 2.5	17 P 24 P 23 P	(21) 3.8	6 P 7 P 9 P	(7) 1.5
	5000 µg	84 P 136 P 86 P	(102) 29.5	16 P 14 P 11 P	(14) 2.5	16 P 9 P 13 P		(13) 3.5	12 P 11 P 10 P	(11) 1.0	8 P 6 P 5 P	(6) 1.5
Positive controls S9-Mix (-)	Name	ENNG		ENNG		ENNG			4NQO		9AA
	Dose Level	3 µg		5 µg		2 µg			0.2 µg		80 µg
	No. of Revertants	738 829 793	(787) 45.8	1347 1693 1423	(1488) 181.8	321 322 316		(320) 3.2	133 140 137	(137) 3.5	492 414 715	(540) 156.2

ENNG        N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO         4-Nitroquinoline-1-oxide

9AA           9-Aminoacridine

P               Test item precipitate

#         Standard deviation

Table 2: Experiment 1 – With Metabolic Activation (Plate Incorporation)

Test Period		From: 26 October 2021					To: 29 October 2021
S9-Mix (+)	Dose Level Per Plate	Number of revertants (rounded mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (DMSO)	174 177 172	(174) 2.5#	12 10 13	(12) 1.5	16 13 18		(16) 2.5	16 25 25	(22) 5.2	14 11 14	(13) 1.7
	1.5 µg	177 152 176	(168) 14.2	13 7 16	(12) 4.6	21 14 18		(18) 3.5	21 20 24	(22) 2.1	8 6 10	(8) 2.0
	5 µg	162 165 153	(160) 6.2	22 12 11	(15) 6.1	15 21 18		(18) 3.0	27 29 21	(26) 4.2	8 8 12	(9) 2.3
	15 µg	170 129 148	(149) 20.5	13 12 11	(12) 1.0	19 15 16		(17) 2.1	28 28 16	(24) 6.9	8 10 8	(9) 1.2
	50 µg	176 177 180	(178) 2.1	14 14 16	(15) 1.2	20 16 19		(18) 2.1	23 25 22	(23) 1.5	8 21 14	(14) 6.5
	150 µg	166 169 166	(167) 1.7	5 19 22	(15) 9.1	16 25 15		(19) 5.5	21 23 24	(23) 1.5	12 13 13	(13) 0.6
	500 µg	175 P 158 P 174 P	(169) 9.5	20 P 12 P 16 P	(16) 4.0	8 P 14 P 13 P		(12) 3.2	23 P 27 P 18 P	(23) 4.5	13 P 15 P 11 P	(13) 2.0
	1500 µg	173 P 147 P 142 P	(154) 16.6	10 P 13 P 16 P	(13) 3.0	17 P 22 P 24 P		(21) 3.6	25 P 16 P 27 P	(23) 5.9	16 P 12 P 12 P	(13) 2.3
	5000 µg	124 P 97 P 125 P	(115) 15.9	14 P 11 P 13 P	(13) 1.5	15 P 14 P 16 P		(15) 1.0	24 P 22 P 22 P	(23) 1.2	3 P 10 P 14 P	(9) 5.6
Positive controls S9-Mix (+)	Name	2AA		2AA		2AA			BP		2AA
	Dose Level	1 µg		2 µg		10 µg			5 µg		2 µg
	No. of Revertants	1473 1507 1393	(1458) 58.5	177 181 179	(179) 2.0	250 254 260		(255) 5.0	361 376 365	(367) 7.8	205 211 225	(214) 10.3

BP          Benzo(a)pyrene

2AA        2-Aminoanthracene

P            Test item precipitate

#         Standard deviation

Table 3: Experiment 2 – Without Metabolic Activation (Pre-Incubation)

Test Period		From: 16 November 2021					To: 19 November 2021
S9-Mix (-)	Dose Level Per Plate	Number of revertants (rounded mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (DMSO)	84 96 106	(95) 11.0#	7 11 13	(10) 3.1	21 12 14		(16) 4.7	18 21 8	(16) 6.8	8 16 24	(16) 8.0
	15 µg	92 93 93	(93) 0.6	6 12 9	(9) 3.0	8 10 21		(13) 7.0	11 15 9	(12) 3.1	8 18 21	(16) 6.8
	50 µg	98 95 106	(100) 5.7	15 10 12	(12) 2.5	18 19 14		(17) 2.6	17 15 15	(16) 1.2	8 8 20	(12) 6.9
	150 µg	98 90 97	(95) 4.4	15 12 15	(14) 1.7	21 8 15		(15) 6.5	20 17 16	(18) 2.1	13 13 13	(13) 0.0
	500 µg	110 97 100	(102) 6.8	14 10 15	(13) 2.6	17 17 16		(17) 0.6	24 24 22	(23) 1.2	23 24 21	(23) 1.5
	1500 µg	85 P 99 P 81 P	(88) 9.5	13 P 8 P 11 P	(11) 2.5	13 P 14 P 13 P		(13) 0.6	16 P 23 P 16 P	(18) 4.0	20 P 25 P 17 P	(21) 4.0
	5000 µg	89 P 91 P 96 P	(92) 3.6	12 P 8 P 16 P	(12) 4.0	22 P 23 P 23 P		(23) 0.6	27 P 15 P 16 P	(19) 6.7	16 P 16 P 18 P	(17) 1.2
Positive controls S9-Mix (-)	Name	ENNG		ENNG		ENNG			4NQO		9AA
	Dose Level	3 µg		5 µg		2 µg			0.2 µg		80 µg
	No. of Revertants	755 728 720	(734) 18.3	2264 2046 1728	(2013) 269.6	575 528 491		(531) 42.1	227 219 238	(228) 9.5	176 315 374	(288) 101.7

ENNG        N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO         4-Nitroquinoline-1-oxide

9AA           9-Aminoacridine

P               Test item precipitate

#         Standard deviation

Table 4: Experiment 2 – With Metabolic Activation (Pre-Incubation)

Test Period		From: 16 November 2021					To: 19 November 2021
S9-Mix (+)	Dose Level Per Plate	Number of revertants (rounded mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (DMSO)	95 99 99	(98) 2.3#	7 11 17	(12) 5.0	27 16 21		(21) 5.5	23 20 28	(24) 4.0	10 18 6	(11) 6.1
	15 µg	100 98 84	(94) 8.7	14 15 6	(12) 4.9	19 15 23		(19) 4.0	25 24 25	(25) 0.6	13 10 15	(13) 2.5
	50 µg	96 92 93	(94) 2.1	16 15 14	(15) 1.0	21 17 20		(19) 2.1	29 22 23	(25) 3.8	14 10 16	(13) 3.1
	150 µg	101 100 103	(101) 1.5	12 11 10	(11) 1.0	16 24 17		(19) 4.4	20 26 19	(22) 3.8	10 12 16	(13) 3.1
	500 µg	97 106 98	(100) 4.9	10 18 16	(15) 4.2	33 25 23		(27) 5.3	33 25 29	(29) 4.0	21 12 13	(15) 4.9
	1500 µg	93 P 101 P 95 P	(96) 4.2	15 P 17 P 17 P	(16) 1.2	21 P 23 P 21 P		(22) 1.2	30 P 31 P 30 P	(30) 0.6	15 P 23 P 23 P	(20) 4.6
	5000 µg	92 P 96 P 82 P	(90) 7.2	17 P 16 P 17 P	(17) 0.6	29 P 30 P 22 P		(27) 4.4	33 P 30 P 21 P	(28) 6.2	18 P 20 P 15 P	(18) 2.5
Positive controls S9-Mix (+)	Name	2AA		2AA		2AA			BP		2AA
	Dose Level	1 µg		2 µg		10 µg			5 µg		2 µg
	No. of Revertants	562 591 531	(561) 30.0	132 115 102	(116) 15.0	87 122 153		(121) 33.0	203 148 176	(176) 27.5	103 123 107	(111) 10.6

BP          Benzo(a)pyrene

2AA        2-Aminoanthracene

P            Test item precipitate

#         Standard deviation

Conclusions:

The in vitro gene mutation potential in bacteria of A003 was determined to be negative.

Executive summary:

The in vitro gene mutation potential in bacteria of A003 was tested in an Ames test (OECD 471) and was determined to be negative. Bacterial S. typhimurium and E. coli strains were treated with suspensions of A003 at eight dose levels in triplicate, both with and without metabolic activation, via plate incorporation and pre-incubation assays. The vehicle, positive and negative controls confirmed the sensitivity of the assay and the efficacy of the S9-mix.

Reference 8

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

other information

Justification for type of information:

Experimental study is on-going. This record will be updated with the results of the study once completed.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Reference 9

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13/10/2021 - 12/11/2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997, corrected 2020

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2008

Deviations:

no

Principles of method if other than guideline:

Based on provisions in the guidelines:
The Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy, Trade and Industry (METI), and Ministry of the Environment (MOE) Guidelines of 31 March 2011
ICH S2(R1) guideline adopted June 2012 (ICH S2(R1) Federal Register. Adopted 2012; 77:33748-33749)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

- Salmonella: Histidine
- E. Coli: Tryptophan

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

Bacteria

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Bacteria

Metabolic activation:

with and without

Metabolic activation system:

- Type of metabolic activation system: Lyophilized phenobarbital/beta-naphthoflavone induced rat liver S9 and cofactors mix (MutazymeTM)
- Source of S9: Prepared in lab before use
- S9 mix: Final concentrations of S9-mix of approximately 10% (v/v), MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium phosphate buffer (100 mM, pH 7.4)
- Concentration: 0.5 mL of S9-mix was added to the molten, trace amino-acid supplemented media

Test concentrations with justification for top dose:

Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate
Pre-Incubation Method: 15, 50, 150, 500, 1500 and 5000 μg/plate.
The maximum concentration was 5000 μg/plate (the OECD TG 471 maximum recommended dose level).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl formamide (DMF)
- Justification for choice of solvent/vehicle: The test item was insoluble in sterile distilled water, dimethyl sulphoxide, dimethyl formamide and acetonitrile at 50 mg/mL, acetone at 100 mg/mL and tetrahydrofuran at 200 mg/mL in solubility checks performed in–house. The test item formed the best doseable suspension in dimethyl formamide, therefore, this solvent was selected.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments: Two - plate incorporation and pre-incubation methods

METHOD OF TREATMENT/ EXPOSURE:
- Test substance in agar (plate incorporation) and pre-incubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 20 minutes (pre-incubation method)
- Exposure duration/duration of treatment: 48 to 72 hours

Evaluation criteria:

Evaluation criteria -
There are several criteria for determining a positive result. Any one, or all, of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537.
A test item is considered non-mutagenic (negative) in the test system if the above criteria are not met.

