Benzenepropanoic acid, 3,5-bis(1,1-dimethylethyl)-4-hydroxy-, 2-ethylhexyl ester

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test article was negative in an OECD 471 compliant Ames test. Neither in an OECD 471 compliant bacterial reverse mutation test nor in an OECD 473 compliant chromosome aberration study appling CHO cells a genotoxic potential of a structural analogue stubstance could be found.

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:

10 Nov 2017 - 24 Nov 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

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

Metabolic activation:

with and without

Metabolic activation system:

liver S9 mix from induced rats

Test concentrations with justification for top dose:

0; 33; 100; 333; 1000; 2500 and 5000 μg/plate

Vehicle / solvent:

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

The test substance was weighed and topped up with the chosen vehicle to achieve the required concentration of the stock solution. To achieve a clear solution of the test substance in the vehicle, the test substance preparation was shaken thoroughly. The further concentrations were diluted from the stock solution according to the planned doses. All test substance formulations were prepared immediately before administration.

Untreated negative controls:

yes

Remarks:

sterility control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see below

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicates
- Number of independent experiments: two

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants) were counted.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
Toxicity detected by a
• decrease in the number of revertants (factor ≤ 0.6)
• clearing or diminution of the background lawn (= reduced his- or trp- background growth)


POSITIVE CONTROLS
The following positive controls were used to check the mutability of the bacteria and the activity of the S9 mix:
With S9 mix
• 2-aminoanthracene (2-AA)
- 2.5 μg/plate, dissolved in DMSO
- strains: TA 1535, TA 100, TA 1537, TA 98
- 60 μg/plate, dissolved in DMSO
- strain: Escherichia coli WP2 uvrA

Without S9 mix
• N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)
- 5 μg/plate, dissolved in DMSO
- strains: TA 1535, TA 100
• 4-nitro-o-phenylenediamine (NOPD)
- 10 μg/plate, dissolved in DMSO
- strain: TA 98
• 9-aminoacridine (AAC)
- 100 μg/plate, dissolved in DMSO
- strain: TA 1537
• 4-nitroquinoline-N-oxide (4-NQO)
- 5 μg/plate, dissolved in DMSO
- strain: E. coli WP2 uvrA

Evaluation criteria:

The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other.

Species / strain:

other: all strains used

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A weak bacteriotox effect (slight decrease in the number of his+ revertants) was observed only with tester strain TA 1535 with S9 mix at a concentration of 5000 μg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: Test substance precipitation was found from about 1000 μg/plate onward both with and without S9 mix.

Ames test:
- Signs of toxicity : A weak bacteriotox effect (slight decrease in the number of his+ revertants) was observed only with tester strain TA 1535 with S9 mix at a concentration of 5000 μg/plate. In the preincaubation assay no bacteriotoxic effect (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants) was observed up to the highest concentration

EXPERIMENTAL RESULTS

Standard Plate Test:

	Mean revertants per plate
Strain	TA 1535		TA 100		TA 1537		TA 98		WP2uvrA
Dose (µg/plate)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
DMSO	9.3	8.7	106.7	120.3	8.3	7.3	19.7	23.0	24.0	22.0
33	10.3	10.7	105.7	102.3	7.0	7.3	14.3	22.0	22.0	21.0
100	13.0	9.7	98.7	119.0	6.3	5.3	17.7	24.3	22.7	26.7
333	9.0	11.0	95.0	116.3	9.7	11.7	16.3	25.7	24.7	24.7
1000	9.7	12.0	100.3	107.0	10.3	8.7	16.3	23.7	21.7	26.7
2500	8.0	7.0	106.3	104.0	8.3	10.7	14.3	28.7	24.7	25.3
5000	10.3	4.3	103.3	98.7	10.3	9.7	15.7	23.7	23.7	23.3
positive control	4514.0	209.3	3176.7	1659.7	763.3	85.3	922.0	854.3	740.0	86.7

