2,2'-[(1-methylethylidene)bis(cyclohexane-4,1-diyloxymethylene)]bisoxirane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames (Engelhardt, 2004)

Under the conditions of the study the test material was a mutagenic agent in the bacterial reverse mutation test.

Ames (Kato, 2019)

Under the conditions of the study the test material was positive for inducing gene mutations.

Mouse lymphoma assay (Kajiwara, 2019)

The test material was considered to have the potential to induce gene mutation in mouse lymphoma cells under the conditions employed in this study.

In vitro cytogenicity study in mammalian cells (waived)

An in vitro cytogenicity study in mammalian cells or an in vitro micronucleus study does not need to be conducted because adequate data from an in vivo cytogenicity test are available - (study scientifically not necessary / other information available).

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:

16 June 2004 to 24 June 2004

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

- Histidine requirement in the Salmonella typhimurium strains (Histidine operon).
- Tryptophan requirement in the Escherichia coli strain (Tryptophan operon).

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Deep-frozen (-70 to -80 °C) bacterial cultures (Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 98) were thawed at room temperature, and 0.1 mL of this bacterial suspension was inoculated in nutrient broth solution (8 g Difco nutrient broth + 5 g NaCI/litre) and incubated in the shaking water bath at 37 °C for about 12-16 hours. As a rule, a germ density of ≥ 10^8 bacteria/mL is reached. These cultures were grown overnight and kept in iced water from the beginning of the experiment until the end in order to prevent further growth.
- The use of the strains mentioned is in accordance with the current scientific recommendations for the conduct of this assay.
- The Salmonella strains were obtained from KNOLL Aktiengesellschaft on October 30, 1989.
- The Salmonella strains were checked for the following characteristics at regular intervals: deep rough character (rfa) ; UV sensitivity (n uvrB); ampicillin resistance (R factor plasmid).

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

- Deep-frozen (-70 to -80 °C) bacterial cultures (E . coli WP2 uvrA) were thawed at room temperature, and 0.1 mL of this bacterial suspension was inoculated in nutrient broth solution (8 g Difco nutrient broth + 5 g NaCI/litre) and incubated in the shaking water bath at 37°C for about 12-16 hours. As a rule, a germ density of ≥ 10^8 bacteria/mL is reached. These cultures were grown overnight and kept in iced water from the beginning of the experiment until the end in order to prevent further growth.
- The use of the strains mentioned is in accordance with the current scientific recommendations for the conduct of this assay.
- The Escherichia coli strain was obtained from Merck on September 9, 1991.
- E.coli WP2 uvrA is checked for UV sensitivity.

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix

Test concentrations with justification for top dose:

1st Experiment (all strains): 0; 32; 160; 800; 4 000 and 8 000 µg/plate
2nd Experiment (TA 1535 only): 0; 100 ; 200 ; 400; 800 and 1 000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the insolubility of the test material in water, DMSO was selected as the vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

other: 2-aminoanthracene (2-AA) - 2.5 µg/plate: TA 1535, 100, 1537, 98 - 60 µg/plate: E.coli WP2 uvrA (+ S-9 mix) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) 5 µg/plate: TA 1535, TA 100 and 4-nitro-o-phenylendiamine (NOPD) 10 µg/plate: TA 98 (- S-9 mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation

NUMBER OF REPLICATIONS: 3 test plates per dose or per control

SALMONELLA TYPHIMURIUM
- Test tubes containing 2 mL portions of soft agar (overlay agar), which consisted of 100 mL agar (0.8% agar + 0.6% NaCI) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0 .5 mM biotin) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution or vehicle
0.1 mL fresh bacterial culture
0.5 mL S-9 mix (in tests with metabolic activation) or 0.5 mL phosphate buffer (in tests without metabolic activation)
- After mixing, the samples were poured onto Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds.
- Composition of the minimal glucose agar: 980 mL aqua dest., 20 mL Vogel-Bonner E medium, 15 g Difco bacto agar and 20 g D-glucose, monohydrate.
- After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies (his+ revertants) were counted.

ESCHERICHIA COLI
- Test tubes containing 2 mL portions of soft agar (overlay agar), which consisted of 100 mL agar (0 .8% agar + 0.6% NaCI) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution or vehicle
0.1 mL fresh bacterial culture
0.5 mL S-9 mix (in tests with metabolic activation) or 0.5 mL phosphate buffer (in tests without metabolic activation)
- After mixing, the samples were poured onto minimal agar plates within approx. 30 seconds.
- Composition of the minimal agar: The composition of the minimal agar (SA1 selective agar) was based on the description of Green, M .H .L. and Muriel, W .J., with the exception of solution E (tryptophan solution), which has previously been added to the soft agar: 300 mL solution B (agar), 100 mL solution A (saline solution), 8 mL solution C (glucose solution) and 10 mL solution D (casein solution).
- After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies (trp+ revertants) were counted.

TITRE DETERMINATION
- 0.1 mL of the overnight cultures was diluted to 10^-6 in each case. Test tubes containing 2 mL portions of soft agar containing maximal amino acid solution (5 mM tryptophan or 5 mM histidine + 0 .5 mM biotin) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order: 0.1 mL vehicle (without and with test material), 0.1 mL fresh bacterial culture (dilution: 10^-6) and 0.5 mL S-9 mix.
- After mixing, the samples were poured onto the agar plates within approx. 30 seconds. After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies were counted.

EVALUATION
- Mutagenicity: Individual plate counts, the mean number of revertant colonies per plate and the standard deviations were given for all dose groups as well as for the positive and negative (vehicle) controls in all experiments . In general, five doses of the test material were tested with a maximum of 5 mg/plate, and triplicate plating was used for all test groups at least in the 1st experiment. Dose selection and evaluation as well as the number of plates used in repeat studies or further experiments were based on the findings of the 1st experiment.
- Titre: The titre was generally determined only in the experimental parts with S-9 mix both for the negative controls (vehicle only) and for the two highest doses in all experiments.
- Toxicity: Toxicity detected by a decrease in the number of revertants, clearing or diminution of the background lawn (= reduced his- or trp- background growth) or reduction in the titre, was recorded for all test groups both with and without S-9 mix in all experiments.
- Solubility: Precipitation of the test material was recorded. As long as precipitation did not interfere with the colony scoring, 5 mg/plate was generally selected and analysed (in cases of non-toxic compounds) as the maximum dose at least in the 1st experiment even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate may also have been tested in repeat experiments for further clarification/substantiation.

ACCEPTANCE CRITERIA
Generally, the experiment is considered valid if the following criteria are met:
- The number of revertant colonies in the negative controls was within the normal range of the historical control data for each tester strain.
- The sterility controls revealed no indication of bacterial contamination.
- The positive control articles both with and without S-9 mix induced a significant increase in the number of revertant colonies within the range of the historical control data or above.
- The titre of viable bacteria was ≥ 10^8/mL.

Evaluation criteria:

EVALUATION CRITERIA
- The test material is considered positive in this assay if: A dose-related and reproducible increase in the number of revertant colonies, i .e. about doubling of the spontaneous mutation rate in at least one tester strain either without S-9 mix or after adding a metabolising system.
- A test material is generally considered non-mutagenic in this test if: The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.

Key result

Species / strain:

S. typhimurium, other: TA 98 and TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium, other: TA 100 and TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

Mutagenicity was observed from about 100 µg/plate onward (factor 2 4) with an increase in the number of his+ revertants by a factor of 13.5 at 1 000 µg/plate

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

Slight increase in the number of revertant colonies at 800 µg/plate (factor 2.2)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

MUTAGENICITY TESTS
- Tests without S-9 mix: No increase in the number of his+ or trp+ revertants in any strain.
- Tests with S-9 mix:
TA 1535 : Mutagenicity was observed from about 100 µg/plate onward (factor 2.4) with an increase in the number of his+ revertants by a factor of 13.5 at 1 000 µg/plate.
TA 100: Slight increase in the number of revertant colonies at 800 µg/plate (factor 2.2).
TA 1537, TA 98 and E. coli WP2 uvrA: No increase in the number of his+ or trp+ revertants.

TOXICITY
- A bacteriotoxic effect (reduced his- or trp- background growth and / or decrease in the number of his+ revertants, reduction in the titre) was observed depending on the strain and test conditions from about 4 000 µg/plate onward.

SOLUBILITY
- Test material precipitation was found from about 4 000 µg/plate onward.

DISCUSSION
- According to the results of the present study, the test material led to a dose-dependent increase in the number of his+ revertants with the strains TA 1535 and TA 100 with S-9 mix.

Table 1: Summary of Experiment 1

± S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type			Frameshift Type
		TA100	TA1535	WP2uvrA	TA98	TA1537
-	Solvent 32 160 800 4000 8000	108 107 109 118 61 54	18 16 17 15 10 7	37 37 33 35 27 27	28 23 27 23 13 9	11 7 6 6 3 2
+	Solvent 32 160 800 4000 8000	110 124 150 242 40 19	17 26 63 182 39 20	39 38 35 32 36 37	40 37 43 33 25 14	12 10 11 8 2 2
Positive Controls
-	Name	MNNG	MNNG	4-NQO	NOPD	ACC
	Concentration (µg/plate)	5	5	5	10	100
	Mean no. colonies/plate	1036	962	701	717	384
+	Name	2AA	2AA	2AA	2AA	2AA
	Concentration (µg/plate)	2.5	2.5	60	2.5	2.5
	Mean no. colonies/plate	1103	151	262	911	166

MNNG = N-methyl-N’-nitro-N-nitrosoguanidine

4-NQO = 4 -nitroquinoline-1-oxide

ACC = 9-aminoacridine

2AA = 2-aminoanthracene

NOPD = 4-nitro-o-phenylendiamine

Table 2: Summary of Experiment 2

± S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		TA1535
-	Solvent 100 200 400 800 1000	18 22 20 19 20 15
+	Solvent 100 200 400 800 1000	18 44 61 127 212 247
-	Name	MNNG
	Concentration (µg/plate)	5.0
	Mean no. colonies/plate	1414
+	Name	2AA
	Concentration (µg/plate)	2.5
	Mean no. colonies/plate	124

MNNG = N-methyl-N’-nitro-N-nitrosoguanidine

2AA = 2-aminoanthracene

Conclusions:

Under the conditions of this study the test material was a mutagenic agent in the bacterial reverse mutation test.

Executive summary:

The genetic toxicity of the test material was investigated in accordance with the standardised guidelines OECD 471, EU Method B.13/B.14 and OPPTS 870.5100, under GLP conditions.

The test material was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay.

S. typhinurium TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA were treated at 32 - 8 000 µg/plate in a standard plate test both with and without metabolic activation (Aroclor-induced rat liver S-9 mix).