Statistics:

Statistical significance was not included as part of the result evaluation.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Checks: Prior to use, the relevant strains were checked for characteristics (deep rough character, ampicillin resistance, UV light sensitivity and histidine or tryptophan auxotrophy), viability and spontaneous reversion rate (all checks were found to be satisfactory).
- Sterility: The amino acid supplemented top agar and the S9-mix used in both experiments were shown to be sterile. The test item formulation was also shown to be sterile.
- Precipitation: A precipitate of the test item (off-white and particulate in appearance) was noted at and above 500 μg/plate in both the presence and absence of S9-mix. This precipitate did not prevent the scoring of revertant colonies.

STUDY RESULTS
- Concurrent vehicle control data: The solvent (dimethyl formamide) control plates gave counts of revertant colonies generally within the normal range. In the second experiment, a single solvent count for Wp2uvrA in the presence of S9 was slightly below the historical range however this was considered acceptable as the average and other individual solvent counts were within the expected range. Two further individual plate counts in the second experiment (TA1535 in the absence of S9 and TA98 in the presence S9) were slightly outside the 95% confidence limits but within the overall expected range, the average count was within the expected ranges, therefore these counts were also considered acceptable.
- Concurrent negative control data: Results for the untreated controls (spontaneous mutation rates) and viability were considered to be acceptable.
- Concurrent positive control data: All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation.
- Validity: As the controls were valid, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Remarks on result:

other: all strains/cell types tested

Table 1: Experiment 1 – Without Metabolic Activation (Plate Incorporation)

Test Period		From: 22 October 2021					To: 25 October 2021
S9-Mix (-)	Dose Level Per Plate	Number of revertants (rounded mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (DMF)	82 119 111	(104) 19.5#	12 13 8	(11) 2.6	20 17 13		(17) 3.5	20 20 21	(20) 0.6	8 5 13	(9) 4.0
	1.5 µg	110 116 86	(104) 15.9	9 10 15	(11) 3.2	15 15 15		(15) 0.0	12 16 12	(13) 2.3	6 9 11	(9) 2.5
	5 µg	91 85 88	(88) 3.0	13 16 15	(15) 1.5	20 19 19		(19) 0.6	15 11 29	(18) 9.5	8 9 9	(9) 0.6
	15 µg	107 77 99	(94) 15.5	14 15 15	(15) 0.6	14 22 11		(16) 5.7	22 20 8	(17) 7.6	8 10 8	(9) 1.2
	50 µg	110 85 113	(103) 15.4	13 13 10	(12) 1.7	16 19 14		(16) 2.5	23 14 11	(16) 6.2	8 5 5	(6) 1.7
	150 µg	96 87 100	(94) 6.7	16 12 14	(14) 2.0	21 13 16		(17) 4.0	12 14 12	(13) 1.2	9 6 7	(7) 1.5
	500 µg	100 P 117 P 76 P	(98) 20.6	15 P 8 P 10 P	(11) 3.6	12 P 19 P 19 P		(17) 4.0	15 P 20 P 21 P	(19) 3.2	3 P 8 P 7 P	(6) 2.6
	1500 µg	114 P 100 P 93 P	(102) 10.7	13 P 6 P 13 P	(11) 4.0	22 P 9 P 18 P		(16) 6.7	20 P 26 P 23 P	(23) 3.0	9 P 14 P 5 P	(9) 4.5
	5000 µg	59 P 64 P 73 P	(65) 7.1	11 P 10 P 12 P	(11) 1.0	20 P 11 P 13 P		(15) 4.7	17 P 10 P 8 P	(12) 4.7	7 P 6 P 4 P	(6) 1.5
Positive controls S9-Mix (-)	Name	ENNG		ENNG		ENNG			4NQO		9AA
	Dose Level	3 µg		5 µg		2 µg			0.2 µg		80 µg
	No. of Revertants	602 580 571	(584) 15.9	787 910 623	(773) 144.0	342 420 381		(381) 39.0	94 97 103	(98) 4.6	222 384 246	(284) 87.4

ENNG        N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO         4-Nitroquinoline-1-oxide

9AA           9-Aminoacridine

P               Test item precipitate

#         Standard deviation

Table 2: Experiment 1 – With Metabolic Activation (Plate Incorporation)

Test Period		From: 22 October 2021					To: 25 October 2021
S9-Mix (+)	Dose Level Per Plate	Number of revertants (rounded mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (DMF)	125 101 116	(114) 12.1#	8 13 11	(11) 2.5	27 25 23		(25) 2.0	24 17 33	(25) 8.0	16 10 11	(12) 3.2
	1.5 µg	100 128 137	(122) 19.3	7 12 11	(10) 2.6	31 32 22		(28) 5.5	27 14 20	(20) 6.5	7 5 7	(6) 1.2
	5 µg	133 139 127	(133) 6.0	15 14 11	(13) 2.1	22 23 25		(23) 1.5	16 18 19	(18) 1.5	10 9 10	(10) 0.6
	15 µg	126 131 145	(134) 9.8	15 14 11	(13) 2.1	33 22 33		(29) 6.4	18 13 21	(17) 4.0	7 14 12	(11) 3.6
	50 µg	115 113 142	(123) 16.2	17 14 13	(15) 2.1	24 15 31		(23) 8.0	20 22 24	(22) 2.0	8 14 8	(10) 3.5
	150 µg	136 142 119	(132) 11.9	9 9 14	(11) 2.9	28 18 23		(23) 5.0	13 18 18	(16) 2.9	8 11 12	(10) 2.1
	500 µg	126 P 136 P 143 P	(135) 8.5	9 P 10 P 13 P	(11) 2.1	19 P 28 P 28 P		(25) 5.2	24 P 16 P 15 P	(18) 4.9	9 P 8 P 10 P	(9) 1.0
	1500 µg	141 P 135 P 110 P	(129) 16.4	6 P 8 P 8 P	(7) 1.2	34 P 28 P 24 P		(29) 5.0	20 P 13 P 19 P	(17) 3.8	11 P 2 P 6 P	(6) 4.5
	5000 µg	99 P 86 P 90 P	(92) 6.7	17 P 18 P 10 P	(15) 4.4	21 P 15 P 14 P		(17) 3.8	18 P 12 P 23 P	(18) 5.5	11 P 12 P 14 P	(12) 1.5
Positive controls S9-Mix (+)	Name	2AA		2AA		2AA			BP		2AA
	Dose Level	1 µg		2 µg		10 µg			5 µg		2 µg
	No. of Revertants	1055 1214 1182	(1150) 84.1	205 223 199	(209) 12.5	160 164 149		(158) 7.8	212 220 220	(217) 4.6	171 190 174	(178) 10.2

BP          Benzo(a)pyrene

2AA        2-Aminoanthracene

P            Test item precipitate

#         Standard deviation

Table 3: Experiment 2 – Without Metabolic Activation (Pre-Incubation)

Test Period		From: 09 November 2021					To: 12 November 2021
S9-Mix (-)	Dose Level Per Plate	Number of revertants (rounded mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (DMF)	139 149 148	(145) 5.5#	10 7 9	(9) 1.5	17 18 17		(17) 0.6	14 13 13	(13) 0.6	8 6 8	(7) 1.2
	15 µg	133 132 139	(135) 3.8	12 10 10	(11) 1.2	12 16 15		(14) 2.1	14 10 24	(16) 7.2	7 5 9	(7) 2.0
	50 µg	151 147 148	(149) 2.1	12 12 16	(13) 2.3	13 15 21		(16) 4.2	22 13 12	(16) 5.5	7 7 7	(7) 0.0
	150 µg	141 143 117	(134) 14.5	10 9 9	(9) 0.6	10 12 21		(14) 5.9	10 10 9	(10) 0.6	6 6 7	(6) 0.6
	500 µg	153 P 136 P 135 P	(141) 10.1	10 P 9 P 13 P	(11) 2.1	20 P 11 P 14 P		(15) 4.6	15 P 19 P 10 P	(15) 4.5	6 P 8 P 12 P	(9) 3.1
	1500 µg	142 P 139 P 142 P	(141) 1.7	13 P 9 P 12 P	(11) 2.1	10 P 20 P 11 P		(14) 5.5	15 P 17 P 12 P	(15) 2.5	9 P 7 P 5 P	(7) 2.0
	5000 µg	124 P 134 P 143 P	(134) 9.5	11 P 7 P 13 P	(10) 3.1	18 P 18 P 20 P		(19) 1.2	16 P 11 P 12 P	(13) 2.6	9 P 9 P 13 P	(10) 2.3
Positive controls S9-Mix (-)	Name	ENNG		ENNG		ENNG			4NQO		9AA
	Dose Level	3 µg		5 µg		2 µg			0.2 µg		80 µg
	No. of Revertants	587 652 791	(677) 104.2	239 328 207	(258) 62.7	156 179 162		(166) 11.9	88 99 92	(93) 5.6	145 202 216	(188) 37.6

ENNG        N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO         4-Nitroquinoline-1-oxide

9AA           9-Aminoacridine

P               Test item precipitate

#         Standard deviation

Table 4: Experiment 2 – With Metabolic Activation (Pre-Incubation)

Test Period		From: 09 November 2021					To: 12 November 2021
S9-Mix (+)	Dose Level Per Plate	Number of revertants (rounded mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (DMF)	157 150 159	(155) 4.7#	9 9 10	(9) 0.6	17 16 12		(15) 2.6	19 14 16	(16) 2.5	10 8 9	(9) 1.0
	15 µg	147 145 141	(144) 3.1	11 15 14	(13) 2.1	21 16 15		(17) 3.2	14 18 23	(18) 4.5	11 12 12	(12) 0.6
	50 µg	144 144 152	(147) 4.6	14 11 7	(11) 3.5	12 24 17		(18) 6.0	11 13 27	(17) 8.7	13 11 11	(12) 1.2
	150 µg	146 137 120	(134) 13.2	11 11 15	(12) 2.3	21 12 15		(16) 4.6	10 21 12	(14) 5.9	11 7 8	(9) 2.1
	500 µg	153 P 133 P 125 P	(137) 14.4	14 P 12 P 11 P	(12) 1.5	13 P 15 P 15 P		(14) 1.2	13 P 22 P 19 P	(18) 4.6	11 P 11 P 10 P	(11) 0.6
	1500 µg	143 P 154 P 144 P	(147) 6.1	8 P 17 P 7 P	(11) 5.5	17 P 15 P 15 P		(16) 1.2	22 P 21 P 29 P	(24) 4.4	10 P 9 P 10 P	(10) 0.6
	5000 µg	149 P 135 P 133 P	(139) 8.7	8 P 14 P 15 P	(12) 3.8	20 P 13 P 29 P		(21) 8.0	17 P 25 P 21 P	(21) 4.0	11 P 15 P 9 P	(12) 3.1
Positive controls S9-Mix (+)	Name	2AA		2AA		2AA			BP		2AA
	Dose Level	1 µg		2 µg		10 µg			5 µg		2 µg
	No. of Revertants	916 796 802	(838) 67.6	138 131 132	(134) 3.8	94 94 92		(93) 1.2	116 102 113	(110) 7.4	121 118 125	(121) 3.5

BP          Benzo(a)pyrene

2AA        2-Aminoanthracene

P            Test item precipitate

#         Standard deviation

Conclusions:

The in vitro gene mutation potential in bacteria of PU08 was determined to be negative.