Preincubation Test

	Mean revertants per plate
Strain	TA 1535		TA 100		TA 1537		TA 98		WP2uvrA
Dose (µg/plate)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
DMSO	9.0	10.3	98.7	109.7	7.0	9.3	15.7	21.7	22.0	22.3
33	12.3	8.0	105.0	102.7	9.7	9.7	18.7	19.7	20.7	17.0
100	10.0	9.3	98.7	109.0	9.7	7.3	13.3	23.7	19.3	25.0
333	8.0	11.0	110.0	97.7	9.7	8.7	13.0	25.0	18.3	33.7
1000	7.7	8.7	97.7	107.7	8.7	7.3	18.3	29.3	16.7	23.7
2600	10.7	12.3	81.7	80.7	10.3	9.7	15.0	22.0	22.3	22.3
5200	8.7	9.0	103.7	97.0	8.3	13.3	16.7	30.0	19.0	24.0
positive control	3321.3	227.0	2361.3	1677.3	842.7	85.0	1057.0	872.0	238.3	88.7

Conclusions:

Under the experimental conditions of this study, the test substance XPDL 920 is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

Executive summary:

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of bacterial strains Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA in a reverse mutation assay. The dose range was 33 μg - 5000 μg/plate in standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats). Precipitation of the test substance was found from about 1000 μg/plate onward both with and without S9 mix. A weak bacteriotoxic effect was observed only in the SPT with tester strain TA 1535 after the addition of S9 mix at a concentration of 5000 μg/plate. A relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test without S9 mix or after the addition of a metabolizing system. Under the experimental conditions of this study, the test substance XPDL 920 is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From June 20th, 1989 to October 20th, 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Deviations:

yes

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

yes

Principles of method if other than guideline:

exception of statistical analysis

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine (his- to his+) and tryptophan (tryp- to tryp+) genes in Salmonella typhimurium and Escherichia coli, respectively

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of liver from rats induced with Aroclor 1254 and cofactors.

Test concentrations with justification for top dose:

313, 625, 1250, 2500 and 5000 µg/0.1 mLl (with and without microsomal activation)

Vehicle / solvent:

Acetone (p.a., Merck, Darmstadt, Germany)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Remarks:

without S9 mix

Positive control substance:

sodium azide

Remarks:

Strain: TA100 and TA1535

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Remarks:

without S9 mix

Positive control substance:

2-nitrofluorene

Remarks:

Strain: TA98

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Remarks:

without S9 mix

Positive control substance:

other: (5)-aminoacridine hydrochloride monohydrate

Remarks:

Strain: TA1537

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Remarks:

without S9 mix

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Strain: E. coli WP2uvrA

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Remarks:

with S9 mix

Positive control substance:

other: 2-aminoanthracene

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Remarks:

with S9 mix

Positive control substance:

cyclophosphamide

Remarks:

Strain: TA1535

Details on test system and experimental conditions:

The tests are carried out in accordance with the method described by Ames et al. (Ames BN et al. (1973) Proc. Natl. Acad. Sci. USA 70, 782-786; Ames BN et al. (1973) Proc. Natl. Acad. Sci. USA 20, 2281-2285; Ames BN et al. (1975) Mutation Res. 31, 347-364).
The test concentrations were selected on the basis of a preliminary toxicity test which had been carried out with concentrations ranging from 20 to 5000 µg/0.1 mL; accordingly, the concentration of 5000 µg/0.1 mL was retained as the highest in the mutagenicity test and the remaining test concentrations were set at 313, 625, 1250 and 2500 µg/mL.
Each Petri dish contained: 1) approx. 20 mL of minimum agar, 2) 0.1 mL of a solution of the test substance or the vehicle and 0.1 mL of a bacterial culture (in 2.5% nutrient broth) in 2.0 mL of soft agar. For the Salmonella strains the soft agar was supplemented with histidine; for E. coli, the soft agar was supplemented with tryptophan.
In the experiments without microsomal activation 0.5 mL of sodium phosphate buffer was added. In the experiments in which the substance is metabolically activated, the buffer is replaced by 0.5 ml of an activation mixture (S9 mix), which, as already indicated, consisted of the S9 fraction of liver from Sprague-Dawley rats induced with Aroclor 1254 and a solution of co-factors.
In the experiments without and with the addition of microsomal activation mixture three Petri dishes were prepared per strain and per group (i.e. per concentration or per control group). The plates were incubated for about 48 hours at 37 +/-0.5 °C in darkness. At test ending, the number of counted back-mutant colonies in the cultures treated with the various concentrations was compared with controls.

