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

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997, corrected 2020

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Test guideline 3

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

Test material

Test material information

Constituent 1

Reference substance name:

Reaction product of MDI (202-966-0), Octadecylamine (204-695-3) and magnesium hydroxide (215-170-3)

EC Number:

944-730-6

IUPAC Name:

Reaction product of MDI (202-966-0), Octadecylamine (204-695-3) and magnesium hydroxide (215-170-3)

Method

Target gene:

- Salmonella: Histidine
- E. Coli: Tryptophan

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

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.

Controls

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.

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.