Executive summary:

The in vitro gene mutation potential in bacteria of PU08 was tested in an Ames test (OECD 471) and was determined to be negative. Bacterial S. typhimurium and E. coli strains were treated with suspensions of PU08 at eight dose levels in triplicate, both with and without metabolic activation, via plate incorporation and pre-incubation assays. The vehicle, positive and negative controls confirmed the sensitivity of the assay and the efficacy of the S9-mix.

Reference 10

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12/10/2021 - 11/11/2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997, corrected 2020

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2008

Deviations:

no

Principles of method if other than guideline:

Based on provisions in the guidelines:
The Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy, Trade and Industry (METI), and Ministry of the Environment (MOE) Guidelines of 31 March 2011
ICH S2(R1) guideline adopted June 2012 (ICH S2(R1) Federal Register. Adopted 2012; 77:33748-33749)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

- Salmonella: Histidine
- E. Coli: Tryptophan

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

Bacteria

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Bacteria

Metabolic activation:

with and without

Metabolic activation system:

- Type of metabolic activation system: Lyophilized phenobarbital/beta-naphthoflavone induced rat liver S9 and cofactors mix (MutazymeTM)
- Source of S9: Prepared in lab before use
- S9 mix: Final concentrations of S9-mix of approximately 10% (v/v), MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium phosphate buffer (100 mM, pH 7.4)
- Concentration: 0.5 mL of S9-mix was added to the molten, trace amino-acid supplemented media

Test concentrations with justification for top dose:

Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate
Pre-Incubation Method: 15, 50, 150, 500, 1500 and 5000 μg/plate.
The maximum concentration was 5000 μg/plate (the OECD TG 471 maximum recommended dose level).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl formamide (DMF)
- Justification for choice of solvent/vehicle: The test item was insoluble in sterile distilled water, dimethyl sulphoxide, dimethyl formamide and acetonitrile at 50 mg/mL, acetone at 100 mg/mL and tetrahydrofuran at 200 mg/mL in solubility checks performed in–house. The test item formed the best doseable suspension in dimethyl formamide, therefore, this solvent was selected.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments: Two - plate incorporation and pre-incubation methods

METHOD OF TREATMENT/ EXPOSURE:
- Test substance in agar (plate incorporation) and pre-incubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 20 minutes (pre-incubation method)
- Exposure duration/duration of treatment: 48 to 72 hours

Evaluation criteria:

Evaluation criteria -
There are several criteria for determining a positive result. Any one, or all, of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537.
A test item is considered non-mutagenic (negative) in the test system if the above criteria are not met.

Statistics:

Statistical significance was not included as part of the result evaluation.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Checks: Prior to use, the relevant strains were checked for characteristics (deep rough character, ampicillin resistance, UV light sensitivity and histidine or tryptophan auxotrophy), viability and spontaneous reversion rate (all checks were found to be satisfactory).
- Sterility: The amino acid supplemented top agar and the S9-mix used in both experiments were shown to be sterile. The test item formulation was also shown to be sterile.
- Precipitation: A precipitate of the test item (off-white and particulate in appearance) was noted at and above 500 μg/plate in both the presence and absence of S9-mix (plate incorporation method), at and above 150 μg/plate in the absence of S9 and from 500 μg/plate in the presence of S9-mix pre-incubation method). This precipitate did not prevent the scoring of revertant colonies.

STUDY RESULTS
- Concurrent vehicle control data: The solvent (dimethyl formamide) control plates gave counts of revertant colonies within the normal range.Results for the untreated controls (spontaneous mutation rates) and viability were considered to be acceptable.
- Concurrent negative control data: Results for the untreated controls (spontaneous mutation rates) and viability were considered to be acceptable.
- Concurrent positive control data: All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation.
- Validity: As the controls were valid, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Remarks on result:

other: all strains/cell types tested

Table 1: Experiment 1 – Without Metabolic Activation (Plate Incorporation)

Test Period		From: 21 October2021					To: 24 October 2021
S9-Mix (-)	Dose Level Per Plate	Number of revertants (rounded mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (DMF)	99 96 97	(97) 1.5#	20 12 17	(16) 4.0	24 18 22		(21) 3.1	26 17 13	(19) 6.7	8 9 8	(8) 0.6
	1.5 µg	102 96 85	(94) 8.6	17 16 14	(16) 1.5	15 19 17		(17) 2.0	14 11 14	(13) 1.7	12 12 19	(14) 4.0
	5 µg	90 76 93	(86) 9.1	16 17 15	(16) 1.0	10 27 13		(17) 9.1	18 10 9	(12) 4.9	10 15 11	(12) 2.6
	15 µg	114 122 112	(116) 5.3	17 13 11	(14) 3.1	14 18 12		(15) 3.1	27 13 13	(18) 8.1	4 13 8	(8) 4.5
	50 µg	106 108 108	(107) 1.2	15 14 15	(15) 0.6	19 18 22		(20) 2.1	14 13 17	(15) 2.1	7 9 11	(9) 2.0
	150 µg	82 89 86	(86) 3.5	16 12 14	(14) 2.0	15 28 21		(21) 6.5	9 15 11	(12) 3.1	10 13 14	(12) 2.1
	500 µg	101 P 97 P 99 P	(99) 2.0	15 P 16 P 9 P	(13) 3.8	12 P 25 P 20 P		(19) 6.6	19 P 20 P 12 P	(17) 4.4	16 P 14 P 9 P	(13) 3.6
	1500 µg	128 P 97 P 115 P	(113) 15.6	15 P 7 P 8 P	(10) 4.4	18 P 23 P 21 P		(21) 2.5	14 P 11 P 15 P	(13) 2.1	16 P 3 P 13 P	(11) 6.8
	5000 µg	91 P 97 P 97 P	(95) 3.5	12 P 14 P 11 P	(12) 1.5	26 P 12 P 18 P		(19) 7.0	7 P 12 P 19 P	(13) 6.0	18 P 16 P 12 P	(15) 3.1
Positive controls S9-Mix (-)	Name	ENNG		ENNG		ENNG			4NQO		9AA
	Dose Level	3 µg		5 µg		2 µg			0.2 µg		80 µg
	No. of Revertants	554 578 608	(580) 27.1	724 881 767	(791) 81.1	309 334 342		(328) 17.2	108 100 109	(106) 4.9	502 759 760	(674) 148.7

ENNG        N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO         4-Nitroquinoline-1-oxide

9AA           9-Aminoacridine

P               Test item precipitate

#         Standard deviation

Table 2: Experiment 1 – With Metabolic Activation (Plate Incorporation)

Test Period		From: 21 October2021					To: 24 October 2021
S9-Mix (+)	Dose Level Per Plate	Number of revertants (rounded mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (DMF)	107 127 124	(119) 10.8#	15 7 10	(11) 4.0	19 26 20		(22) 3.8	15 20 22	(19) 3.6	13 13 7	(11) 3.5
	1.5 µg	105 117 153	(125) 25.0	6 11 18	(12) 6.0	29 26 17		(24) 6.2	19 17 18	(18) 1.0	12 14 11	(12) 1.5
	5 µg	119 134 110	(121) 12.1	11 11 8	(10) 1.7	22 21 24		(22) 1.5	12 16 13	(14) 2.1	11 11 16	(13) 2.9
	15 µg	112 108 100	(107) 6.1	16 19 6	(14) 6.8	27 12 21		(20) 7.5	26 22 26	(25) 2.3	7 14 15	(12) 4.4
	50 µg	103 104 119	(109) 9.0	9 7 7	(8) 1.2	19 21 17		(19) 2.0	20 25 24	(23) 2.6	13 15 15	(14) 1.2
	150 µg	115 103 95	(104) 10.1	14 7 7	(9) 4.0	25 31 21		(26) 5.0	26 18 18	(21) 4.6	12 7 6	(8) 3.2
	500 µg	98 P 136 P 117 P	(117) 19.0	12 P 13 P 11 P	(12) 1.0	22 P 20 P 21 P		(21) 1.0	19 P 21 P 21 P	(20) 1.2	8 P 8 P 16 P	(11) 4.6
	1500 µg	126 P 148 P 120 P	(131) 14.7	9 P 15 P 12 P	(12) 3.0	21 P 21 P 25 P		(22) 2.3	11 P 11 P 17 P	(13) 3.5	17 P 11 P 9 P	(12) 4.2
	5000 µg	111 P 101 P 107 P	(106) 5.0	14 P 15 P 8 P	(12) 3.8	22 P 14 P 19 P		(18) 4.0	21 P 17 P 15 P	(18) 3.1	13 P 8 P 11 P	(11) 2.5
Positive controls S9-Mix (+)	Name	2AA		2AA		2AA			BP		2AA
	Dose Level	1 µg		2 µg		10 µg			5 µg		2 µg
	No. of Revertants	921 964 894	(926) 35.3	194 164 213	(190) 24.7	125 137 111		(124) 13.0	284 256 272	(271) 14.0	109 109 129	(116) 11.5

BP          Benzo(a)pyrene

2AA        2-Aminoanthracene

P            Test item precipitate

#         Standard deviation

Table 3: Experiment 2 – Without Metabolic Activation (Pre-Incubation)