Evaluation criteria:

The test substance is considered to be positive in this test system if one or both of the following conditions are met:
- at least a reproducible doubling of the mean number of revertants per plate above that of the negative control at any concentration level for one or more of the tester strains TA 98, TA 1535, TA 1537 and E. coli WP2uvrA;
- a reproducible increase of the mean number of revertants per plate for any concentration above that of the negative control by at least a factor of 1.5 for strain TA 100. Generally a concentration-related effect should be demonstrable.

Statistics:

The data reported include individual numbers of revertants per plate, mean values and standard deviation for each bacterial strain and each concentration.

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

(at the highest test concentration of 5000 µg/0.1 mL the substance precipitated in soft agar)

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 and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

(at the highest test concentration of 5000 µg/0.1 mL the substance precipitated in soft agar)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

(at the highest test concentration of 5000 µg/0.1 mL the substance precipitated in soft agar)

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 and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

(at the highest test concentration of 5000 µg/0.1 mL the substance precipitated in soft agar)

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:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

(at the highest test concentration of 5000 µg/0.1 mL the substance precipitated in soft agar)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

In the experiments performed without and with microsomal activation, comparison of the number of back-mutant colonies in the controls and the cultures treated with the various concentrations revealed no marked deviations. No evidence of the induction of point mutations by the substance or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium and Escherichia coli used in these experiments.

SUMMARY OF RESULTS

Experiment I

	TA 1535		TA 100		TA 1537		TA 98		WP2 uvra
Dose (µg/plate)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Control	9	7	137	139	4	7	14	27	15	19
313	9	6	125	133	4	7	17	22	15	16
625	12	9	139	128	6	5	21	35	18	20
1250	8	5	101	120	3	9	19	33	19	19
2500	9	5	112	118	4	10	16	34	19	18
5000	7	6	108	123	3	7	19	28	16	16
positive control	510	234	580	646	446	123	1107	1584	240	480

Experiment II

	TA 1535		TA 100		TA 1537		TA 98		WP2 uvra
Dose (µg/plate)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Control	11	18	126	151	6	16	18	30	27	24
313	11	16	109	162	5	20	16	37	23	25
625	13	14	118	151	8	22	20	35	26	22
1250	11	10	127	159	6	16	21	36	34	24
2500	15	14	137	145	6	13	25	36	22	25
5000	7	13	136	168	6	13	20	24	24	23
positive control	201	352	737	944	1198	311	963	1430	397	558

Conclusions:

No evidence of the induction of point mutations by the test item or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium and Escherichia coli used in these experiments.

Executive summary:

The test substance was tested for mutagenic effects on histidine-auxotrophicmutants of Salmonella typhimurium (strain TA 1535, TA 1537, TA 98 and TA 100) and on a tryptophan-auxotrophic strain of E. coli (WP2 uvrA) according to the OECD TG 471 (1983). The investigations were performed with the following concentrations of the trial substance without and with microsomal activation: 313, 625, 1250, 2500 and 5000 µg/0.1 mL. In order to confirm the results, the experiments were repeated.

For all tested concentrations, without and with microsomal activation,comparison of the number of back-mutant colonies in the controls and the treated bacteria cultures revealed no marked deviations. No evidence of the induction of point mutations by the substance or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium and Escherichia coli used in these experiments. No cytotoxicity was observed up to the highest tested concentration of 5000 µg/0.1 mL, which precipitated in soft agar.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From August 28th, 1992 to February 03rd, 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5375 (In Vitro Mammalian Chromosome Aberration)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

yes

Remarks:

requirements of Article 23 (1,3).