Precipitation of the test material and a bacteriotoxic effect was found from about 4 000 µg/plate onward.

No increase in the number of his+ or trp+ revertants was observed in any strain without S9 mix.

With S9 mix a positive reaction from about 100 µg/plate (factor 2.4) onward with an increase in the number of mutant colonies by a factor of 13.5 at 1 000 µg/plate was observed in TA 1535. In TA 100 with S9 mix, a slight increase in the number of revertant colonies at 800 µg/plate (factor 2.2) was observed. No increase in the number of his+ or trp+ revertants were seen in TA 1537, TA 98 or E.coli WP2 uvrA with S9 mix.

Under the conditions of this study the test material was a mutagenic agent in the bacterial reverse mutation test.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 May 2018 to 10 April 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

Notification of testing methods relating to the new chemical substances (Notification No. 7 of Japanese Ministry of Health, Labour and Welfare, No. 5 of Japanese Ministry of Economy, Trade and Industry, No. 110331009 of Japanese Ministry of the Environment on March 31, 2011) and Notification on partial revision of the standard (Notification No. 1 of Japanese Ministry of Health, Labour and Welfare, No. 1 of Japanese Ministry of Economy, Trade and Industry, No. 1512211 of Japanese Ministry of the Environment on December 21, 2015).
The standard that the Minister of Labor establishes based on the Industrial Safety and Health Law Article 57-3 Paragraph 1 (Notification No. 77 of Japanese Ministry of Labor on September 1, 1988) and Notification on partial revision of the standard (Notification No. 67 of Japanese Ministry of Labor on June 2, 1997).
Notification for concrete operation for bacterial mutagenicity tests and methods for evaluation of test results (Administrative notice, Japanese Ministry of Labor on February 8, 1999).

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells
Salmonella typhimurium strains: Japan Bioassay Research Center
Escherichia coli: National Institution of Genetics
Bacterial strains have been stored at -80 °C until use

MEDIA USED
- Type and identity of media: 25 g/L Nutirent broth no. 2 (Oxoid)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Dose-finding study
In the dose-finding study, 6 doses including 5 000 µg/plate as the highest dose obtained using a common ratio of 4.

Main Study
In Salmonella typhimurium TA1535 in the absence of metabolic activation two different dose ranges were used in the main study, from 313 to 5 000 µg/plate and from 2.44 to 78.1 µg/plate using a ratio of 2, were tested.
In Salmonella typhimurium TA100 in the absence of metabolic activation and in Salmonella typhimurium TA100 and TA1535 in the presence of metabolic activation, the doses for the main study were set with reference to the dose at which increases were observed irrespective of growth-inhibition. The maximum dose was set at 2 500 or 5 000 µg/plate and a total of 5 or 6 doses obtained using a common ratio of 2 were tested to confirm the dose-dependency.
The maximum dose for the main study was set at 78.1, 313, or 1 250 µg/plate with reference to the growth-inhibition dose and total 6 doses using a common ratio of 2 were tested.
A confirmatory study was performed to confirm the reproducibility of the results for the main study. In Salmonella typhimurium TA100 and TA1535, the maximum dose was set at 625 or 5 000 µg/plate in the absence of metabolic activation and at 1 250 or 2 500 µg/plate in the presence of metabolic activation and total 5 to 7 doses using a common ratio of 2 were tested in order to observe the dose-dependency.
The other bacterial strains were at the same doses as the main study.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulfoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide; 2-Aminoanthracene.

Details on test system and experimental conditions:

METHOD OF APPLICATION: The dose-finding, main, confirmatory, and additional confirmatory studies were performed by a pre-incubation method.
- Cell density at seeding: 1.0 × 10^9 cells/mL or more
- Preparation of bacterial suspension: Each 50 µL thawed bacterial suspension was added on 25 mL of culture medium in a 200 mL Erlenmeyer flask. Each flask was shake-cultured by 100 reciprocations/minute for 10 hours at 37 °C. The nutrient broth culture medium was kept at 4 °C until the start of shake culture. The optical density of bacterial suspension was then measured at 660 nm by a spectrophotometer. The number of bacterial cells was confirmed to be 1.0 × 10^9 cells/mL or more.
- The studies were performed in the dose-finding, main, and confirmatory studies in the presence and absence of metabolic activation in each strain including the positive and negative controls. In Salmonella typhimurium TA1535 in the absence of metabolic activation, an additional confirmatory study was performed. Based on the data obtained from the dose-finding study, doses for the main, confirmatory, and additional confirmatory studies were set. Positive controls were shared in plural experiments. Minimum glucose agar plates were prepared in duplicate per dose for the test substance, negative and positive controls in the dose-finding study and in triplicate in the main, confirmatory, and additional confirmatory studies.


DURATION
- Preincubation period: 20 minutes at 37 °C
- A volume of 0.1 mL of negative control, test substance, and positive control solution, was put in a test tube. The 0.5 mL of 1/15 mol/L sodium phosphate buffer (pH 7.4) in the absence of metabolic activation and 0.5 mL of S9 mix in the presence of metabolic activation, and 0.1 mL of bacterial suspension were added into each tube. After mixing, the mixture was shake-cultured by 105 to 116 reciprocations/minute (variation range) at 37 °C for 20 minutes. The test tube was then taken out and 2.0 mL of top agar was added and mixed. The solution was overlaid on a minimum glucose agar plate.
- Expression time: Each minimum glucose agar plate was inverted and cultivated for 48 hours at 37 °C.


NUMBER OF CELLS EVALUATED
Precipitation was checked macroscopically and observation of the presence or absence for the growth of the background lawn was performed with the stereomicroscope at colony counting. The evidence was recorded when any precipitation or growth-inhibition was produced. The number of revertant colonies produced after treatment of Salmonella typhimurium TA100 and positive control substance in all bacterial strains were counted using a colony analyser CA-11D and the others were counted manually. Since the test substance precipitated, for the plates in which precipitation affected colony counting using a colony analyser, the number of revertant colonies was counted manually. When using a colony analyser, the observed colony counts were corrected (correction value: 1.20).


STERILITY TEST
The sterility test was conducted to examine for contamination in the test substance or S9 mix. A volume of the test substance solution and S9 mix were the same as the treatment of bacterial strain with the test substance solution. The maximum concentration of the test substance solution or S9 mix, and 2.0 mL of top agar was mixed and overlaid on a minimum glucose agar plate. Each minimum glucose agar plate was inverted and cultivated for 48 hours at 37 °C.

Evaluation criteria:

The test results were judged as positive when mean value of revertant colonies treated with the test substance exceeded the upper limit of background data of the negative control value and also biologically meaningful increase in the number of revertant colonies due to mutagenicity of the test substance such as statistically significant increases with dose-dependency and reproducibility were observed.

The study was judged as a compatible condition in the following cases.
1. The mean values of revertant colonies in the negative and positive controls are within the range of background data. When the values deviate from the range, the deviation can be judged to be accidental by comparison with the background data.
2. The mean values of revertant colonies in positive controls are twice or more than the corresponding negative controls.
3. No contaminants are found in the sterility test.
4. No lack of the colony counts value.

Statistics:

Two statistical analyses of Dunnett’s multiple comparison method (one-side test) and linear regression method were prepared in this study. The number of revertant colonies of each bacterial strain at each dose in the main and the confirmatory studies were compared with that of the negative control both in presence and absence of metabolic activation, respectively, and statistically significant difference in the number of revertant colonies was analysed first by the multiple comparison method (p < 0.05). The dose-reactivity was analysed by linear regression method (p < 0.05) when the statistically significant difference was detected by the multiple comparison method. In linear regression analysis, the dose of negative control was replaced with the calculated value as:

X0: X1-[(Xr-X1)/(r-1)]

X1: log (lowest dose)
Xr: log (highest dose of no growth inhibition)
r: number of doses between X1 and Xr

Application usage:
Mean and standard deviation; Microsoft® EXCEL 2010 for Windows (Version 14)
Statistical analysis; SAS® system for Window (Release 9.3)

Key result

Species / strain:

other: Salmonella typhimurium TA100, TA1535, TA98, and TA1537, and Escherichia coli WP2 uvrA.

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The induction of gene mutation in the test substance was evaluated by the reverse mutation test with a pre-incubation method at 37 °C for 20 minutes using base-pair substitution type strains of Salmonella typhimurium TA100, TA1535, Escherichia coli WP2uvrA, and frameshift mutation type strains of Salmonella typhimurium TA98, TA1537. The test was composed of the dose-finding, main, and confirmatory studies, which were performed in the presence and absence of metabolic activation and reproducibility of the results of the main and confirmatory studies was examined. In addition for Salmonella typhimurium TA1535 in the absence of metabolic activation, an additional confirmatory study was performed to confirm the reproducibility of the positive response. Based on the data obtained from the dose-finding study, doses for the main, confirmatory, and additional confirmatory studies were set. Reproducibility of the results of the main, confirmatory, and additional confirmatory studies was examined.
The test substance increased the number of revertant mutant colonies in Salmonella typhimurium TA100 and TA1535 both in the presence and absence of metabolic activation at the dose at which growth inhibition was observed in the main and confirmatory studies, in three studies with an additional confirmatory study. Statistically significant increases in the number of revertant colonies were observed in both bacterial strains except for Salmonella typhimurium TA1535 in the absence of metabolic activation in the confirmatory study. The mean number of revertant colonies were exceeded the upper limit of variation range of the negative control calculated from background data and the mean number of revertant colonies for each bacterial strains were approximately from 1.8 to 8.8-fold compared with the respective negative control values. Based on observation of the background lawn confirmed that all observed colonies were revertant colonies, not surviving bacteria grown by cytotoxicity. In the main study, statistically significant dose-dependency was confirmed only in Salmonella typhimurium TA100 in the presence of metabolic activation, and was confirmed only in Salmonella typhimurium TA100 in the presence and absence of metabolic activation in the confirmatory study. Statistically significant dose-dependency was not observed in the other strains of Salmonella typhimurium TA100 and TA1535 as statistical analysis is based on the doses which did not show growth inhibition, the number of revertant colonies had shown a clear dose-dependency. Thus, it was judged that the dose-dependent increase in the number of revertant colonies was reproducible, and also judged that statistically significant increases in the number of revertant colonies exceeding background data were confirmed. In Salmonella typhimurium TA1535 in the absence of metabolic activation in the main study, since cytotoxicity have strongly affected, it seemed that positive response was not observed. These results revealed that biologically meaningful increase in the number of revertant colonies due to mutagenicity of the test substance was shown. The number of revertant colonies in the positive controls were twice or more than that of the respective negative controls in all bacterial strains both in the presence and absence of metabolic activation. 
The mean values of revertant colonies of the negative and positive controls were within the variation range calculated from background data in all studies. No contaminants were found in the sterility test. These results demonstrated that the test was properly performed. No suspected factor that affected the reliability of the studies was observed.
Based on the above results, the induction of gene mutation in the test substance was determined as positive.
The test substance showed growth-inhibition in all bacterial strains at 78.1 or 156 µg/plate or more in the absence of metabolic activation and at 156 or 625 µg/plate or more in the presence of metabolic activation. The test substance precipitated at 5 000 µg/plate in the absence of metabolic activation.