Test Period		From: 08 November 2021					To: 11 November 2021
S9-Mix (-)	Dose Level Per Plate	Number of revertants (rounded mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (DMF)	101 85 96	(94) 8.2#	9 14 18	(14) 4.5	19 19 17		(18) 1.2	24 11 21	(19) 6.8	9 13 10	(11) 2.1
	15 µg	91 94 92	(92) 1.5	14 17 14	(15) 1.7	12 22 13		(16) 5.5	11 20 17	(16) 4.6	15 8 9	(11) 3.8
	50 µg	89 106 92	(96) 9.1	12 10 16	(13) 3.1	14 10 22		(15) 6.1	18 23 24	(22) 3.2	7 8 14	(10) 3.8
	150 µg	89 P 87 P 111 P	(96) 13.3	18 P 13 P 12 P	(14) 3.2	14 P 23 P 13 P		(17) 5.5	24 P 20 P 13 P	(19) 5.6	8 P 7 P 7 P	(7) 0.6
	500 µg	108 P 105 P 104 P	(106) 2.1	14 P 12 P 14 P	(13) 1.2	17 P 23 P 21 P		(20) 3.1	20 P 12 P 21 P	(18) 4.9	9 P 11 P 10 P	(10) 1.0
	1500 µg	108 P 109 P 107 P	(108) 1.0	13 P 12 P 11 P	(12) 1.0	19 P 13 P 10 P		(14) 4.6	19 P 14 P 11 P	(15) 4.0	12 P 9 P 10 P	(10) 1.5
	5000 µg	106 P 102 P 91 P	(100) 7.8	16 P 9 P 13 P	(13) 3.5	14 P 15 P 21 P		(17) 3.8	10 P 20 P 17 P	(16) 5.1	11 P 8 P 14 P	(11) 3.0
Positive controls S9-Mix (-)	Name	ENNG		ENNG		ENNG			4NQO		9AA
	Dose Level	3 µg		5 µg		2 µg			0.2 µg		80 µg
	No. of Revertants	613 621 627	(620) 7.0	1422 1243 1420	(1362) 102.8	237 280 338		(285) 50.7	78 79 103	(87) 14.2	136 79 184	(133) 52.6

4NQO         4-Nitroquinoline-1-oxide

9AA           9-Aminoacridine

P               Test item precipitate

#         Standard deviation

Table 4: Experiment 2 – With Metabolic Activation (Pre-Incubation)

Test Period		From: 08 November 2021					To: 11 November 2021
S9-Mix (+)	Dose Level Per Plate	Number of revertants (rounded mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (DMF)	127 115 158	(133) 22.2#	11 10 13	(11) 1.5	20 26 18		(21) 4.2	20 32 25	(26) 6.0	8 18 14	(13) 5.0
	15 µg	114 132 140	(129) 13.3	8 12 13	(11) 2.6	13 17 12		(14) 2.6	23 31 28	(27) 4.0	13 9 12	(11) 2.1
	50 µg	130 111 144	(128) 16.6	13 9 8	(10) 2.6	20 13 17		(17) 3.5	29 29 28	(29) 0.6	11 6 9	(9) 2.5
	150 µg	130 126 121	(126) 4.5	15 14 11	(13) 2.1	21 18 16		(18) 2.5	28 23 29	(27) 3.2	13 14 15	(14) 1.0
	500 µg	129 P 138 P 134 P	(134) 4.5	14 P 13 P 15 P	(14) 1.0	17 P 15 P 18 P		(17) 1.5	24 P 23 P 29 P	(25) 3.2	14 P 17 P 12 P	(14) 2.5
	1500 µg	124 P 121 P 123 P	(123) 1.5	16 P 9 P 9 P	(11) 4.0	15 P 16 P 17 P		(16) 1.0	21 P 26 P 17 P	(21) 4.5	7 P 11 P 9 P	(9) 2.0
	5000 µg	120 P 118 P 130 P	(123) 6.4	15 P 9 P 11 P	(12) 3.1	23 P 25 P 17 P		(22) 4.2	18 P 23 P 28 P	(23) 5.0	15 P 13 P 7 P	(12) 4.2
Positive controls S9-Mix (+)	Name	2AA		2AA		2AA			BP		2AA
	Dose Level	1 µg		2 µg		10 µg			5 µg		2 µg
	No. of Revertants	1070 979 1046	(1032) 47.2	158 145 173	(159) 14.0	170 153 122		(148) 24.3	108 116 125	(116) 8.5	132 162 127	(140) 18.9

BP          Benzo(a)pyrene

2AA        2-Aminoanthracene

P            Test item precipitate

#         Standard deviation

Conclusions:

The in vitro gene mutation potential in bacteria of PU09 was determined to be negative.

Executive summary:

The in vitro gene mutation potential in bacteria of PU09 was tested in an Ames test (OECD 471) and was determined to be negative. Bacterial S. typhimurium and E. coli strains were treated with suspensions of PU09 at eight dose levels in triplicate, both with and without metabolic activation, via plate incorporation and pre-incubation assays. The vehicle, positive and negative controls confirmed the sensitivity of the assay and the efficacy of the S9-mix.

Reference 11

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12/10/2021 - 08/11/2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997, corrected 2020

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2008

Deviations:

no

Principles of method if other than guideline:

Based on provisions in the guidelines:
The Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy, Trade and Industry (METI), and Ministry of the Environment (MOE) Guidelines of 31 March 2011
ICH S2(R1) guideline adopted June 2012 (ICH S2(R1) Federal Register. Adopted 2012; 77:33748-33749)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

- Salmonella: Histidine
- E. Coli: Tryptophan

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

Bacteria

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Bacteria

Metabolic activation:

with and without

Metabolic activation system:

- Type of metabolic activation system: Lyophilized phenobarbital/beta-naphthoflavone induced rat liver S9 and cofactors mix (MutazymeTM)
- Source of S9: Prepared in lab before use
- S9 mix: Final concentrations of S9-mix of approximately 10% (v/v), MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium phosphate buffer (100 mM, pH 7.4)
- Concentration: 0.5 mL of S9-mix was added to the molten, trace amino-acid supplemented media

Test concentrations with justification for top dose:

Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate
Pre-Incubation Method: 15, 50, 150, 500, 1500 and 5000 μg/plate.
The maximum concentration was 5000 μg/plate (the OECD TG 471 maximum recommended dose level).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl formamide (DMF)
- Justification for choice of solvent/vehicle: The test item was insoluble in sterile distilled water, dimethyl sulphoxide, dimethyl formamide and acetonitrile at 50 mg/mL, acetone at 100 mg/mL and tetrahydrofuran at 200 mg/mL in solubility checks performed in–house. The test item formed the best doseable suspension in dimethyl formamide, therefore, this solvent was selected.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments: Two - plate incorporation and pre-incubation methods

METHOD OF TREATMENT/ EXPOSURE:
- Test substance in agar (plate incorporation) and pre-incubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 20 minutes (pre-incubation method)
- Exposure duration/duration of treatment: 48 to 72 hours

Evaluation criteria:

Evaluation criteria -
There are several criteria for determining a positive result. Any one, or all, of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537.
A test item is considered non-mutagenic (negative) in the test system if the above criteria are not met.

Statistics:

Statistical significance was not included as part of the result evaluation.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Checks: Prior to use, the relevant strains were checked for characteristics (deep rough character, ampicillin resistance, UV light sensitivity and histidine or tryptophan auxotrophy), viability and spontaneous reversion rate (all checks were found to be satisfactory).
- Sterility: The amino acid supplemented top agar and the S9-mix used in both experiments were shown to be sterile. The test item formulation was also shown to be sterile.
- Precipitation: A test item precipitate (crystalline in appearance) was noted in both the presence and absence of S9-mix from 1500 g/plate in Experiment 1 (plate incorporation) and from 500 µg/plate in Experiment 2 (pre-incubation). The precipitate did not prevent the scoring of revertant colonies.

STUDY RESULTS
- Concurrent vehicle control data: The solvent (dimethyl formamide) control plates gave counts of revertant colonies within the normal range. Five single counts (WP2uvrA untreated control, TA98 and TA1537, vehicle controls dosed in the presence of S9-mix after the first mutation test and TA98, vehicle control dosed in the presence of S9-mix after the second mutation test) were marginally below/above the minimum/maximum level (95% confidence limit) of the in-house historical untreated/solvent controls for the tester strains. These counts were considered acceptable as the other solvent and untreated control counts were within the expected range and the tester strains responded very well to the respective positive controls in both the presence and absence of S9 mix.
- Concurrent positive control data: All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation.
- Concurrent negative control data: Results for the untreated controls (spontaneous mutation rates) and viability were considered to be acceptable.
- Validity: As the controls were valid, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Remarks on result:

other: all strains/cell types tested

Table 1: Experiment 1 – Without Metabolic Activation (Plate Incorporation)

Test Period		From: 19 October 2021					To: 22 October 2021
S9-Mix (-)	Dose Level Per Plate	Number of revertants (rounded mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (DMF)	119 131 121	(124) 6.4#	30 9 15	(18) 10.8	23 31 26		(27) 4.0	24 23 22	(23) 1.0	12 19 20	(17) 4.4
	1.5 µg	129 139 125	(131) 7.2	9 16 10	(12) 3.8	19 19 26		(21) 4.0	15 16 20	(17) 2.6	25 24 24	(24) 0.6
	5 µg	134 141 109	(128) 16.8	17 21 9	(16) 6.1	28 22 19		(23) 4.6	22 18 12	(17) 5.0	24 23 15	(21) 4.9
	15 µg	142 130 120	(131) 11.0	16 17 12	(15) 2.6	22 20 24		(22) 2.0	18 19 19	(19) 0.6	18 13 26	(19) 6.6
	50 µg	132 112 138	(127) 13.6	11 9 12	(11) 1.5	26 19 19		(21) 4.0	13 16 19	(16) 3.0	23 19 7	(16) 8.3
	150 µg	122 123 129	(125) 3.8	16 14 22	(17) 4.2	17 16 16		(16) 0.6	17 20 22	(20) 2.5	11 19 14	(15) 4.0
	500 µg	129 136 126	(130) 5.1	8 24 16	(16) 8.0	24 22 27		(24) 2.5	27 26 18	(24) 4.9	17 13 10	(13) 3.5
	1500 µg	126 P 127 P 134 P	(129) 4.4	22 P 20 P 20 P	(21) 1.2	20 P 18 P 17 P		(18) 1.5	18 P 23 P 14 P	(18) 4.5	10 P 25 P 22 P	(19) 7.9
	5000 µg	121 P 133 P 123P	(126) 6.4	12 P 13 P 10 P	(12) 1.5	18 P 13 P 14 P		(15) 2.6	14 P 14 P 16 P	(15) 1.2	23 P 16 P 19 P	(19) 3.5
Positive controls S9-Mix (-)	Name	ENNG		ENNG		ENNG			4NQO		9AA
	Dose Level	3 µg		5 µg		2 µg			0.2 µg		80 µg
	No. of Revertants	511 434 328	(424) 91.9	149 141 214	(168) 40.0	809 767 653		(743) 80.7	106 100 105	(104) 3.2	964 563 931	(819) 222.6