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

The test article was investigated for clastogenic effects on Chinese hamster ovary cells in vitro with and without the addition of an extrinsic metabolic activation system.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Cell line: ATCC (Amercian Type Culture Collection) CCL 61 (ovary, Chinese hamster, CHO K1)

Metabolic activation:

with and without

Metabolic activation system:

Post mitochondrial fraction S9 from Aroclor 1254 induced rat liver

Test concentrations with justification for top dose:

Experiments without metabolic activation:
- 18 hours incubation time: 250, 500 and 1000 /µg/mL
- 42 hours incubation time: 15.63, 31.25 and 62.5 /µg/mL
Higher concentrations were not scored according to SOP (strong inhibition of mitotic activity).

Experiments with metabolic activation:
- 3 hours incubation followed by 15 hours recovery period: 250, 500 and 1000 µg/mL
- 3 hours incubation followed by 39 hours recovery period: 250, 500 and 1000 µg/mL

Vehicle / solvent:

Dimethylsulfoxide

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

The CHO cell line CCL 61 (Chinese hamster ovary cells, CHO) was maintained in culture medium consisting of Nutrient Mixture F-12 supplemented with 10% fetal calf serum and Penicillin/Streptomycin in 75 cm2 tissue-culture (plastic) flasks. The cultures were incubated at 37°C in a humidified atmosphere containing 5% CO2. The cells were passaged twice weekly. The duration of a normal cell cycle determined in the laboratory was 12 - 13 hours. The cell cultures were periodically checked for mycoplasma contamination.
First experiment:
The cells were exposed for 18 hours without metabolic activation to eight concentrations of the test substance. The cells were harvested immediately after treatment. Concentration range: 7.81 - 1000 µg/mL.
Second experiment:
The cells were exposed for three hours with metabolic activation to eight concentrations of the test substance. After removal of the test substance the cells were washed and incubated in new complete culture medium for 15 hours. Concentration range: 7.81 - 1000 µg/mL.
Third experiment:
The cells were exposed for 42 hours without metabolic activation to eight concentrations of the test substance. The cells were harvested immediately after treatment. Concentration range: 7.81 - 1000 µg/mL.
Fourth experiment:
The cells were exposed for three hours with metabolic activation to eight concentrations of the test substance. After removal of the test substance the cells were washed and incubated in new complete culture medium for 39 hours. Concentration range: 7.81 - 1000 µg/mL.
Two hours prior to harvesting, the cultures were treated with Colcemide 0.4 µg/mL to arrest cells in metaphase. The experiment was terminated by hypotonic treatment (0.075 M KCl solution) of the cells, followed by fixation (methanol:acetic acid, 3:1). Slides were air-dried and stained with orcein.

Evaluation criteria:

Whenever possible two hundred well spread metaphase figures with 19 to 21 centromeres from two cultures (100 metaphases/replicate culture) in the vehicle control and in the treated groups were scored. At least fifty metaphases were scored in the positive controls (25/replicate culture).
The test substance is generally considered to be active in the Chinese Hamster cells if the following conditions are met:
- The percentage of metaphases containing specific aberrations in a treatment group is higher than 6.0 (based on historical negative control range) and differs statistically significant from the respective value of the negative control.
- A concentration-related response should be demonstrable.
The test substance is generally considered to be inactive in the Chinese Hamster cells if the following conditions are met:
- The percentage of metaphases containing specific aberrations in all treatment groups is less than or equal to 6.0 (based on historical negative control range) and does not differ statistically significant from the respective value of the negative control.