Dose Range-Finding Study

In the dose-finding study, the test substance showed growth-inhibition in all bacterial strains at 78.1 or 313 µg/plate or more in the absence of metabolic activation and at 313 or 1 250 µg/plate or more in the presence of metabolic activation. The test substance increased the number of revertant colonies with dose dependence in the dose range showing growth-inhibition in Salmonella typhimurium TA100 in the absence of metabolic activation and Salmonella typhimurium TA100 and TA1535 in the presence of metabolic activation. In Salmonella typhimuium TA1535 in the absence of metabolic activation, a slightly increase in the number of revertant colonies was observed in the dose range showing growth-inhibition. The test substance precipitated in the absence of metabolic activation at 5 000 µg/plate, but did not affect the colony counting.

Results of the Dose Range-Finding Study Without Metabolic Activation

Compound	Dose (µg/plate)	Revertants Per Plate (Mean Number of Revertants per Dose)
		Base-Pair Substitution Type			Frameshift Mutation Type
		TA 100	TA 1535	WP2 uvrA	TA 98	TA 1537
Negative control: Dimethyl sulfoxide	-	108 121 (115)	13 19 (16)	26 22 (24)	34 30 (32)	6 11 (9)
Test substance	4.88	116 124 (120)	10 16 (13)	20 21 (21)	20 17 (19)	6 5 (6)
	19.5	147 133 (140)	16 13 (15)	23 26 (25)	22 13 (18)	6 9 (8)
	78.1	175* 199* (187)	11* 10* (11)	31 27 (29)	18* 26* (22)	5* 7* (6)
	313	233* 217* (225)	11* 9* (10)	25* 16* (21)	20* 13* (17)	8* 6* (7)
	1250	303* 262* (333)	17* 11* (14)	18* 23* (21)	21* 14* (18)	2* 4* (3)
	5000	461*# 436*# (449)	19*# 20*# (20)	28*# 26*# (27)	14*# 15*# (15)	1*# 2*# (2)
2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide	0.01	505 475 (490)	-	120 110 (115)	-	-
	0.1	-	-	-	363 366 (365)
Sodium azide	0.5	-	618 648 (633)	-	-	-
9-Aminoacridine	80.0	-	-	-	-	301 244 (273)

* Growth inhibition

# Precipitation

 

Results of the Dose Range-Finding Study With Metabolic Activation

Compound	Dose (µg/plate)	Revertants Per Plate (Mean Number of Revertants per Dose)
		Base-Pair Substitution Type			Frameshift Mutation Type
		TA 100	TA 1535	WP2 uvrA	TA 98	TA 1537
Negative control: Dimethyl sulfoxide	-	127 128 (128)	9 8 (9)	31 33 (32)	24 25 (25)	17 16 (17)
Test material	4.88	113 118 (116)	7 13 (10)	31 20 (26)	23 33 (28)	15 15 (15)
	19.5	126 132 (129)	14 15 (15)	21 20 (21)	21 28 (25)	17 13 (15)
	78.1	145 155 (150)	16 20 (18)	33 25 (29)	19 32 (26)	12 14 (13)
	313	175 172 (174)	24* 23* (24)	22 38 (30)	20 20 (20)	12* 14* (13)
	1 250	179* 188* (184)	95* 90* (93)	20* 28* (24)	24* 24* (24)	9* 11* (10)
	5 000	469* 472* (471)	212* 235* (224)	31* 14* (23)	31* 14* (23)	7* 10* (9)
2-Aminoanthracine	0.5	-	-	-	548 546 (547)	-
	1.0	1234 1197 (1216)	-	-	-	-
	2.0	-	499 466 (483)	-	-	234 195 (215)
	10.0	-	-	1065 987 (1026)	-	-

* Growth inhibition

 

Main Study Results Without Metabolic Activation

Compound	Dose (µg/plate)	Revertants Per Plate (Mean Number of Revertants per Dose)
		Base-Pair Substitution Type
		TA 100	TA 1535
Negative control: Dimethyl sulfoxide	-	124 130 111 (122) [9.7]	12 8 9 (10) [2.1]
Test material	78.1	181* 170* 199* (183) [14.6]†	-
	156	175* 206* 180* (187) [16.6]†	-
	313	199* 181* 253* (211) [37.5]†	6* 9* 15* (10) [4.6]
	625	294* 220* 204* (239) [48.0]†	10* 12* 13* (12) [1.5]
	1 250	327* 307* 330* (321) [12.5]†	19* 19* 10* (16) [5.2]
	2 500	404* 460* 428* (431) [28.1]†	21* 17* 19* (19) [2.0]†
	5 000	-	34*# 31*# 24*# (30) [5.1]†
2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide	0.01	524 507 545 (525) [19.0]	-
Sodium azide	0.5	-	641 589 601 (610) [27.2]

* Growth inhibition

# Precipitation

[ ] Standard deviation

† p < 0.05

 

Main Study Results Without Metabolic Activation

Compound	Dose (µg/plate)	Revertants Per Plate (Mean Number of Revertants per Dose)
		Base-Pair Substitution Type		Frameshift Mutation Type
		TA 1535	WP2 uvrA	TA98	TA1537
Negative control: Dimethyl sulfoxide	-	12 8 9 (10 [2.1]	38 30 42 (37) [6.1]	27 22 19 (23) [4.0]	8 8 6 (7) [1.2]
Test material	2.44	13 9 12 (11) [2.1]	-	24 24 21 (23) [1.7]	7 10 8 (8) [1.5]
	4.88	13 15 6 (11) [4.7]	-	26 20 23 (23) [3.0]	6 9 8 (8) [1.5]
	9.77	8 9 8 (8) [0.6]	37 47 33 (39) [7.2]	18 26 21 (22) [4.0]	5 8 6 (6) [1.5]
	19.5	6 6 10 (7) [2.3]	35 26 37 (33) [5.9]	15 26 16 (19) [1.6]	7 5 7 (6) [1.2]
	39.1	9 15 14 (13) [3.2]	38 29 38 (35) [5.2]	23 17 24 (21) [3.8]	7 6 9 (7) [1.5]
	78.1	9* 13* 13* (12) [2.3]	45 43 29 (39) [8.7]	17* 20* 18* (18) [1.5]	8* 7* 8* (8) [0.6]
	156	-	28* 35* 40* (34) [6.0]	-	-
	313	-	29* 34* 33* (29) [4.5]	-	-
2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide	0.01	-	141 139 137 (139) [2.0]	-	-
	0.1	-	-	402 433 375 (403) [29.0]	-
Sodium azide	0.5	641 589 601 (610) [27.2]	-	-	-
9-Aminoacridine	80.0	-	-	-	245 232 259 (245) [13.5]

* Growth inhibition

 [ ] Standard deviation

 

Main Study Results With Metabolic Activation

Compound	Dose (µg/plate)	Revertants Per Plate (Mean Number of Revertants per Dose)
		Base-Pair Substitution Type			Frameshift Mutation Type
		TA 100	TA 1535	WP2 uvrA	TA 98	TA 1537
Negative control: Dimethyl sulfoxide	-	128 131 113 (124) [9.6]	13 11 12 (12) [1.0]	43 39 35 (39) [4.0]	34 25 26 (28) [4.9]	14 16 18 (16) [2.0]
Test material	9.77	-	-	-	-	61 16 13 (15) [1.7]
	19.5	-	-	-	-	15 15 16 (15) [1.0]
	39.1	-	-	49 39 36 (41) [6.8]	24 39 25 (29) [8.4]	17 14 16 (16) [1.5]
	78.1	-	17 12 19 (16) [3.6]	48 38 37 (41) [6.1]	35 35 27 (32) [4.6]	18 10 12 (13) [4.2]
	156	158 195 153 (169) [22.9]†	18* 17* 23* (19) [3.2]	38 40 33 (37) [3.6]	34 36 33 (34) [1.5]	9* 12* 12* (11) [1.7]
	313	189 201 164 (185) [18.9]†	17* 23* 24* 21 [3.8]	42 32 37 (37) [5.0]	31 27 25 (28) [3.1]	17* 11* 14* (14) [3.0]
	625	220* 198* 183* (200) [18.6]†	44* 43* 35* (41) [4.9]†	29* 33* 28* (27) [3.2]	32* 25* 23* (27) [4.7]	-
	1 250	234* 202* 249* (228) [24.0]†	71* 63* 73* (69) [5.3]†	29* 22* 33* (28) [5.6]	28* 22* 20* (23) [4.2]	-
	2 500	289* 270* 253* (271) [18.0]†	120* 104* 92* (105) [14.0]†	-	-	-
	5 000	401* 382* 443* (409) [31.2]†	-	-	-	-
2-Aminoanthracine	0.5	-	-	-	541 526 599 (555) [38.6]	-
	1.0	1432 1350 1354 (1379) [46.2]	-	-	-	-
	2.0	-	480 511 580 (524) [51.2]	-	-	246 218 216 (227) [16.8]
	10.0	-	-	1225 1272 1173 (1223) [49.5]	-	-

* Growth inhibition

[ ] Standard deviation

† p < 0.05

 

Main Study Results With Metabolic Activation

Compound	Dose (µg/plate)	Revertants Per Plate (Mean Number of Revertants per Dose)
		Base-Pair Substitution Type
		TA 100	TA 1535	WP2 uvrA
Negative control	-	153 139 140 (144) [7.8]	13 12 14 (13) [1.0]	31 39 36 (35) [4.0]
Test material	9.77	145 153 159 (152) [7.0]	-	32 29 38 (33) [4.6]
	19.5	139 135 177 (150) [23.2]	-	32 31 32 (32) [0.6]
	39.1	173 167 169 (170) [3.1]†	-	26 27 32 (28) [3.2]
	78.1	194* 185* 164* (181) [15.4]†	-	31 33 32 (32) [1.0]
	156	235* 249* 234* (239) [8.4]†	-	23* 28* 20* (24) [4.0]
	313	253* 248* 238* (246) [7.6]	10* 9* 5* (8) [2.6]	19* 21* 23* (21) [2.0]
	625	254* 261* 265* (260) [5.6]†	9* 7* 10* (9) [1.5]	-
	1 250	-	7* 9* 13* (10) [3.1]	-
	2 500	-	13* 12* 6* (10) [3.8]	-
	5 000	-	18*# 17*# 13*# (16) [2.6]	-
2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide	0.01	525 498 565 (529) [33.7]	-	117 96 104 (106) [10.6]
Sodium azide	0.5	-	498 511 534 (514) [18.2]	-