ENNG        N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO         4-Nitroquinoline-1-oxide

9AA           9-Aminoacridine

P               Test item precipitate

#         Standard deviation

Table 2: Experiment 1 – With Metabolic Activation (Plate Incorporation)

Test Period		From: 19 October 2021					To: 22 October 2021
S9-Mix (+)	Dose Level Per Plate	Number of revertants (rounded mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (DMF)	94 87 93	(91) 3.8#	22 11 11	(15) 6.4	28 26 26		(27) 1.2	24 26 13	(21) 7.0	24 24 12	(20) 6.9
	1.5 µg	89 107 95	(97) 9.2	10 11 16	(12) 3.2	32 29 21		(27) 5.7	20 31 17	(23) 7.4	14 14 8	(12) 3.5
	5 µg	107 97 104	(103) 5.1	7 10 22	(13) 7.9	25 16 23		(21) 4.7	19 26 20	(22) 3.8	15 14 11	(13) 2.1
	15 µg	105 105 110	(107) 2.9	11 13 8	(11) 2.5	29 27 31		(29) 2.0	22 20 28	(23) 4.2	12 16 11	(13) 2.6
	50 µg	93 87 87	(89) 3.5	19 11 10	(13) 4.9	26 15 23		(21) 5.7	17 19 17	(18) 1.2	11 7 13	(10) 3.1
	150 µg	96 119 89	(101) 15.7	16 8 13	(12) 4.0	30 20 27		(26) 5.1	20 17 27	(21) 5.1	15 10 21	(15) 5.5
	500 µg	87 92 82	(87) 5.0	13 13 14	(13) 0.6	30 23 25		(26) 3.6	32 32 34	(33) 1.2	14 21 9	(15) 6.0
	1500 µg	109 P 101 P 91 P	(100) 9.0	16 P 9 P 12 P	(12) 3.5	27 P 18 P 20 P		(22) 4.7	26 P 14 P 27 P	(22) 7.2	12 P 6 P 18 P	(12) 6.0
	5000 µg	89 P 97 P 124 P	(103) 18.3	22 P 15 P 15 P	(17) 4.0	25 P 25 P 21 P		(24) 2.3	30 P 25 P 25 P	(27) 2.9	22 P 11 P 9 P	(14) 7.0
Positive controls S9-Mix (+)	Name	2AA		2AA		2AA			BP		2AA
	Dose Level	1 µg		2 µg		10 µg			5 µg		2 µg
	No. of Revertants	1310 1279 1010	(1200) 165.0	191 195 193	(193) 2.0	168 112 149		(143) 28.5	220 277 222	(240) 32.3	189 166 191	(182) 13.9

BP          Benzo(a)pyrene

2AA        2-Aminoanthracene

P            Test item precipitate

#         Standard deviation

Table 3: Experiment 2 – Without Metabolic Activation (Pre-Incubation)

Test Period		From: 04 November 2021					To: 07 November 2021
S9-Mix (-)	Dose Level Per Plate	Number of revertants (rounded mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (DMF)	86 95 96	(92) 5.5#	9 12 12	(11) 1.7	18 18 18		(18) 0.0	33 11 12	(19) 12.4	6 6 6	(6) 0.0
	15 µg	94 95 83	(91) 6.7	7 13 11	(10) 3.1	22 20 17		(20) 2.5	19 13 16	(16) 3.0	5 7 9	(7) 2.0
	50 µg	102 108 102	(104) 3.5	14 9 14	(12) 2.9	14 19 11		(15) 4.0	17 9 22	(16) 6.6	7 7 6	(7) 0.6
	150 µg	90 78 96	(88) 9.2	17 12 13	(14) 2.6	17 23 18		(19) 3.2	15 17 21	(18) 3.1	13 3 3	(6) 5.8
	500 µg	125 P 89 P 107 P	(107) 18.0	10 P 11 P 10 P	(10) 0.6	24 P 24 P 28 P		(25) 2.3	9 P 12 P 10 P	(10) 1.5	6 P 11 P 12 P	(10) 3.2
	1500 µg	93 P 82 P 85 P	(87) 5.7	17 P 10 P 5 P	(11) 6.0	18 P 13 P 10 P		(14) 4.0	15 P 13 P 19 P	(16) 3.1	6 P 7 P 2 P	(5) 2.6
	5000 µg	72 P 70 P 66 P	(69) 3.1	10 P 14 P 8 P	(11) 3.1	16 P 19 P 15 P		(17) 2.1	19 P 21 P 15 P	(18) 3.1	9 P 9 P 4 P	(7) 2.9
Positive controls S9-Mix (-)	Name	ENNG		ENNG		ENNG			4NQO		9AA
	Dose Level	3 µg		5 µg		2 µg			0.2 µg		80 µg
	No. of Revertants	927 1015 987	(976) 45.0	1203 1126 1284	(1204) 79.0	585 541 535		(554) 27.3	125 114 121	(120) 5.6	144 185 160	(163) 20.7

ENNG        N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO         4-Nitroquinoline-1-oxide

9AA           9-Aminoacridine

P               Test item precipitate

#         Standard deviation

Table 4: Experiment 2 – With Metabolic Activation (Pre-Incubation)

Test Period		From: 04 November 2021					To: 07 November 2021
S9-Mix (+)	Dose Level Per Plate	Number of revertants (rounded mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (DMF)	127 103 98	(109) 15.5#	8 9 16	(11) 4.4	22 25 17		(21) 4.0	16 28 24	(23) 6.1	6 6 11	(8) 2.9
	15 µg	112 122 123	(119) 6.1	11 15 13	(13) 2.0	29 21 17		(22) 6.1	15 25 28	(23) 6.8	11 13 10	(11) 1.5
	50 µg	106 125 122	(118) 10.2	7 8 14	(10) 3.8	20 14 19		(18) 3.2	25 28 28	(27) 1.7	12 14 10	(12) 2.0
	150 µg	117 121 103	(114) 9.5	12 12 19	(14) 4.0	19 21 21		(20) 1.2	22 15 24	(20) 4.7	15 10 10	(12) 2.9
	500 µg	111 P 142 P 126 P	(126) 15.5	17 P 13 P 17 P	(16) 2.3	16 P 25 P 14 P		(18) 5.9	15 P 15 P 25 P	(18) 5.8	7 P 12 P 11 P	(10) 2.6
	1500 µg	102 P 86 P 89 P	(92) 8.5	9 P 16 P 16 P	(14) 4.0	17 P 18 P 15 P		(17) 1.5	20 P 18 P 22 P	(20) 2.0	12 P 7 P 13 P	(11) 3.2
	5000 µg	88 P 97 P 80 P	(88) 8.5	12 P 9 P 10 P	(10) 1.5	18 P 18 P 16 P		(17) 1.2	20 P 19 P 17 P	(19) 1.5	6 P 11 P 5 P	(7) 3.2
Positive controls S9-Mix (+)	Name	2AA		2AA		2AA			BP		2AA
	Dose Level	1 µg		2 µg		10 µg			5 µg		2 µg
	No. of Revertants	1042 969 996	(1002) 36.9	144 145 114	(134) 17.6	93 102 85		(93) 8.5	129 135 146	(137) 8.6	175 160 159	(165) 9.0

BP          Benzo(a)pyrene

2AA        2-Aminoanthracene

P            Test item precipitate

#         Standard deviation

Conclusions:

The in vitro gene mutation potential in bacteria of R03 was determined to be negative.

Executive summary:

The in vitro gene mutation potential in bacteria of R03 was tested in an Ames test (OECD 471) and was determined to be negative. Bacterial S. typhimurium and E. coli strains were treated with suspensions of R03 at eight dose levels in triplicate, both with and without metabolic activation, via plate incorporation and pre-incubation assays. The vehicle, positive and negative controls confirmed the sensitivity of the assay and the efficacy of the S9-mix.

Reference 12

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12/10/2021 - 09/11/2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997, corrected 2020

Deviations:

no

Principles of method if other than guideline:

Based on provisions in the guidelines:
The Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy, Trade and Industry (METI), and Ministry of the Environment (MOE) Guidelines of 31 March 2011
ICH S2(R1) guideline adopted June 2012 (ICH S2(R1) Federal Register. Adopted 2012; 77:33748-33749)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

- Salmonella: Histidine
- E. coli: Tryptophan

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: Lyophilized phenobarbital/beta-naphthoflavone induced rat liver S9 and cofactors mix (MutazymeTM)
- source of S9: Prepared in lab before use
- method of preparation of S9 mix
- S9 mix: Final concentrations of S9-mix of approximately 10% (v/v) MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium phosphate buffer (100 mM, pH 7.4)
- Concentration: 0.5 mL of S9-mix was added to the molten, trace amino-acid supplemented media

Test concentrations with justification for top dose:

Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate
Pre-Incubation Method: 15, 50, 150, 500, 1500 and 5000 μg/plate.
The maximum concentration was 5000 μg/plate (the OECD TG 471 maximum recommended dose level).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl formamide (DMF)
- Justification for choice of solvent/vehicle: The test item was insoluble in sterile distilled water, dimethyl sulphoxide, dimethyl formamide and acetonitrile at 50 mg/mL, acetone at 100 mg/mL and tetrahydrofuran at 200 mg/mL in solubility checks performed in–house. The test item formed the best doseable suspension in dimethyl formamide, therefore, this solvent was selected.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-aminoanthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments: Two - plate incorporation and pre-incubation methods

METHOD OF TREATMENT/ EXPOSURE:
- Test substance in agar (plate incorporation) and preincubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 20 minutes (pre-incubation method)
- Exposure duration/duration of treatment: 48 to 72 hours

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537
A test item is considered non-mutagenic (negative) in the test system if the above criteria are not met.

Statistics:

Statistical significance was not included as part of the result evaluation.

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Checks: Prior to use, the relevant strains were checked for characteristics (deep rough character, ampicillin resistance, UV light sensitivity and histidine or tryptophan auxotrophy), viability and spontaneous reversion rate (all checks were found to be satisfactory).
- Sterility: The amino acid supplemented top agar and the S9-mix used in both experiments were shown to be sterile. The test item formulation was also shown to be sterile.
- Precipitation: A test item precipitate (particulate in appearance) was noted in both the presence and absence of S9-mix from 500 μg/plate in Experiment 1 (plate incorporation). In Experiment 2 (pre-incubation), a precipitate was noted from 150 μg/plate in all of the strains dosed in the both the absence and presence of S9 except for TA100 (presence of S9) where a precipitate was first noted at 500 μg/plate. The precipitate did not prevent the scoring of revertant colonies.