Statistics:

The evaluated numbers of specific aberrations were subjected to statistical analysis. In the preliminary tests the data were assessed for flask effects dependence of cells within each culture) using a chi-square test. The non-significant result of this test means there is no substantial evidence to conclude a flask effect (although a flask effect still might exist). Accordingly a chi-square test for trend was performed modelling all cells in a given experiment as independent. That is, the individual cell is taken as the experimental unit. Consequently the power of the test is substantially increased, resulting in a rather save judgement of the observed effects. The tests were performed based upon the presence of any specific aberration.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

not clastogenic

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Toxicity test
The results obtained from the toxicity test (mitotic index) are the highest concentration of 1000 µg/mL selected for analysis in the first experiment caused 63.83% suppression of mitotic activity. The highest concentration of 1000 µg/mL selected for analysis in the second experiment caused 13.98% suppression of mitotic activity. In the third experiment with a 42 hours treatment period the highest concentration of 62.5 µg/mL selected for analysis caused 68.80% suppression of mitotic activity. The next higher concentration caused 72% suppression of mitotic activity and was not scored according to SOP. In the fourth experiment (3 hours treatment / 39 hours recovery) the highest concentration of 1000 µg/mL selected for analysis caused no suppression of mitotic activity. Concentrations higher than 1000 µg/mL could not be tested because of strong precipitates in the culture medium.

Mutagenicity study
In the first experiment performed without metabolic activation , 3% of metaphases with specific chromosomal aberrations were detected in the negative control. At the concentrations of 250, 500 and 1000 µg/mL, 3.5%, 1.5% and 2% of cells showed specific chromosomal aberrations.
In the second experiment performed with metabolic activation, 2% of metaphases with specific chromosomal aberrations were seen in the negative control. At the concentrations of 250, 500 and 1000 µg/mL the respective values were 3%, 2% and 1,5%. In the third experiment performed without metabolic activation, 0% of metaphases with specific chromosomal aberrations were detected in the negative control cultures. At the concentrations of 15.63, 31.25 and 62.5 µg/mL the corresponding values were 2%, 0.5% and 3.5%.
In the fourth experiment performed with metabolic activation, 2.5% of metaphases with specific chromosomal aberrations were registered in the negative control cultures. At the concentrations of 250, 500 and 1000 µg/mL the corresponding values were 2.5%, 2% and 3%.
1 out of these 12 values reported above showed no statistically significant difference when compared with their respective negative control. In the third experiment (42 hours treatment without metabolic activation) at the concentration of 62.5 µg/mL a slight, but statistically significant increase in specific chromosomal aberrations was observed (p< 0.01). The incidence of metaphases with aberrations (3.5%) was, however, within the historical negative control range and the criteria for a positive response were not fulfilled. In addition specific chromosomal aberrations like those obtained at the concentration of 62.5 µg/mL are also occasionally found in the negative control experiments. The observed specific chromosomal aberrations were therefore considered to be spontaneous in origin and not related to treatment. Unspecific chromosomal aberrations in the form of chromatid gaps found in all experiments were within the frequency generally observed.

Conclusions:

No evidence of clastogenic effects was obtained in Chinese hamster ovary cells under in vitro conditions.

Executive summary:

The test substance was investigated for clastogenic effects on Chinese hamster ovary cells in vitro with and without metabolic activation, according to the OECD TG 473 (1983). In experiments performed without metabolic activation no biologically significant increase in the number of specific chromosome aberrations was observed. In the experiments performed in the presence of a metabolic activation system, no biologically significant increase in the number of specific chromosome aberrations was observed. The number of chromosome aberrations was within the historical control range at all doses assessed. It was concluded that no evidence of clastogenic effects was obtained in Chinese hamster ovary cells under in vitro conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No mutagenic potential of a structural analogue substance was observed in an OECD 474 and GLP compliant study with mice.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From July 03rd, 1989 to November 01st, 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Official Journal of the European Communities No L 251 137-139

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)

Version / remarks:

Guidelines 52 FR 19080 (May 20, 1987; effective June 19, 1987), 798.5395.