* Growth inhibition

# Precipitation

[ ] Standard deviation

† p < 0.05

 

Results of the Confirmatory Test Without Metabolic Activation

Compound	Dose (µg/plate)	Revertants Per Plate (Mean Number of Revertants per Dose)
		Frameshift Mutation Type
		TA 98	TA 1537
Negative control: Dimethyl sulfoxide	-	25 19 13 (19) [6.0]	10 9 13 (11) [2.1]
Test material	2.44	25 17 23 (22) [4.2]	15 11 11 (12) [2.3]
	4.88	19 22 16 (19) [3.0]	14 9 13 (12) [2.6]
	9.77	22 16 19 (19) [3.0]	11 13 9 (11) [2.0]
	19.5	15 17 15 (16) [1.2]	8 11 9 (9) [1.5]
	39.1	13 17 20 (17) [3.5]	13 9 12 (11) [2.1]
	78.1	16* 14* 20* (17) [3.1]	5* 10* 6* (7) [2.6]
2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide	0.1	379 352 322 (351) [28.5]	-
9-Aminoacridine	80.0	-	244 222 199 (222) [22.5]

 

Results of the Confirmatory Test With Metabolic Activation

Compound	Dose (µg/plate)	Revertants Per Plate (Mean Number of Revertants per Dose)
		Base-Pair Substitution Type			Frameshift Mutation Type
		TA 100	TA 1535	WP2 uvrA	TA 98	TA 1537
Negative control: Dimethyl sulfoxide	-	143 140 148 (144) [4.0]	15 16 15 (15) [0.6]	33 38 31 (34) [3.6]	21 32 28 (27) [5.6]	19 14 16 (16) [2.5]
Test material	9.77	-	-	-	-	13 16 13 (14) [1.7]
	19.5	144 153 132 (143) [10.5]	-	-	-	16 19 19 (18) [1.7]
	39.1	144 132 145 (140) [7.2]	-	40 29 32 (34) [5.7]	26 26 31 (28) [2.9]	16 11 15 (14) [2.6]
	78.1	154 156 152 (153) [1.0]	15 19 20 (18) [2.6]	35 31 32 (33) [2/1]	21 18 31 (23) [6.8]	15 14 12 (14) [1.5]
	156	167 164 187 (173) [12.5]	21* 17* 30* (23) [6.7]	45 28 38 (37) [8.5]	33 21 32 (29) [6.7]	13* 16* 14* (14) [1.5]
	313	184 182 245 (204) [35.8]†	23* 26* 29* (26) [3.0]	28 34 31 (31) [3.0]	23 19 24 (22) [2.6]	11* 9* 10* (10) [1.0]
	625	247* 247* 243* (246) [2.3]†	58* 53* 61* (57) [4.0]†	41* 38* 28* (36) [6.8]	25* 22* 18* (22) [3.5]	-
	1 250	275* 306* 277* (286) [17.3]†	96* 94* 107* (99) [7.0]†	37* 28* 29 (31*) [4.9]	20* 21* 22* (21) [1.0]	-
	2 500	-	89* 117* 118* (108) [16.5]†
2-Aminoanthracene	0.5	-	-	-	588 516 538 (547) [36.9]	-
	1.0	1125 1022 1320 (1156) [151.3]	-	-	-	-
	2.0	-	493 523 471 (496) [26.1]	-	-	226 196 294 (239) [50.2]
	10.0	-	-	1091 1112 1235 (1146) [77.8]	-	-

* Growth inhibition

[ ] Standard deviation

†p <0.05

 

Additional Confirmatory Study Results Without Metabolic Activation

Compound	Dose (µg/plate)	Revertants Per Plate (Mean Number of Revertants per Dose)
		Base-pair substitution type
		TA 1535
Negative control: Dimethyl sulfoxide	-	13 18 12 (14) [3.2]
Test material	313	6* 8* 16* (10) [5.3]
	625	12* 6* 7* (8) [3.2]
	1 250	18* 16* 13* (16) [2.5]
	2 500	20* 29* 15* (21) [7.1]
	5 000	29*# 25*# 36*# (30) [5.6]†
Sodium azide	5.0	571 592 561 (575) [15.8]

* Growth inhibition

# Precipitation

[ ] Standard deviation

† p < 0.05

 

Specific Activity Values

Examination	Strain	Absence of Metabolic Activation		Presence of Metabolic Activation
		Specific Activity	Calculated Dose	Specific Activity	Calculated Dose
Main study	TA 100	78	78.1 µg/plate	288	156 µg/plate
	TA 1535	4.0	5 000 µg/plate	46.4	625 µg/plate
	WP2 uvrA
	TA 98
	TA 1537
Confirmatory Study	TA 100	665	39.1 µg/plate	192	313 µg/plate
	TA 1535			67.2	625 µg/plate
	WP2 uvrA
	TA 98
	TA 1537
Additional confirmatory study	TA 1535	3.2	5 000 µg/plate

Specific activity: (Mean number of revertants per dose = mean number of revertants in negative control) / Dose (mg)

Conclusions:

Under the conditions of the study the test material was positive for inducing gene mutations.

Executive summary:

The genotoxicity of the test material was assessed according to Japanese Guidelines for Screening Mutagenicity Testing Of Chemicals as well as the standardised guideline OECD 471, under GLP conditions.

Genotoxicity was evaluated by the reverse mutation test with a pre-incubation method at 37 °C for 20 minutes using base-pair substitution type strains of Salmonella typhimurium TA100, TA1535, Escherichia coli WP2uvrA, and frameshift mutation type strains of Salmonella typhimurium TA98, TA1537. The test was composed of the dose-finding, main, and confirmatory studies, which were performed in the presence and absence of metabolic activation and reproducibility of the results of the main and confirmatory studies was examined. In addition for Salmonella typhimurium TA1535 in the absence of metabolic activation, an additional confirmatory study was performed to confirm the reproducibility of the observed positive response. Based on the data obtained from the dose-finding study, doses for the main, confirmatory, and additional confirmatory studies were set. As a result, the test substance increased the number of revertant mutant colonies in Salmonella typhimurium TA100 and TA1535 both in the presence and absence of metabolic activation at the dose at which growth inhibition was observed in the main and confirmatory studies. Statistical significance was admitted in all bacterial strains except for Salmonella typhimurium TA1535 in the absence of metabolic activation in the confirmatory study. The mean number of revertant colonies exceeded the upper limit of variation range of the negative control calculated from background data and the mean number of revertant colonies for each bacterial strain was approximately from 1.8 to 8.8-fold compared with the respective negative control values. 

In the main study, statistical dose-dependency was confirmed only in Salmonella typhimurium TA100 in the presence of metabolic activation, and was confirmed only in Salmonella typhimurium TA100 in the presence and absence of metabolic activation in the confirmatory study. Statistical dose-dependency was not observed in other strains of Salmonella typhimurium TA100 and TA1535 as statistical analysis is based on the doses which did not show growth inhibition, the number of revertant colonies had shown a clear dose-dependency. Thus, it was judged that reproducibility of dose-dependent increase in the number of revertant colonies was admitted, and also judged that statistically significant increase in the number of revertant colonies exceeding background data were confirmed. In Salmonella typhimurium TA1535 in the absence of metabolic activation in the main study, since cytotoxicity have strongly affected, it seemed that positive response was not observed. These results revealed that biologically meaningful increase in the number of revertant colonies due to mutagenicity of the test substance was shown. The number of revertant colonies in the positive controls were twice or more than that of the respective negative controls in all bacterial strains both in the presence and absence of metabolic activation. The mean values of revertant colonies of the negative and positive controls were within the variation range calculated from background data in all studies. No contaminants were found in the sterility test. These results demonstrated that the test was properly performed. No suspected factor that affected the reliability of the studies was observed.

Under the conditions of the study the test material was positive for inducing gene mutations.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 September 2018 to 30 October 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Partial Revision of Testing Methods Concerning New Chemical Substances

Version / remarks:

(Notification No. 0329-B , PSEHB, MHLW; No. 1 of 20180326, MIB, METI; No. 201803293, EPB, MOE; dated March 29, 2018)

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

The test material solutions were corrected for purity.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Remarks:

tk± -3.7.2C

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: National Institute of Health Sciences
- Suitability of cells: This cell line is recommended in the applied guideline.

For cell lines:
- Absence of Mycoplasma contamination: yes
- Doubling time: 9.0 hours
- Spontaneous mutant frequency: Less than 170 x 10^6
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- RPMl-0 medium (1000 mL of medium contained , 10 mL of penicillin/ streptomycin solution, 5 mL of 4% w/v solution of sodium pyruvate, 5 mL of 10% volume pluronic solution, and 980 mL of RPMI-1640 liquid medium.
- RPMl-10 medium (heat -inactivated (56°C, 30 minutes) horse serum was added to RPMI-0 medium at 10% volume).
- RPMl-20 medium (10 mL of penicillin/streptomycin solution and 5 mL of 4% w/v sodium pyruvate solution were added to 985 mL of RPMI1640 liquid medium. Heat-inactivated horse serum was added to the above medium at 20% volume).

Metabolic activation:

with and without

Metabolic activation system:

The S9 was purchased. It had been prepared from from the livers of 7-week-old male Sprague-Dawley rats (body weight: 198 to 242 g) treated with phenobarbital (PB, 4 times at 24-hour intervals at i.p. doses of 30, 60, 60, and 60 mg/kg, respectively) and 5,6-benzoflavone (once at an i.p. dose of 80 mg/kg on the day of the third PB injection).
The S9 mix consisted of 1.05 mL of S9 and 2.45 mL of cofactor mix solution (1 mL of cofactor mix solution contained: 5 µmol D-glucose 6-phosphate, 4 µmol NADP, 4 µmol HEPES buffer (pH 7.2), 5 µmol MgCl2, and 33 µmol KCl).

Test concentrations with justification for top dose:

- Dose-finding test:
7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000, and 2000 µg/mL (3-hour treatment with and without S9 mix and 24-hour treatment without S9 mix)
Rationale for concentration selection: 2000 µg/mL is the highest concentration specified in the applied guideline.