STUDY RESULTS
- Concurrent vehicle control data: The solvent (DMF) control plates gave counts of revertant colonies within the normal range.
- Concurrent negative control data: Results for the untreated controls (spontaneous mutation rates) and viability were considered to be acceptable.
- Concurrent positive control data: All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation.
- Validity: As the controls were valid, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Remarks on result:

other: All strains/cell types tested

Table 1: Experiment 1 (Plate Incorporation) without Metabolic Activation

Test Period		From: 21 October 2021					To: 24 October 2021
S9-Mix (-)	Dose Level Per Plate	Number of revertants (rounded mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (DMF)	124 104 106	(111) 11.0#	13 12 10	(12) 1.5	21 24 22		(22) 1.5	17 16 16	(16) 0.6	12 17 13	(14) 2.6
	1.5 µg	109 109 98	(105) 6.4	11 10 11	(11) 0.6	24 19 22		(22) 2.5	16 16 17	(16) 0.6	6 11 10	(9) 2.6
	5 µg	98 103 91	(97) 6.0	14 16 13	(14) 1.5	24 20 22		(22) 2.0	12 13 18	(14) 3.2	5 13 12	(10) 4.4
	15 µg	120 109 95	(108) 12.5	11 9 10	(10) 1.0	20 21 17		(19) 2.1	22 16 19	(19) 3.0	16 6 8	(10) 5.3
	50 µg	89 110 101	(100) 10.5	9 15 16	(13) 3.8	24 17 17		(19) 4.0	19 12 20	(17) 4.4	8 7 8	(8) 0.6
	150 µg	90 105 94	(96) 7.8	14 8 15	(12) 3.8	24 20 23		(22) 2.1	20 19 20	(20) 0.6	11 8 11	(10) 1.7
	500 µg	100 P 119 P 103 P	(107) 10.2	14 P 13 P 18 P	(15) 2.6	24 P 23 P 20 P		(22) 2.1	17 P 14 P 16 P	(16) 1.5	13 P 10 P 5 P	(9) 4.0
	1500 µg	102 P 97 P 97 P	(99) 2.9	9 P 12 P 11 P	(11) 1.5	21 P 27 P 21 P		(23) 3.5	19 P 16 P 18 P	(18) 1.5	10 P 5 P 14 P	(10) 4.5
	5000 µg	90 P 115 P 112 P	(106) 13.7	11 P 10 P 12 P	(11) 1.0	20 P 25 P 29 P		(25) 4.5	16 P 14 P 17 P	(16) 1.5	6 P 13 P 10 P	(10) 3.5
Positive controls S9-Mix (-)	Name	ENNG		ENNG		ENNG			4NQO		9AA
	Dose Level	3 µg		5 µg		2 µg			0.2 µg		80 µg
	No. of Revertants	686 489 660	(612) 107.0	909 278 905	(697) 363.2	736 661 646		(681) 48.2	107 121 104	(111) 9.1	312 504 235	(350) 138.5

ENNG          N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO           4-Nitroquinoline-1-oxide

9AA             9-Aminoacridine

P                 Test item precipitate

#           Standard deviation

 

Table 2: Experiment 1 (Plate Incorporation) with Metabolic Activation

Test Period		From: 21 October 2021					To: 24 October 2021
S9-Mix (+)	Dose Level Per Plate	Number of revertants (rounded mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (DMF)	127 115 115	(119) 6.9#	17 16 16	(16) 0.6	27 31 27		(28) 2.3	24 20 19	(21) 2.6	13 14 7	(11) 3.8
	1.5 µg	135 136 137	(136) 1.0	9 16 13	(13) 3.5	26 26 32		(28) 3.5	27 28 19	(25) 4.9	15 19 13	(16) 3.1
	5 µg	127 122 109	(119) 9.3	18 21 14	(18) 3.5	19 21 30		(23) 5.9	21 30 19	(23) 5.9	10 16 16	(14) 3.5
	15 µg	104 105 108	(106) 2.1	15 18 10	(14) 4.0	22 31 23		(25) 4.9	25 28 23	(25) 2.5	11 13 11	(12) 1.2
	50 µg	127 140 131	(133) 6.7	8 8 16	(11) 4.6	20 22 24		(22) 2.0	26 27 30	(28) 2.1	13 14 15	(14) 1.0
	150 µg	134 134 123	(130) 6.4	13 14 15	(14) 1.0	20 19 22		(20) 1.5	24 31 29	(28) 3.6	13 16 17	(15) 2.1
	500 µg	135 P 131 P 126 P	(131) 4.5	13 P 11 P 7 P	(10) 3.1	28 P 23 P 23 P		(25) 2.9	22 P 20 P 19 P	(20) 1.5	16 P 11 P 10 P	(12) 3.2
	1500 µg	136 P 131 P 141 P	(136) 5.0	9 P 8 P 10 P	(9) 1.0	21 P 19 P 23 P		(21) 2.0	23 P 20 P 26 P	(23) 3.0	13 P 18 P 16 P	(16) 2.5
	5000 µg	132 P 137 P 133 P	(134) 2.6	11 P 9 P 9 P	(10) 1.2	21 P 22 P 29 P		(24) 4.4	19 P 29 P 28 P	(25) 5.5	17 P 17 P 17 P	(17) 0.0
Positive controls S9-Mix (+)	Name	2AA		2AA		2AA			BP		2AA
	Dose Level	1 µg		2 µg		10 µg			5 µg		2 µg
	No. of Revertants	1244 1174 867	(1095) 200.5	185 164 170	(173) 10.8	121 119 118		(119) 1.5	344 374 337	(352) 19.7	119 131 132	(127) 7.2

BP            Benzo(a)pyrene

2AA          2-Aminoanthracene

P              Test item precipitate

#           Standard deviation

 

Table 3: Experiment 2 (Pre-Incubation) without Metabolic Activation

Test Period		From: 05 November 2021					To: 08 November 2021
S9-Mix (-)	Dose Level Per Plate	Number of revertants (rounded mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (DMF)	98 99 120	(106) 12.4#	13 13 10	(12) 1.7	26 25 21		(24) 2.6	19 12 20	(17) 4.4	7 7 4	(6) 1.7
	15 µg	99 116 103	(106) 8.9	13 12 13	(13) 0.6	17 26 24		(22) 4.7	20 16 12	(16) 4.0	10 10 13	(11) 1.7
	50 µg	85 88 99	(91) 7.4	10 12 22	(15) 6.4	15 22 34		(24) 9.6	18 14 22	(18) 4.0	8 14 12	(11) 3.1
	150 µg	88 P 76 P 95 P	(86) 9.6	10 P 16 P 11 P	(12) 3.2	21 P 26 P 18 P		(22) 4.0	25 P 20 P 24 P	(23) 2.6	9 P 5 P 18 P	(11) 6.7
	500 µg	93 P 95 P 95 P	(94) 1.2	10 P 7 P 11 P	(9) 2.1	19 P 16 P 15 P		(17) 2.1	19 P 17 P 14 P	(17) 2.5	7 P 4 P 8 P	(6) 2.1
	1500 µg	81 P 84 P 94 P	(86) 6.8	13 P 16 P 19 P	(16) 3.0	15 P 20 P 14 P		(16) 3.2	11 P 14 P 16 P	(14) 2.5	11 P 5 P 4 P	(7) 3.8
	5000 µg	82 P 90 P 77 P	(83) 6.6	11 P 12 P 9 P	(11) 1.5	14 P 11 P 14 P		(13) 1.7	12 P 12 P 16 P	(13) 2.3	11 P 7 P 9 P	(9) 2.0
Positive controls S9-Mix (-)	Name	ENNG		ENNG		ENNG			4NQO		9AA
	Dose Level	3 µg		5 µg		2 µg			0.2 µg		80 µg
	No. of Revertants	790 739 760	(763) 25.6	2285 2055 2593	(2311) 269.9	335 293 274		(301) 31.2	93 97 146	(112) 29.5	334 340 141	(272) 113.2

ENNG          N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO           4-Nitroquinoline-1-oxide

9AA             9-Aminoacridine

P                 Test item precipitate

#           Standard deviation

 

Table 4: Experiment 2 (Pre-Incubation) with Metabolic Activation

Test Period		From: 05 November 2021					To: 08 November 2021
S9-Mix (+)	Dose Level Per Plate	Number of revertants (rounded mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (DMF)	155 118 133	(135) 18.6#	11 13 12	(12) 1.0	31 28 35		(31) 3.5	20 14 27	(20) 6.5	7 8 6	(7) 1.0
	15 µg	145 135 153	(144) 9.0	12 12 11	(12) 0.6	39 30 26		(32) 6.7	15 23 17	(18) 4.2	16 7 10	(11) 4.6
	50 µg	121 138 116	(125) 11.5	14 20 13	(16) 3.8	35 28 30		(31) 3.6	24 20 18	(21) 3.1	10 13 13	(12) 1.7
	150 µg	146 133 126	(135) 10.1	9 P 16 P 15 P	(13) 3.8	26 P 28 P 23 P		(26) 2.5	22 P 29 P 20 P	(24) 4.7	9 P 14 P 9 P	(11) 2.9
	500 µg	137 P 145 P 121 P	(134) 12.2	9 P 16 P 14 P	(13) 3.6	22 P 22 P 17 P		(20) 2.9	20 P 17 P 18 P	(18) 1.5	10 P 8 P 8 P	(9) 1.2
	1500 µg	117 P 107 P 122 P	(115) 7.6	8 P 8 P 21 P	(12) 7.5	16 P 19 P 22 P		(19) 3.0	16 P 10 P 18 P	(15) 4.2	8 P 9 P 8 P	(8) 0.6
	5000 µg	93 P 93 P 89 P	(92) 2.3	11 P 8 P 14 P	(11) 3.0	14 P 17 P 14 P		(15) 1.7	17 P 16 P 13 P	(15) 2.1	5 P 6 P 7 P	(6) 1.0
Positive controls S9-Mix (+)	Name	2AA		2AA		2AA			BP		2AA
	Dose Level	1 µg		2 µg		10 µg			5 µg		2 µg
	No. of Revertants	845 956 1043	(948) 99.2	140 118 123	(127) 11.5	127 135 130		(131) 4.0	122 128 137	(129) 7.5	157 163 169	(163) 6.0

BP            Benzo(a)pyrene

2AA          2-Aminoanthracene

P              Test item precipitate

#           Standard deviation

 

Conclusions:

The in vitro gene mutation potential of R04 was determined to be negative.

Executive summary:

The in vitro gene mutation potential in bacteria of R04 was tested in an Ames test (OECD 471) and was determined to be negative. Bacterial S. typhimurium and E. coli strains were treated with suspensions of R04 at eight dose levels in triplicate, both with and without metabolic activation, via plate incorporation and pre-incubation assays. The vehicle, positive and negative controls confirmed the sensitivity of the assay and the efficacy of the S9-mix.

Reference 13

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12/10/2021 - 04/11/2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997, corrected 2020

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2008

Deviations:

no

Principles of method if other than guideline:

Based on provisions in the guidelines:
The Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy, Trade and Industry (METI), and Ministry of the Environment (MOE) Guidelines of 31 March 2011
ICH S2(R1) guideline adopted June 2012 (ICH S2(R1) Federal Register. Adopted 2012; 77:33748-33749)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

- Salmonella: Histidine
- E. Coli: Tryptophan

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

Bacteria

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Bacteria

Metabolic activation:

with and without

Metabolic activation system:

- Type of metabolic activation system: Lyophilized phenobarbital/beta-naphthoflavone induced rat liver S9 and cofactors mix (MutazymeTM)
- Source of S9: Prepared in lab before use
- S9 mix: Final concentrations of S9-mix of approximately 10% (v/v) MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium phosphate buffer (100 mM, pH 7.4)
- Concentration: 0.5 mL of S9-mix was added to the molten, trace amino-acid supplemented media

Test concentrations with justification for top dose:

Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate
Pre-Incubation Method: 15, 50, 150, 500, 1500 and 5000 μg/plate.
The maximum concentration was 5000 μg/plate (the OECD TG 471 maximum recommended dose level).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl formamide (DMF)
- Justification for choice of solvent/vehicle: The test item was insoluble in sterile distilled water, dimethyl sulphoxide, dimethyl formamide and acetonitrile at 50 mg/mL, acetone at 100 mg/mL and tetrahydrofuran at 200 mg/mL in solubility checks performed in–house. The test item formed the best doseable suspension in dimethyl formamide, therefore, this solvent was selected.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments: Two - plate incorporation and pre-incubation methods

METHOD OF TREATMENT/ EXPOSURE:
- Test substance in agar (plate incorporation) and pre-incubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 20 minutes (pre-incubation method)
- Exposure duration/duration of treatment: 48 to 72 hours

Evaluation criteria:

Evaluation criteria -
There are several criteria for determining a positive result. Any one, or all, of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537.
A test item is considered non-mutagenic (negative) in the test system if the above criteria are not met.

Statistics:

Statistical significance was not included as part of the result evaluation.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Checks: Prior to use, the relevant strains were checked for characteristics (deep rough character, ampicillin resistance, UV light sensitivity and histidine or tryptophan auxotrophy), viability and spontaneous reversion rate (all checks were found to be satisfactory).
- Sterility: The amino acid supplemented top agar and the S9-mix used in both experiments were shown to be sterile. The test item formulation was also shown to be sterile.
- Precipitation: A test item precipitate (particulate in appearance) was noted in both the presence and absence of S9-mix from 1500 μg/plate in Experiment 1 (plate incorporation) and from 500 µg/plate in Experiment 2 (pre-incubation). The precipitate did not prevent the scoring of revertant colonies.

STUDY RESULTS
- Concurrent vehicle control data: The solvent (dimethyl formamide) control plates gave counts of revertant colonies within the normal range.
- Concurrent negative control data: Results for the untreated controls (spontaneous mutation rates) and viability were considered to be acceptable.
- Concurrent positive control data: All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation.
- Validity: As the controls were valid, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Remarks on result:

other: all strains/cell types tested

Table 1: Experiment 1 – Without Metabolic Activation (Plate Incorporation)

Test Period		From: 15 October 2021					To: 18 October 2021
S9-Mix (-)	Dose Level Per Plate	Number of revertants (rounded mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (DMF)	105 111 130	(115) 13.1#	13 10 14	(12) 2.1	25 20 24		(23) 2.6	19 15 18	(17) 2.1	5 11 17	(11) 6.0
	1.5 µg	127 126 127	(127) 0.6	12 18 13	(14) 3.2	16 20 16		(17) 2.3	16 17 16	(16) 0.6	6 9 14	(10) 4.0
	5 µg	107 117 131	(118) 12.1	18 20 16	(18) 2.0	15 14 16		(15) 1.0	21 13 19	(18) 4.2	9 8 9	(9) 0.6
	15 µg	118 119 132	(123) 7.8	17 14 19	(17) 2.5	23 15 15		(18) 4.6	21 16 18	(18) 2.5	8 9 12	(10) 2.1
	50 µg	107 126 128	(120) 11.6	14 16 13	(14) 1.5	23 22 16		(20) 3.8	21 14 20	(18) 3.8	11 12 7	(10) 2.6
	150 µg	128 108 131	(122) 12.5	15 17 16	(16) 1.0	11 16 19		(15) 4.0	15 21 16	(17) 3.2	6 14 12	(11) 4.2
	500 µg	134 128 104	(122) 15.9	12 18 14	(15) 3.1	24 14 23		(20) 5.5	22 16 13	(17) 4.6	11 12 16	(13) 2.6
	1500 µg	119 P 135 P 107 P	(120) 14.0	21 P 16 P 23 P	(20) 3.6	18 P 25 P 25 P		(23) 4.0	19 P 20 P 16 P	(18) 2.1	9 P 16 P 10 P	(12) 3.8
	5000 µg	105 P 125 P 112 P	(114) 10.1	23 P 24 P 22 P	(23) 1.0	18 P 14 P 16 P		(16) 2.0	15 P 18 P 12 P	(15) 3.0	9 P 7 P 13 P	(10) 3.1
Positive controls S9-Mix (-)	Name	ENNG		ENNG		ENNG			4NQO		9AA
	Dose Level	3 µg		5 µg		2 µg			0.2 µg		80 µg
	No. of Revertants	347 388 417	(384) 35.2	1162 955 1201	(1106) 132.2	738 714 692		(715) 23.0	119 119 110	(116) 5.2	279 555 464	(433) 140.6

ENNG        N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO         4-Nitroquinoline-1-oxide

9AA           9-Aminoacridine

P               Test item precipitate

#         Standard deviation

Table 2: Experiment 1 – With Metabolic Activation (Plate Incorporation)

Test Period		From: 15 October 2021					To: 18 October 2021
S9-Mix (+)	Dose Level Per Plate	Number of revertants (rounded mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (DMF)	109 124 129	(121) 10.4#	11 24 23	(19) 7.2	25 33 18		(25) 7.5	19 26 21	(22) 3.6	11 7 13	(10) 3.1
	1.5 µg	114 101 115	(110) 7.8	16 14 19	(16) 2.5	30 19 24		(24) 5.5	14 19 22	(18) 4.0	10 7 11	(9) 2.1
	5 µg	125 124 127	(125) 1.5	18 17 13	(16) 2.6	35 19 15		(23) 10.6	20 24 17	(20) 3.5	10 11 18	(13) 4.4
	15 µg	131 119 124	(125) 6.0	19 18 20	(19) 1.0	23 35 15		(24) 10.1	16 27 23	(22) 5.6	12 10 11	(11) 1.0
	50 µg	123 134 125	(127) 5.9	18 17 13	(16) 2.6	18 17 20		(18) 1.5	22 23 18	(21) 2.6	11 10 13	(11) 1.5
	150 µg	116 112 122	(117) 5.0	15 18 21	(18) 3.0	22 20 21		(21) 1.0	21 24 28	(24) 3.5	9 15 12	(12) 3.0
	500 µg	121 129 122	(124) 4.4	21 13 20	(18) 4.4	30 27 20		(26) 5.1	25 22 18	(22) 3.5	13 16 19	(16) 3.0
	1500 µg	133 P 133 P 124 P	(130) 5.2	20 P 21 P 23 P	(21) 1.5	26 P 25 P 27 P		(26) 1.0	23 P 16 P 22 P	(20) 3.8	13 P 13 P 9 P	(12) 2.3
	5000 µg	119 P 132 P 120 P	(124) 7.2	16 P 15 P 22 P	(18) 3.8	24 P 15 P 20 P		(20) 4.5	24 P 28 P 30 P	(27) 3.1	12 P 14 P 16 P	(14) 2.0
Positive controls S9-Mix (+)	Name	2AA		2AA		2AA			BP		2AA
	Dose Level	1 µg		2 µg		10 µg			5 µg		2 µg
	No. of Revertants	828 1029 961	(939) 102.2	137 141 115	(131) 14.0	121 133 143		(132) 11.0	197 249 242	(229) 28.2	108 152 107	(122) 25.7

BP          Benzo(a)pyrene

2AA        2-Aminoanthracene

P            Test item precipitate

#         Standard deviation

Table 3: Experiment 2 – Without Metabolic Activation (Pre-Incubation)

Test Period		From: 01 November 2021					To: 04 November 2021
S9-Mix (-)	Dose Level Per Plate	Number of revertants (rounded mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (DMF)	128 148 141	(139) 10.1#	15 13 23	(17) 5.3	23 17 16		(19) 3.8	21 21 24	(22) 1.7	7 4 4	(5) 1.7
	15 µg	145 158 135	(146) 11.5	14 9 10	(11) 2.6	19 16 14		(16) 2.5	17 15 23	(18) 4.2	6 6 11	(8) 2.9
	50 µg	127 147 135	(136) 10.1	12 16 15	(14) 2.1	25 11 17		(18) 7.0	15 14 17	(15) 1.5	5 5 9	(6) 2.3
	150 µg	143 137 129	(136) 7.0	14 9 14	(12) 2.9	12 14 14		(13) 1.2	18 19 19	(19) 0.6	9 10 9	(9) 0.6
	500 µg	146 P 143 P 140 P	(143) 3.0	15 P 19 P 26 P	(20) 5.6	13 P 18 P 18 P		(16) 2.9	15 P 21 P 21 P	(19) 3.5	7 P 10 P 8 P	(8) 1.5
	1500 µg	141 P 149 P 159 P	(150) 9.0	20 P 16 P 15 P	(17) 2.6	26 P 17 P 16 P		(20) 5.5	25 P 19 P 20 P	(21) 3.2	11 P 5 P 8 P	(8) 3.0
	5000 µg	148 P 141 P 147 P	(145) 3.8	13 P 15 P 18 P	(15) 2.5	14 P 16 P 16 P		(15) 1.2	23 P 20 P 23 P	(22) 1.7	7 P 6 P 4 P	(6) 1.5
Positive controls S9-Mix (-)	Name	ENNG		ENNG		ENNG			4NQO		9AA
	Dose Level	3 µg		5 µg		2 µg			0.2 µg		80 µg
	No. of Revertants	754 772 949	(825) 107.8	2451 2532 2368	(2450) 82.0	506 421 390		(439) 60.1	134 143 131	(136) 6.2	165 125 652	(314) 293.4