- Main test:
15, 20, 25, 30, 35, 40, 45, 50, and 60 µg/mL (3-hour treatment, without S9 mix)
60, 70, 80, 90, 100, 110, 120, and 130 µg/mL (3-hour treatment, with S9 mix)
6, 9, 12, 15, 18, 21, 24, 27, 30, and 35 µg/mL (24-hour treatment, without S9 mix)
Rationale for concentration selection: test concentrations were selected on the basis of the findings of the dose-finding test.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: In the solvent selection test, the test material was not dissolved or suspended at 20 mg/mL in physiological saline and was dissolved at 200 mg/mL in DMSO. Exothermic reaction, discolouration and foaming were not observed in the preparations. Therefore, DMSO was selected as the vehicle for the test material and was used as the negative control substance in this study.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

- Cell treatment:
Preliminary cultured L5178Y cells were suspended in RPMI-10 medium at 1 x 10^6 cells/mL. For the 3-hour treatments without S9 mix, 0.2 mL test material solution was added to 10 mL cell suspension and 9.8 mL RPMI-0 medium. For the 3-hour treatments with S9 mix, 0.2 mL test material solution was added to 10 mL cell suspension, 1 mL S9 mix and 8.8 mL RPMI-0 medium. For the 24-hour treatments without S9 mix, 0.5 mL test material solution was added to 10 mL cell suspension and 39.5 mL RPMI-10 medium. For the negative control groups, the cells were similarly treated but with DMSO instead of the test material solution.
Two centrifuge tubes (or flasks) were used for each concentration in the main test.
In the short-term (3-hour) treatment assay, the treatment mixture was incubated in the centrifuge tube with shaking (tilt angle: about 8°, rotation speed: ca. 30 rpm) by a shaker shielded from light for 3 hours. In the long-term (24-hour) treatment assay, the treatment mixtures were incubated in 75 cm² flask shielded from light for 24 hours without shaking.
At the beginning and the end of the treatment, all tubes (or flasks) were examined for precipitation of the test substance with unaided eye.

- Measurement of cell concentration during the mutation expression period
After the treatment, the cell suspensions were centrifuged at 110 g for 5 minutes. The supernatants were removed. The cells were washed with RPMI-0 medium and the cell suspensions were centrifuged under the same condition. The supernatants were removed. The cells were suspended in RPMI-10 medium and were counted with a cell counter.
14 to 30 mL of the 2 x 10^5 cells/mL suspensions were prepared with RPMI-10 medium (main test).
The cell suspensions were incubated in CO2 programmable incubator.
Approximately 24 hours after the end of the treatment, the cells were counted with a cell counter. Then, the cell suspensions were diluted with RPMI-10 medium to keep the cell concentration at 2.5 x 10^5 cells/mL or below. At 45 (one of duplicate cultures), 50, and 60 µg/mL in the -S9 mix assay, at 130 µg/mL in the +S9 mix assay, and at 35 µg /mL (one of duplicate cultures) in the 24-hour assay, the cell concentration was less than 2.5 x 10^5 cells/mL, therefore, the cell suspensions were not diluted.
The cells were incubated in CO2 programmable incubator for approximately 48 hours after the end of the treatment.

- Preparation and observation of PE (plating efficiency) plate
The cells incubated for approximately 48 hours after the end of the treatment were suspended by mixing with a pipette. For observation of mutant frequency, cell suspensions were placed at 200 µL per well of two (four in the negative control) 96-well plates (2000 cells/well).
With the remainder of the cell suspension, the cells were counted with a cell counter. The cell suspensions were diluted with RPMI-20 medium to prepare 50 mL (100 mL in the negative control) of the 1 x 10^4 cells/mL suspensions.
The 1x 10^4 cells/mL suspensionswere diluted by two step dilution with RPMI-10 medium and RPMI-20 medium to prepare 8 cells/mL suspension. The resultant cell suspensions were placed at 200 µL per well (1.6 cells/well) of two 96-well plates.
The plates were incubated in CO2 programmable incubator for 11 or 12 days. After the incubation, the plates were observed with unaided eye, and numbers of Normal well and Empty well were recorded for each plate.

- Preparation and observation of MF (mutant frequency) plate
The rest of the 1 x 10^4 cells/mL suspensions prepared were supplemented with 100 µL (200 µL in the negative control) of 3-mg/mL trifluorothymidine solution.
The plates were incubated in CO2 programmable incubator for 12 days. After the incubation, the plates were observed with an inverted microscope, and the numbers of well observed with large and/or small colon ies were recorded for each plate.

- Criteria for identification of the colony
The colonies were identified according to the criteria shown below. The colonies were classified by size in principle; however, when this was too difficult, the colonies were classified additionally by density.
Large colony: the diameter is 1/4 or more of the well, and packed loosely Small colony: the diameter is less than 1/4 of the well, and tightly packed

- Calculations
Plating efficiency (PE), relative survival rate (%RS), the daily cell growth at day 1 and day 2 (DCG-1 and DCG-2), suspension growth (SG), relative suspension growth (RSG), and relative total growth (RTG) were calculated according to the following:

PE = {-In (EW / TW)} / 1.6
where
EW: number of empty wells (wells without colonies)
TW: total number of wells
PE% = PE x 100

%RS = {PE (treatment group) / PE (negative control group)} x 100

DCG-1 = (cell concentration at approximately 24 hours after cell treatment) / (cell concentration at the beginning of the incubation)
DCG-2 = (cell concentration at approximately 48 hours after cell treatment) / (cell concentration adjusted at approximately 24 hours after cell treatment)

SG (short-term treatment) =DCG-1 x DCG-2
SG (long-term treatment) = (cell concentration at end of cell treatment) / (cell concentration at beginning of cell treatment) x DCG-1 x DCG-2

RSG = SG (treatment group) / SG (negative control group)
RSG%= RSG x 100

RTG = RSG x %RS

The mutant frequency (MF) was calculated by the following formula as an index of the mutation frequency.
MF= [{-In (EW / TW)} / 2000] / PE

Total-MF (T-MF), large colony-MF (L-MF), and small colony-MF (S-MF) were calculated separately. TEW, LEW, and SEW, which were used as EW in the calculation of each MF mentioned above, were calculated by the following formula:
TEW = TW - (A + B + C)
LEW = TW - (A + C) SEW = TW - (B + C)
where:
A: number of wells containing large colony only
B: numb er of wells containing small colony only
C: number of wells contai ning both large and small colonies

The percentage of small colony (%SC) was calculated by the following formula for each treatment condition:
%SC = (S-MF I T -MF) x 100

To evaluate the heterogeneity between duplicate treatment cultures, the heterogeneity factor (HGF) was calculated for the data used in the calculation of PE and MF in each treatment.

Evaluation criteria:

The following comparison with the negative control value was conducted for T-MF in the test material treatment groups.
GN = T-MF (negative control value) + (126 x 10^-6)
GT = T-MF (test material treatment group)
When all GT values were lower than values, the test material was judged to be negative.

When GT values were equal to or higher than GN values, the dose-dependency was examined for T-MF. When a significant increase is detected, the test material was judged to be positive.
When a significant increase is not detected, the test material was judged to be inconclusive.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

- Dose-finding test
Precipitation of the test materialwas observed in the treatment mixture at the beginning and the end of the treatment at 250 µg/mL or more in the -S9 mix assay and +S9 mix assay and at 500 µg/mL or more in the 24-hour assay, and at the beginning of the treatment at 250 µg/mL in the 24-hour assay.
RTG was less than 20% at 62.5 µg/mL or more in the -S9 mix assay, at 125 µg/mL or more in the +S9 mix assay, and at 31.3 µg/mL or more in the 24-hour assay.

- Main test
Precipitation of the test substance was not observed in the treatment mixture in any treatment condition.
The data fulfilled all of the criteria for an acceptable study under all treatment conditions. Therefore, main test was judged to be acceptable.
RTG was less than 10% at 45 µg/mL or more in the -S9 mix assay, at 130 µg/mL in the +S9 mix assay, and at 27 µg/mL or more in the 24-hour assay. Therefore, the data at above concentrations was not accepted, and was not used for GEF evaluation.
As a result of GEF evaluation, the GR values were higher than GN values at all accepted concentrations in the -S9 mix assay and 24-hour assay and at 70 µg/mL or more in the +S9 mix assay. Therefore, statistical analysis of dose-dependent increase was conducted in the all treatment conditions. As a result, significant difference was recognised in the all treatment conditions.

Summary of findings from main study - short-term treatment

Test material conc (µg/mL)	without S9 mix
	PE%	%RS	SG	RSG%	RTG (%)	T-MF	L-MF	S-MF	%SC	Judgment
DMSO	111.0	100.0	21.2	100.0	100.0	91.4	46.3	41.8	45.7	Positive
15	98.0	88.3	19.4	91.8	81.1	372.4	172.1	161.1	43.3
20	79.9	71.9	17.1	80.6	58.0	560.0	241.7	227.5	40.6
25	84.7	76.3	13.3	63.0	48.1	681.6	223.4	320.7	47.1
30	79.3	71.4	11.0	51.7	37.0	640.9	212.9	320.5	50.0
35	75.9	68.4	7.3	34.6	23.7	860.5	259.4	443.0	51.5
40	61.7	55.6	5.7	27.0	15.0	1193.2	297.3	679.2	56.9
45	57.5	51.8	3.8	18.1	9.4	1311.0	287.1	783.1	59.7
50	47.0	42.3	2.7	12.6	5.3	1508.3	276.8	1021.3	67.7
60	29.6	26.7	0.9	4.2	1.1	2253.2	361.2	1541.5	68.4
Positive control (0.3 µg/mL)	53.2	47.9	14.4	67.9	32.5	1407.0	651.8	377.6	26.8
Test material conc (µg/mL)	with S9 mix
	PE%	%RS	SG	RSG%	RTG (%)	T-MF	L-MF	S-MF	%SC	Judgment
DMSO	97.3	100.0	27.6	100.0	100.0	108.4	78.7	26.1	24.1	Positive
60	92.1	94.7	20.4	73.9	70.0	211.7	118.0	75.7	35.8
70	86.6	89.1	18.6	67.4	60.0	342.8	192.7	110.6	32.3
80	83.5	85.8	17.7	64.2	55.1	347.4	204.4	109.2	31.4
90	83.5	85.8	14.9	54.1	46.4	341.9	180.7	124.4	36.4
100	83.5	85.8	11.2	40.6	34.8	460.6	231.3	164.1	35.6
110	75.4	77.5	8.4	30.3	23.5	548.5	287.4	186.3	34.0
120	77.6	79.8	5.7	20.6	16.4	659.7	303.0	231.8	35.1
130	67.2	69.1	2.9	10.3	7.1	709.2	316.3	278.7	39.3
Positive control (3 µg/mL)	61.7	63.5	21.3	77.0	48.9	942.0	528.3	238.6	25.3

Summary of findings from main study - long-term treatment

Test material conc (µg/mL)	without S9 mix
	PE%	%RS	SG	RSG%	RTG (%)	T-MF	L-MF	S-MF	%SC	Judgment
DMSO	96.5	100.0	98.4	100.0	100.0	100.2	58.5	40.7	40.6	Positive
6	92.1	95.4	87.6	89.1	85.0	237.4	126.9	90.6	38.2
9	94.2	97.7	79.6	81.0	79.1	340.8	154.5	139.9	41.0
12	79.9	82.8	70.3	71.5	59.1	450.5	227.5	169.3	37.6
15	95.7	99.2	60.2	61.2	60.7	419.5	157.5	193.7	46.2
18	74.3	77.0	43.7	44.4	34.2	650.9	244.6	283.5	43.6
21	64.9	67.2	32.7	33.3	22.4	922.6	233.4	321.6	34.9
24	60.4	62.6	22.6	23.0	14.4	1373.4	402.7	543.8	39.6
27	59.2	61.3	15.0	15.3	9.4	1722.7	466.9	649.7	37.7
30	46.0	47.7	9.4	9.6	4.6	2307.4	566.9	898.9	39.0
35	28.3	29.4	4.0	4.1	1.2	2270.8	576.2	1260	55.5
Positive control (0.3 µg/mL)	47.4	49.1	44.0	44.7	21.9	1696.6	823.7	403.6	23.8

 

Conclusions:

The test material was considered to have the potential to induce gene mutation in mouse lymphoma cells under the conditions employed in this study.

Executive summary:

The potential of the test material to induce gene mutation in mouse lymphoma cells was investigated in vitro, in a study which was conducted in accordance with the standardised Japanese guidelines and OECD 490, and under GLP conditions.

During the study, a dose-finding test was conducted at 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000, and 2000 µg/mL in the absence of S9 mix (-S9 mix assay) and the presence of S9 mix (+S9 mix assay) in the short-term treatment assay and long-term treatment assay for 24 hours (24-hour assay).

As a result, the relative total growth (RTG) was less than 20% at 62.5 µg/mL or more in the -S9 mix assay, at 125 µg/mL or more in the +S9 mix assay, and at 31.3 µg/mL or more in the 24-hour assay.

Based on the result of the dose-finding test, the main test was conducted at 15, 20, 25, 30, 35, 40, 45, 50, and 60 µg/mL in the - S9 mix assay, at 60, 70, 80, 90, 100, 110, 120, and 130 µg/mL in the +S9 mix assay, and at 6, 9, 12, 15, 18, 21, 24, 27, 30, and 35 µg/mL in the 24-hour assay.

As a result, the mutant frequency (MF) was higher than MF in the negative control group + global evaluation factor (GEF: 126 x 10^6) at 15 µg/mL or more in the - S9 mix assay, at 70 µg/mL or more in the +S9 mix assay, and at 6 µg/mL or more in the 24-hour assay. Moreover, a dose-dependent increase in MF was also detected by statistical analysis in all treatment conditions.

In conclusion, the test material was considered to have the potential to induce gene mutation in mouse lymphoma cells under the conditions employed in this study.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Mouse Micronucleus (Schwind, 2005)

Under the conditions of the study, the test material had no chromosome-damaging (clastogenic) effect nor did it lead to any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo.

Testing proposal

It is proposed that a transgenic rodent gene mutation assay in the liver and glandular stomach, is an appropriate and scientifically justified assay for in vivo evaluation of the mutagenic hazard identified in vitro for YX8000D.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 July 2005 to 16 November 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 5 - 8 weeks
Weight at study initiation: mean: 29 g
- Assigned to test groups randomly: yes, the animals were assigned to the test groups separately according to a randomisation plan prepared with an appropriate computer program.
- Fasting period before study: no
- Housing: the animals were individually housed in Makrolon cages type MI
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 24°C
- Humidity: 30 - 70%
- Photoperiod: 12 hours (12 hours light from 6.00 - 18.00 hours and 12 hours darkness from 18.00 - 6.00 hours)

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: Due to the insolubility of the test material in water, corn oil was selected as the vehicle, which had been demonstrated to be suitable in the in vivo micronucleus test and for which historical data are available.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The substance to be administered twice per kg body weight was dissolved in corn oil:
- The low dose group was given 500 mg test material/kg body weight or 10 mL/kg body weight of a solution with a concentration of 5 g/100 mL.
- The intermediate dose group was given 1000 mg test material/kg body weight or 10 mL/kg body weight of a solution with a concentration of 10 g/100 mL.
- The top dose group was given 2000 mg test material/kg body weight or 10 mL/kg body weight of a solution with a concentration of 20 g/100 mL.
- To achieve a solution of the test material in the vehicle, the test material preparation was shaken thoroughly.
- All test material formulations were prepared immediately before administration.

Duration of treatment / exposure:

The animals of the vehicle control and the dose groups were treated twice at a 24-hour interval and samples of bone marrow were taken 24 hours after the last treatment. Animals of the positive control groups were treated only once and samples of bone marrow were taken after 24 hours.

Frequency of treatment:

Two treatments

Post exposure period:

24 hours after the treatment was complete the animals were sacrificed.

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 males per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide for clastogenicity
- Route of administration: oral
- Doses / concentrations: 20 mg/kg bw (single dose in a volume of 10 mL/kg)

Vincristine for spindle poison effects
- Route of administration: intraperitoneally
- Doses / concentrations: 0.15 mg/kg bw (single dose in a volume of 10 mL/kg)

Tissues and cell types examined:

Slides were prepared from the bone marrow of the femora and stained. The numbers of micronucleated polychromatic erythrocytes and normochromatic erythrocytes were recorded.

Details of tissue and slide preparation:

DOSE SELECTION
- In a pre-test for the determination of the acute oral toxicity, all animals (male and female) survived following two treatments with 2000 mg/kg bw recommended as the highest dose according to the OECD Guideline. The only clinical signs observed were squatting posture and eyelid closure, however, there were no distinct symptomatic differences between the male and female animals. Thus, only male animals were used for the cytogenetic investigations.
- Therefore, a dose of 2000 mg/kg body weight was selected as the highest dose in the present cytogenetic study. 1000 and 500 mg/kg body weight were administered as further doses.

PREPARATION OF THE BONE MARROW
- The two femora of the animals sacrificed by cervical dislocation were prepared by dissection and removing all soft tissues.
- After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis into a centrifuge tube using a cannula filled with foetal calf serum which was at 37°C (about 2 mL/femur).
- The suspension was mixed thoroughly with a pipette, centrifuged at 300 x g for 5 minutes, the supernatant was removed and the precipitate was resuspended in about 50 µL fresh FCS.
- 1 drop of this suspension was dropped onto clean microscopic slides, using a Pasteur pipette. Smears were prepared using slides with ground edges, the preparations were dried in the air and subsequently stained.

STAINING OF THE SLIDES
- The slides were stained in eosin and methylene blue for about 5 minutes.
- After having briefly been rinsed in purified water, the preparations were soaked in purified water for about 2 - 3 minutes.
- Subsequently, the slides were stained in Giemsa solution (15 mL Giemsa, 185 mL purified water) for about 15 minutes.
- After having been rinsed twice in purified water and clarified in xylene, the preparations were mounted in Corbit-Balsam.

EVALUATION
Microscopic evaluation:
- In general, 2000 polychromatic erythrocytes (PCEs) from each of the male animals of every test group were evaluated and investigated for micronuclei (MN). The normochromatic erythrocytes (NCEs) which occurred were also scored. The following parameters were recorded:
- The number of polychromatic erythrocytes.
- The number of polychromatic erythrocytes containing micronuclei were recorded.
The increase in the number of micronuclei in polychromatic erythrocytes of treated animals as compared with the solvent control group provides an index of a chromosome-breaking (clastogenic) effect or damage of the mitotic apparatus (aneugenic activity) of the substance tested.
- Number of normochromatic erythrocytes
- Number of normochromatic erythrocytes containing micronuclei
- Ratio of polychromatic to normochromatic erythrocytes
An alteration of this ratio indicated that the test material actually reached the target Individual animals with pathological bone marrow depression may be identified and excluded from the evaluation.
- Number of small micronuclei (d < D/4) and of large micronuclei (d > D/4) (d = diameter of micronucleus, D = cell diameter)
The size of micronuclei may indicate the possible mode of action of the test material, i.e. a clastogenic or a spindle poison effect.

Slides were coded before microscopic analysis.

Clinical examinations
- After treatment up to the time of sacrifice, the animals were examined for any clinically evident signs of toxicity several times.

Evaluation criteria:

ACCEPTANCE CRITERIA
The mouse micronucleus test is considered valid if the following criteria are met:
- The quality of the slides must allow the identification and evaluation of a sufficient number of analysable cells, i.e. ≥ 2 000 PCEs and a clear differentiation between PCEs and NCEs.
- The ratio of PCEs/NCEs in the untreated animals (negative control) has to be within the normal range for the animal strain selected.
- The number of cells containing micronuclei in negative control animals has to be within the range of the historical control data both for PCEs and for NCEs.
- The two positive control substances have to induce a significant increase in the number of PCEs containing small and large micronuclei within the range of the historical control data or above.

ASSESMENT CRITERIA
A finding is considered positive if the following criteria are met:
- Significant and dose-related increase in the number of PCEs containing micronuclei.
- The number of PCEs containing micronuclei has to exceed both the concurrent negative control and the highest value of the historical control range.
A test material is considered negative if the following criteria are met:
- The number of cells containing micronuclei in the dose groups is not significantly above the negative control and is within the historical control data.

Statistics:

- The statistical evaluation of the data was carried out using the program system MUKERN (BASF Aktiengesellschaft).
- The asymptotic U test according to MANN-WHITNEY (modified rank test according to WILCOXON) was carried out to clarify the question whether there were significant differences between the control group and dose groups with regard to the micronucleus rate in polychromatic erythrocytes. The relative frequencies of cells containing micronuclei of each animal was used as a criterion for the rank determination for the U test. Significances were identified as follows:
* p ≤ 0.05
** p ≤ 0.0 1

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

MICROSCOPIC EVALUATION
- The two oral administrations of corn oil in a volume of 10 mL/kg body weight led to 2.1% polychromatic erythrocytes containing micronuclei after the 24-hour sacrifice interval.
- After two administrations of the highest dose of 2000 mg/kg body weight, 2.5% polychromatic erythrocytes containing micronuclei were found after 24 hours.
- In the two lower dose groups, rates of micronuclei of about 2.0% (1000 mg/kg group) and 1.5% (500 mg/kg group) were detected.
- With 18.8% the positive control substance cyclophosphamide for clastogenicity, as expected, led to a clear increase in the number of polychromatic erythrocytes containing mainly small micronuclei. Vincristine, a spindle poison agent, produced a 51.0% increase in micronuclei in polychromatic erythrocytes. A significant portion increase, 12.5% was attributable to large micronuclei.
- The number of normochromatic erythrocytes containing micronuclei did not differ to any appreciable extent in the negative control or in the various dose groups.
- A slight inhibition of erythropoiesis, induced by the treatment of mice with the test material was detected at doses of 1000 mg/kg body weight and 2000 mg/kg body weight.