ENNG        N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO         4-Nitroquinoline-1-oxide

9AA           9-Aminoacridine

P               Test item precipitate

#         Standard deviation

Table 4: Experiment 2 – With Metabolic Activation (Pre-Incubation)

Test Period		From: 01 November 2021					To: 04 November 2021
S9-Mix (+)	Dose Level Per Plate	Number of revertants (rounded mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (DMF)	166 138 145	(150) 14.6#	10 9 7	(9) 1.5	23 17 22		(21) 3.2	15 20 22	(19) 3.6	8 9 13	(10) 2.6
	15 µg	152 156 163	(157) 5.6	10 12 7	(10) 2.5	22 27 26		(25) 2.6	27 20 21	(23) 3.8	15 10 13	(13) 2.5
	50 µg	154 141 153	(149) 7.2	13 7 17	(12) 5.0	26 23 24		(24) 1.5	23 25 16	(21) 4.7	6 8 12	(9) 3.1
	150 µg	164 162 157	(161) 3.6	13 16 14	(14) 1.5	25 17 28		(23) 5.7	25 23 26	(25) 1.5	11 13 11	(12) 1.2
	500 µg	151 P 152 P 153 P	(152) 1.0	17 P 12 P 16 P	(15) 2.6	24 P 23 P 16 P		(21) 4.4	26 P 16 P 23 P	(22) 5.1	16 P 9 P 15 P	(13) 3.8
	1500 µg	159 P 158 P 151 P	(156) 4.4	12 P 16 P 17 P	(15) 2.6	27 P 27 P 13 P		(22) 8.1	28 P 19 P 26 P	(24) 4.7	16 P 13 P 16 P	(15) 1.7
	5000 µg	163 P 145 P 160 P	(156) 9.6	17 P 18 P 11 P	(15) 3.8	21 P 23 P 21 P		(22) 1.2	28 P 25 P 21 P	(25) 3.5	7 P 13 P 8 P	(9) 3.2
Positive controls S9-Mix (+)	Name	2AA		2AA		2AA			BP		2AA
	Dose Level	1 µg		2 µg		10 µg			5 µg		2 µg
	No. of Revertants	1112 1053 1194	(1120) 70.8	142 138 144	(141) 3.1	206 136 160		(167) 35.6	174 173 165	(171) 4.9	124 139 134	(132) 7.6

BP          Benzo(a)pyrene

2AA        2-Aminoanthracene

P            Test item precipitate

#         Standard deviation

Conclusions:

The in vitro gene mutation potential in bacteria of R05 was determined to be negative.

Executive summary:

The in vitro gene mutation potential in bacteria of R05 was tested in an Ames test (OECD 471) and was determined to be negative. Bacterial S. typhimurium and E. coli strains were treated with suspensions of R05 at eight dose levels in triplicate, both with and without metabolic activation, via plate incorporation and pre-incubation assays. The vehicle, positive and negative controls confirmed the sensitivity of the assay and the efficacy of the S9-mix.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

A number of in vivo genotoxicity tests were included as part of NONS registrations. Three in vivo micronucleus clastogenicity studies conducted in mice were undertaken on 3,3'-dioctadecyl-1,1'- methylenebis(4,1-phenylene)diurea (PU12/A123; EC 406-690-3), 3,3'-dicyclohexyl-1,1'-methylenebis(4,1-phenylene)diurea (R95; EC 406-370-3), and a mixture of: 3,3'-dicyclohexyl-1,1'-methylenebis(4,1-phenylene)diurea; 3-cyclohexyl- 1-(4-(4-(3-octadecylureido)benzyl)phenyl)urea; 3,3'-dioctadecyl-1,1'-methylenebis(4,1-phenylene)diurea (PU10; A002; PU18; EC 406-530-2). No adverse effects were observed and no chromosomal abnormalities detected based on either the P/N ratio or micronucleus formation. Hence it was confirmed that no in vivo clastogenicity was evident as a result of treatment with the test substances.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Remarks:

This study is included in a NONS registration and therefore has been evaluated by a relevant competent authority and is considered to be reliable.

Qualifier:

according to guideline

Guideline:

other: Annex V (Micronucleus)

GLP compliance:

yes

Type of assay:

other: micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Route of administration:

oral: unspecified

Vehicle:

Corn oil

Details on exposure:

The dose of the test substance was judged to be the maximum attainable.

Duration of treatment / exposure:

Male: 5000 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 5000 mg/kg; No. of animals: 5; Sacrifice time: 48 hours
Male: 5000 mg/kg; No. of animals: 5; Sacrifice time: 72 hours
Female: 5000 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
Female: 5000 mg/kg; No. of animals: 5; Sacrifice times: 48 hours
Female: 5000 mg/kg; No. of animals: 5; Sacrifice times: 72 hours

Frequency of treatment:

Not specified

Post exposure period:

Not specified

Dose / conc.:

5 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

15 male and 15 females

Control animals:

not specified

Positive control(s):

Cyclophosphamide

Tissues and cell types examined:

Not specified

Evaluation criteria:

Micronucleus formation

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

No increase in micronucleus formation was observed. The positive control substance cyclophosphamide led to an increase in the incidence of micronuclei.
Toxicity: yes. Doses producing toxicity: Mitotic index: 5000 mg/kg; In a preliminary experiment 2 males and 2 females were treated with 5000 mg/kg of the test substance and exhibited a reduction in spontaneous activity.

Conclusions:

The in vivo genetic toxicity of the test item was assessed in a micronucleus assay. The result was negative for the test item.

Executive summary:

The in vivo genetic toxicity of the test item was assessed in a micronucleus assay. The result was negative for the test item.

In vivo genetic toxicity was assessed in a micronucleus assay following a standard guideline. 15 male and female mice were exposed at a concentration of 5000 mg/kg, with 5 of each sex being sacrificed after 24, 48 and 72 hours. The study was negative, as no increase in micronucleus formation was observed.

In a preliminary experiment, 2 males and 2 females were treated with 5000 mg/kg of the test substance and exhibited a reduction in spontaneous activity.

The study is a GLP compliant, guideline experimental study and is acceptable with restrictions for assessment.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Remarks:

This study is included in a NONS registration and therefore has been evaluated by a relevant competent authority and is considered to be reliable.

Qualifier:

according to guideline

Guideline:

other: Annex V (B12, Micronucleus)

GLP compliance:

yes

Type of assay:

other: micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Route of administration:

oral: unspecified

Vehicle:

Corn oil

Duration of treatment / exposure:

Male: 5000 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 5000 mg/kg; No. of animals: 5; Sacrifice time: 48 hours
Male: 5000 mg/kg; No. of animals: 5; Sacrifice time: 72 hours
Female: 5000 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
Female: 5000 mg/kg; No. of animals: 5; Sacrifice times: 48 hours
Female: 5000 mg/kg; No. of animals: 5; Sacrifice times: 72 hours

Frequency of treatment:

Not specified

Post exposure period:

Not specified

Dose / conc.:

5 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

15 male and 15 females

Control animals:

not specified

Positive control(s):

Not specified

Tissues and cell types examined:

Not specified

Evaluation criteria:

P/N Ratio

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

Doses producing toxicity: No change in the P/N ratio.
In a preliminary toxicity study in which 2 males and 2 females were given 5000 mg/kg, the only sign of toxicity observed was apathy and a decrease in spontaneous activity for up to 24 hours post-treatment

Conclusions:

An in vivo micronucleus assay with the test item was negative, as no change in the P/N ratio was observed.

Executive summary:

The in vivo genetic toxicity of the test item was assessed using NMIR rats (male and female) in a micronucleus assay following a standard guideline. 15 male and female mice were exposed at a concentration of 5000 mg/kg, with 5 of each sex sacrificed after 24, 48 and 72 hours. The P/N ratio was investigated. The study was negative. as no change in the P/N ratio was observed. In a preliminary toxicity study in which 2 males and 2 females were given 5000 mg/kg, the only sign of toxicity observed was apathy and a decrease in spontaneous activity for up to 24 hours post-treatment.

The study is a GLP compliant, guideline experimental study and is acceptable with restrictions for assessment of this endpoint.

Reference 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Remarks:

This study is included in a NONS registration and therefore has been evaluated by a relevant competent authority and is considered to be reliable.

Qualifier:

according to guideline

Guideline:

other: Annex V

GLP compliance:

yes

Type of assay:

mammalian bone marrow chromosome aberration test

Species:

mouse

Strain:

NMRI

Sex:

male/female

Route of administration:

other: oral (not specified)

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil

Duration of treatment / exposure:

24, 48 and 72 hours

Dose / conc.:

5 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 per sex per dose

Control animals:

not specified

Positive control(s):

Not specified

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

Based on P/N ratio

Additional information on results:

Other toxic signs: Dosing at 5000 mg/kg in a preliminary test for toxicity using 20 mL/kg b.w. resulted in a reduction in activity in all animals up to 6 hours post-dosing but these effects are not clearly substance related. No signs of toxicity were stated for the main study.

Conclusions:

The test item was concluded to be negative for mammalian chromosome aberration. No signs of toxicity or cytotoxicity (based on P/N ratio) were observed.

Executive summary:

The test item was investigated for in vivo chromosome aberration in male and female NMRI mice following EU Annex V guidelines (micronucleus test). Test animals were administered 5000 mg/kg test item by oral gavage and sacrificed at 24, 48 and 72 hours. The test item was concluded to be negative for mammalian chromosome aberration. No signs of toxicity or cytotoxicity (based on P/N ratio) were observed.

The study is a GLP compliant, guideline experimental study and is acceptable with restrictions for assessment of this endpoint.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

The substances within the MDI category did not demonstrate a mutagenic or clastogenic response, and thus are considered to be non-mutagenic. Therefore, the substances in the MDI category are not mutagenic or clastogenic and are not classified for this endpoint.