CLINICAL EXAMINATIONS
- The two oral administrations of the vehicle in a volume of 10 mL/kg body weight were tolerated by all animals without any signs or symptoms.
- The administration of the test material led to clinical signs of toxicity (Table 3).
- Neither the single administration of the positive control substance, cyclophosphamide, in a dose of 20 mg/kg body weight nor that of vincristine in a dose of 0.15 mg/kg body weight caused any evident signs of toxicity.

ANALYTICAL INVESTIGATIONS
- The stability of the test material at room temperature in oil over a period of 1 hour was verified analytically. Since a solution was obtained with the vehicle, it was not necessary to verify the homogeneity analytically.
- Depending on the dose, about 84 - 99% of the theoretical values was determined analytically:
200 mg/mL: mean analytical 168.0 mg/mL
100 mg/mL: mean analytical 88.0 mg/mL
50 mg/mL: mean analytical 49.7 mg/mL

DISCUSSION
- There were no biologically relevant, significant differences in the frequency of erythrocytes containing micronuclei between the vehicle control and the 3 dose groups (500 mg/kg, 1 000 mg/kg and 2 000 mg/kg). The number of normochromatic or polychromatic erythrocytes containing small micronuclei (d < D/4) or large micronuclei (d > D/4) did not deviate from the vehicle control value and was within the historical control range.
- Thus, under the experimental conditions chosen here, the test material has no chromosome-damaging (clastogenic) effect nor does it lead to any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo.

Table 1: Polychromatic and normochromatic erythrocytes

Treatment	Total No. of MN (o/oo) in:
	PCE’s	NCE’s	PCE’s	NCE’s
Vehicle (corn oil)	10000	3201	2.1	0.3
500 mg/kg test material	10000	3580	1.5	1.7
1000 mg/kg test material	10000	5005	2.0	0.8
2000 mg/kg test material	10000	4225	2.5	0.7
20 mg/kg Cyclophosphamide	10000	2804	18.8**	1.1
0.15 mg/kg Vincristine	10000	4923	51.0**	0.8

* p ≤ 0.05, ** p ≤ 0.01

 

Table 2: Polychromatic erythrocytes: differentiation between small and large micronuclei

Treatment	Total No. of Cells (o/oo) with:
	PCE’s	MN.d <D/4	MN. d ≥ D/4
Vehicle (corn oil)	10000	2.0	0.1
500 mg/kg test material	10000	1.4	0.1
1000 mg/kg test material	10000	2.0	0.0
2000 mg/kg test material	10000	2.5	0.0
20 mg/kg Cyclophosphamide	10000	18.6**	0.2
0.15 mg/kg Vincristine	10000	38.5**	12.5**

* p ≤ 0.05, ** p ≤ 0.01

 

Table 3: Clinical Signs During the Study

Time	2 x 500 mg/kg Animal No:					2 x 1000 mg/kg Animal No:					2 x 2000 mg/kg Animal No:
	7	10	16	20	24	5	14	18	21	29	2	9	13	19	27
First administration
1 h	1	1	1	1	1	3	3	3	3	3	3 11	3 11	3 11	3 11	3 11
2 h	1	1	1	1	1	3	3	3	3	3	3 11	3 11	3 11	3 11	3 11
4 h	1	1	1	1	1	3	3	3	3	3	3	3	3	3	3
23 h	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1
Second administration
25 h	1	1	1	1	1	3	3	3	3	3	3 11	3 11	3 11	3 11	3 11
26 h	1	1	1	1	1	3	3	3	3	3	3 11	3 11	3 11	3 11	3 11
28 h	1	1	1	1	1	3	3	3	3	3	3	3	3	3	3
2 d	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1

1 = no signs of symptoms

3 = squatting posture

11 = eyelid closure

Conclusions:

Under the conditions of this study, the test material has no chromosome-damaging (clastogenic) effect nor does it lead to any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo.

Executive summary:

The genetic toxicity of the test material was determined in accordance with the standardised guidelines OECD 474, EU Method B.12 and OPPTS 870.5395, under GLP conditions.

The test material was tested for chromosomal damage (clastogenicity) and for its ability to induce spindle poison effects (aneugenic activity) in NMRI mice using the micronucleus test method. For this purpose, the test material, dissolved in corn oil, was administered twice orally, with a 24-hour interval between administrations, to male animals at dose levels of 500, 1000 and 2000 mg/kg bw at a volume of 10 mL/kg bw in each case.

As a negative control, male mice were administered merely the vehicle, corn oil, by the same route and in the same volume as the animals of the dose groups, which gave frequencies of micronucleated polychromatic erythrocytes within the historical control range. Both of the positive control chemicals, i.e. cyclophosphamide for clastogenicity and vincristine for spindle poison effects, led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei.

The animals were sacrificed and the bone marrow of the two femora was prepared 24 hours after the second administration. After staining of the preparations, 2000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes with and without micronuclei occurring per 2000 polychromatic erythrocytes were also recorded.

According to the results of the present study, the two oral administrations of the test material did not lead to any increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was always within the same range as that of the concurrent negative control in all dose groups and within the range of the historical control data.

A slight inhibition of erythropoiesis, determined from the ratio of polychromatic to normochromatic erythrocytes, was detected at doses of 1000 and 2000 mg/kg body weight.

Under the conditions of this study, the test material has no chromosome-damaging (clastogenic) effect nor does it lead to any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo.

Reference 2

Endpoint:

in vivo mammalian somatic and germ cell study: gene mutation

Type of information:

experimental study planned

Study period:

To be determined upon receipt of ECHA final decision

Justification for type of information:

** Please see attached justification **

TESTING PROPOSAL ON VERTEBRATE ANIMALS

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out : 2,2'-[(1-methylethylidene)bis(cyclohexane-4,1-diyloxymethylene)]bisoxirane (YX8000D)

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION :
- Available GLP studies :
The following genotoxicity data are available to support an Annex VIII registration for YX8000D:
Two bacterial reverse mutation test (OECD TG 471): concluded to be positive
An in vitro mouse lymphoma assay (OECD TG 490): concluded to be positive
An in vivo mouse micronucleus test (OECD TG 474): concluded to be negative

In two bacterial reverse mutation assays (Engelhardt, 2004 and Kato, 2019), concentration-related and reproducible increases in revertant colonies were observed in strains TA100 and TA1535 when tested in the presence of exogenous metabolic activation (rat S9). In the most recent study (Kato, 2019), concentration-related and reproducible increases in revertant colonies were also observed in strains TA100 and TA1535 in the absence of exogenous metabolic activation. These data meet all the requirements of OECD 471 for a clearly positive result.
In the mouse lymphoma assay (Kajiwara, 2019), concentration-related increases in mutant frequency (in both large and small colonies) that clearly exceeded the OECD 490-defined global evaluation criteria were observed following 3 hour treatments both in the absence and presence of rat S9, and following continuous (24 hour) treatment in the absence of S9. The data meet all the requirements of OECD 490 for a clearly positive result.
No in vitro data for chromosome damage or chromosome loss endpoints are available, however, YX8000D is clearly negative in a mouse micronucleus test. Although this study (Schwind & Hellwig, 2005) has some deficiencies compared to the current OECD 474, 2016 requirements (most notably that only 2000 immature erythrocytes were scored for micronuclei compared to 4000 cells currently recommended), the study is considered to be reliable. The data obtained in this study provides evidence of bone marrow toxicity (manifesting as a reduction in the ratio of immature to total erythrocytes), thus confirming bone marrow exposure to YX8000D, or its metabolites, following oral administration. The negative conclusion is therefore confirmed to be valid.
The available in vitro data provide clear evidence of an ability of YX8000D to induce gene mutations in both bacterial and mammalian cells. The responses in the absence and presence of S9 are comparable, demonstrating no activation or deactivation of YX8000D mutagenicity by metabolic enzymes present in rat S9. There is no evidence that YX8000D induces chromosome damage or chromosome loss in vivo, however, as the mouse micronucleus is uninformative with respect to gene mutations, there are no data available to evaluate the gene mutagenicity of YX8000D in vivo.
In accordance with Annex VIII of the REACh legislation it is considered necessary to conduct an in vivo gene mutation assay in order to fully evaluate the mutagenic hazard of YX8000D.

- Available non-GLP studies : none available
- Historical human data : there are no historical human data available to address the endpoint
- (Q)SAR : QSAR data is not considered appropriate for conclusively informing on the mutagenic hazard of the substance
- In vitro methods : The REACh information requirements state that for substances demonstrated to be positive for any of the genotoxicity studies in Annex VII or VIII, appropriate in vivo mutagenicity assays shall be considered to ascertain if the genotoxicity potential observed in vitro can be expressed in vivo. Generation of additional in vitro data is therefore not applicable.
- Weight of evidence : The available GLP studies have been considered together and are not adequate for fully informing on mutagenic hazard of the substance. In accordance with Annex VIII of the REACh legislation it is considered necessary to conduct an in vivo gene mutation assay in order to fully evaluate the mutagenic hazard of YX8000D.
- Grouping and read-across : No appropriate substance surrgates have been identified for use in a read-across approach.
- Substance-tailored exposure driven testing: the potential for exposure cannot be excluded and hence it is not considered appropriate to utilise exposure-based waiving

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
The registrant does not consider it possible to utilise any of the adaptations noted in the REACH Annexes to conclusively inform on the mutagenic hazard of the substance. The only method for informing on the mutgaenic hazard of the substance is through additional (in vivo) testing.

FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
The available mutagenicity data for YX8000D indicate an ability to directly induce gene mutations in bacteria and mouse lymphoma L5178Y cells; no activation by an exogenous rat metabolic system (rat S9) is required for YX8000D mutagenicity to be detected. According to the REACh information requirements for mutagenicity, a variety of in vivo studies maybe used to follow-up positive in vitro gene mutation results. These are:
- A transgenic rodent (TGR) mutation assay (OECD TG 488)
- A comet (single cell gel electrophoresis) assay (OECD TG 489)
- Other DNA strand breakage assays as alternatives to the comet assay
- A rat liver Unscheduled DNA synthesis (UDS) test (OECD TG 486).
With the exception of the comet assay (OECD TG 489), DNA strand breakage assays have not been widely validated in international collaborative trials and are not described by any OECD test guideline. The UDS test has been adopted by OECD (TG 486) however, as it is an indicator test measuring DNA repair of primary damage in liver cells, it is not a surrogate test for gene mutations per se.
Of the two remaining assays, the TGR test (OECD TG 488) and the comet assay (OECD TG 489), only the transgenic rodent gene mutation test directly measures gene mutations in both somatic and (male) germ cells and consequently is one of the few available assays that can provide adequate data to enable assessment of mutagenicity according to the CLP criteria.
In contrast, the comet assay is only able to detect DNA strand breaks. DNA strand breaks may be transient and are often fully and correctly repaired thus they do not always result in gene mutation. Consequently, the comet assay is an indicator test and not a direct measure of gene mutation. Furthermore, although modifications to the standard assay that enable detection of strand breaks in germ cells have been reported, these have not been fully validated and as such are not described by OECD 489. A standard comet assay could be conducted in gonadal tissue, which if positive provides evidence of gonad exposure, but this would not provide conclusive evidence of germ cell mutations and therefore may be insufficient for classification purposes. A further disadvantage of the comet assay is that DNA strand breaks can occur as a secondary consequence of cellular toxicity, oxidative stress and general physiological imbalance. Such “indirect” DNA strand breaks are indistinguishable from strand breaks induced by substances that interact directly with DNA, and therefore, in the event of a positive comet assay, it may not be possible to conclude on the mechanism(s) involved in the observed strand breaks. Available toxicological data for YX8000D indicates that the substance causes site of contact irritation in the stomach and liver injury after repeated oral administration (Saitoh, 2020: Combined repeated dose and reproductive/developmental toxicity test of YX8000D by oral administration in rats conducted according to OECD 422). These effects have the potential to confound the results of any comet investigations in these two key tissues, such that the outcome of a comet assay may be inconclusive.
It is therefore proposed that a transgenic rodent gene mutation assay in the liver and glandular stomach, is an appropriate and scientifically justified assay for in vivo evaluation of the mutagenic hazard identified in vitro for YX8000D. Furthermore, it is proposed that male germ cells (i.e. developing germ cells isolated from seminiferous tubules) are retained, such that if positive results are obtained in either liver and/or glandular stomach, the hazard posed to germ cells can be assessed.

The assay would be conducted as follows:
- Species: Rats or mice (According to OECD 488, either species is acceptable. The model selected should be fully validated in the test facility, with appropriate historical control data (HCD) available for all three tissues proposed).
- Strain: To be determined depending on the species selected and test facility experience/HCD. Strains such as BigBlue rat or mouse, MutaMouse or gpt delta rat or mouse are acceptable.
- Sex: Males, although if clear differences in maximum tolerated dose (MTD) are identified between the sexes in a preliminary dose setting phase, then both sexes should be tested in the main experiment.
- Exposure route: Oral gavage (Oral exposure is a potential route of human exposure)
- Duration of treatment: 1 per day for 28 days consecutive days
- Sample time: 28 days after the last dose administration
- Dose levels : 0.25x MTD, 0.5x MTD, MTD (Actual doses to be determined based on the results of a preliminary dose setting phase conducted in the same species of animals and using the same dosing regimen, route and dose volume. Preliminary dose setting maybe conducted in either the transgenic strain or in the non-transgenic parental strain)
- Controls: Concurrent negative (vehicle) controls. Positive control animals may be not be necessary if the laboratory is able to demonstrate suitable proficiency in the study and in each tissue under investigation. Under these circumstances, the positive control animals may be substituted with concurrent processing of tissue-match positive control DNA.
- Group size: N=5-6 for all groups depending on standard practice at test facility
- Tissues for mutation analysis: Liver and glandular stomach
- Tissues retained as a contingency: Developing germ cells isolated from seminiferous tubules. In the event of positive mutant frequencies in the somatic tissues, these cells may be analysed to elucidate mutagenicity in germ cells.
- Additional assessments: Clinical signs, bodyweight and food consumption. At necropsy organ weights for liver and testis to be recorded. Macroscopic observations.

Qualifier:

according to guideline

Guideline:

OECD Guideline 488 (Transgenic Rodent Somatic and Germ Cell Gene Mutation Assays)

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Ames (Engelhardt, 2004)

The genetic toxicity of the test material was investigated in accordance with the standardised guidelines OECD 471, EU Method B.13/B.14 and OPPTS 870.5100, under GLP conditions.

The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay.

S. typhinurium TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA were treated at 32 - 8 000 µg/plate in a standard plate test both with and without metabolic activation (Aroclor-induced rat liver S-9 mix).

Precipitation of the test material and a bacteriotoxic effect was found from about 4 000 µg/plate onward.

No increase in the number of his+ or trp+ revertants was observed in any strain without S9 mix.

With S9 mix a positive reaction from about 100 µg/plate (factor 2.4) onward with an increase in the number of mutant colonies by a factor of 13.5 at 1 000 µg/plate was observed in TA 1535. In TA 100 with S9 mix, a slight increase in the number of revertant colonies at 800 µg/plate (factor 2.2) was observed. No increase in the number of his+ or trp+ revertants were seen in TA 1537, TA 98 or E.coli WP2 uvrA with S9 mix.

Under the conditions of this study the test material was a mutagenic agent in the bacterial reverse mutation test.

Ames (Kato, 2019)

The genotoxicity of the test materialwas assessed according to JapaneseGuidelines for Screening Mutagenicity Testing Of Chemicalsas well as the standardised guideline OECD 471, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Genotoxicity wasevaluated bythe reverse mutation testwitha pre-incubation methodat 37°Cfor 20 minutesusingbase-pair substitution type strains ofSalmonella typhimurium TA100, TA1535,Escherichia coli WP2uvrA, and frameshift mutation type strains of Salmonella typhimurium TA98, TA1537. The test was composed of the dose-finding, main, and confirmatory studies, which were performed in the presence and absence of metabolic activation and reproducibility of the results of the main and confirmatory studies was examined. In addition for Salmonella typhimurium TA1535 in the absence of metabolic activation, an additional confirmatory study was performed to confirm the reproducibility of the observed positive response. Based on the data obtained from the dose-finding study, doses for the main, confirmatory, and additional confirmatory studies were set. As a result, the test substance increased the number of revertant mutant colonies in Salmonella typhimurium TA100 and TA1535 both in the presence and absence of metabolic activation at the dose at which growth inhibition was observed in the main and confirmatory studies. Statistical significance was admitted in all bacterial strains except for Salmonella typhimurium TA1535 in the absence of metabolic activation in the confirmatory study. The mean number of revertant colonies exceeded the upper limit of variation range of the negative control calculated from background data and the mean number of revertant colonies for each bacterial strain was approximately from 1.8 to 8.8-fold compared with the respective negative control values. 

In the main study, statistical dose-dependency was confirmed only in Salmonella typhimurium TA100 in the presence of metabolic activation, and was confirmed only in Salmonella typhimurium TA100 in the presence and absence of metabolic activation in the confirmatory study. Statistical dose-dependency was not observed in other strains of Salmonella typhimurium TA100 and TA1535 as statistical analysis is based on the doses which did not show growth inhibition, the number of revertant colonies had shown a clear dose-dependency. Thus, it was judged that reproducibility of dose-dependent increase in the number of revertant colonies was admitted, and also judged that statistically significant increase in the number of revertant colonies exceeding background data were confirmed. In Salmonella typhimurium TA1535 in the absence of metabolic activation in the main study, since cytotoxicity have strongly affected, it seemed that positive response was not observed. These results revealed that biologically meaningful increase in the number of revertant colonies due to mutagenicity of the test substance was shown. The number of revertant colonies in the positive controls were twice or more than that of the respective negative controls in all bacterial strains both in the presence and absence of metabolic activation. Themean valuesof revertant coloniesofthe negative and positive controlswerewithinthe variation rangecalculated frombackground datain all studies. No contaminantswerefound in the sterility test. These results demonstrated that the test was properly performed. No suspected factorthat affected the reliability of thestudies was observed.

Under the conditions of the study the test material was positive for inducing gene mutations.

Mouse lymphoma assay (Kajiwara, 2019)

The potential of the test material to induce gene mutation in mouse lymphoma cells was investigated in vitro, in a study which was conducted in accordance with the standardised Japanese guidelines and OECD 490, and under GLP conditions.

During the study, a dose-finding test was conducted at 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000, and 2000 µg/mL in the absence of S9 mix (-S9 mix assay) and the presence of S9 mix (+S9 mix assay) in the short-term treatment assay and long-term treatment assay for 24 hours (24-hour assay).

As a result, the relative total growth (RTG) was less than 20% at 62.5 µg/mL or more in the -S9 mix assay, at 125 µg/mL or more in the +S9 mix assay, and at 31.3 µg/mL or more in the 24-hour assay.

Based on the result of the dose-finding test, the main test was conducted at 15, 20, 25, 30, 35, 40, 45, 50, and 60 µg/mL in the - S9 mix assay, at 60, 70, 80, 90, 100, 110, 120, and 130 µg/mL in the +S9 mix assay, and at 6, 9, 12, 15, 18, 21, 24, 27, 30, and 35 µg/mL in the 24-hour assay.

As a result, the mutant frequency (MF) was higher than MF in the negative control group + global evaluation factor (GEF: 126 x 10^6) at 15 µg/mL or more in the - S9 mix assay, at 70 µg/mL or more in the +S9 mix assay, and at 6 µg/mL or more in the 24-hour assay. Moreover, a dose-dependent increase in MF was also detected by statistical analysis in all treatment conditions.

In conclusion, the test material was considered to have the potential to induce gene mutation in mouse lymphoma cells under the conditions employed in this study.

Mouse Micronucleus (Schwind, 2005)

The genetic toxicity of the test material was determined in accordance with the standardised guidelines OECD 474, EU Method B.12 and OPPTS 870.5395, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material was tested for chromosomal damage (clastogenicity) and for its ability to induce spindle poison effects (aneugenic activity) in NMRI mice using the micronucleus test method. For this purpose, the test material, dissolved in corn oil, was administered twice orally, with a 24-hour interval between administrations, to male animals at dose levels of 500, 1000 and 2000 mg/kg bw at a volume of 10 mL/kg bw in each case.

As a negative control, male mice were administered merely the vehicle, corn oil, by the same route and in the same volume as the animals of the dose groups, which gave frequencies of micronucleated polychromatic erythrocytes within the historical control range. Both of the positive control chemicals, i.e. cyclophosphamide for clastogenicity and vincristine for spindle poison effects, led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei.

The animals were sacrificed and the bone marrow of the two femora was prepared 24 hours after the second administration. After staining of the preparations, 2000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes with and without micronuclei occurring per 2000 polychromatic erythrocytes were also recorded.

According to the results of the present study, the two oral administrations of the test material did not lead to any increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was always within the same range as that of the concurrent negative control in all dose groups and within the range of the historical control data.

A slight inhibition of erythropoiesis, determined from the ratio of polychromatic to normochromatic erythrocytes, was detected at doses of 1000 and 2000 mg/kg body weight.

Under the conditions of this study, the test material has no chromosome-damaging (clastogenic) effect nor does it lead to any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to genetic toxicity in the absence of positive in vivo data.