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

hamster, Chinese

Strain:

other: Chinese hamster (Cricetulus griseus) random outbred strain

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain as cited inthe reported: Chinese hamster (Cricetulus griseus) random outbred strain
- Source: CIBA-GEIGY Tierfarm, Sisseln.
- Weights at test initiation:
- tolerability test: females 27-33 g, males 31-33 g
- mutagenicity test: females 23-33 g, males 26-33 g
- Days of acclimatization: at least 3 days
- Diet: standard diet NAFAG No.924, ad libitum
- Water: tap water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 21-23°C
- Humidity: 58-75%
- Air changes: air-conditioned room, no further details
- Photoperiod: 12 hrs/12 hrs

Route of administration:

oral: gavage

Vehicle:

Klucel 0.5%

Duration of treatment / exposure:

The animals were sacrificed after 16, 24 and 48 hours post treatment

Frequency of treatment:

Single administration by gavage

Post exposure period:

16, 24 and 48 hours (8 female and 8 male animals per sampling time point were sacrificed)

Dose / conc.:

5 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

8

Control animals:

yes, concurrent vehicle

Positive control(s):

64 mg/kg bw Cyclophosphamide in 20 mL/kg Klucel 0.5% served for positive control substance. the animals were sacrificed after 24 hours following treatment.

Tissues and cell types examined:

Bone marrow smears.

Details of tissue and slide preparation:

Following sacrifice of the animals, bone marrow was harvested from the shafts of both femurs with fetal calf serum. After centrifugation small drops of the sediment mixture were transferred on the end of a slide, spread out with the aid of a polished cover glass and the preparations were air-dried. Within 24 hours, the slides were stained with undiluted May-Grünwald solution for 3 minutes and then with May-Grünwald solution/water (1:1) for 2 minutes. After being rinsed in distilled water, the slides were left immersed in diluted Giemsa solution (16.6%), for 10 miniutes. After rinsing with distilled water and air-drying, the slides were cleared in Xylene and mounted.
The slides of five animals from each sex showing the best differentiation between mature and polychromatic erythrocytes were selected for scoring. The slides of five female and five male animals each of the negative control group and of the dosage group sacrificed at 16, 24 and 48 hours post-treatment were examined. From the animals of the positive control group which were sacrificed 24 hours after application, the slides of five female and five male animals were scored. 1000 polychromatic erythrocytes per animal each were scored for the incidence of micronuclei. To determine the mitotic activity of the red compartment, the ratio of polychromatic to normochromatic erythrocytes was calculated for each animal by counting a total of 1000 erythrocytes. A low proportion of polychromatic erythrocytes was indicative for a mitosis inhibiting activity of the test substance.

Evaluation criteria:

A test substance is considered to be active in this test system if a statistically significant increase in the number of polychromatic erythrocytes with micronuclei in comparison with the negative control occurs at any sampling time.

Statistics:

Chi-squared test.

Key result

Sex:

male/female

Genotoxicity:

negative

Remarks:

There was no significant increase in the number of micronucleated polychromatic erythrocytes in the animals treated with 5000 mg/kg bw of the test item as compared with the negative control animals at all sampling times.

Toxicity:

yes

Remarks:

one case of mortality (male) was reported in the treatment group of 48 hours.

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Tolerability test:
In the tolerability test the maximum dose of 5000 mg/kg bw caused no death in a group of four animals. Therefore, this dose was taken as the highest in the mutagenicity test.
Mutagenicity test:
The mutagenicity test was performed with the dosage of 5000 mg/kg bw.
One male animal died in the treatment group of 48 hours. The animals were treated once with 5000 mg/kg bw test material and bone marrow was harvested 16, 24 and 48 hours thereafter. There was no significant increase in the number of micronucleated polychromatic erythrocytes in the animals treated with the dose of 5000 mg/kg bw of test material as compared with the negative control animals at all three sampling times.
By contrast, the positive control (cyclophosphamide, 64 mg/kg bw, sampling time 24 hours) yielded a marked increase of the percentage of micronucleated cells. Here the mean percentage of polychromatic erythrocytes with micronuclei was 2.0. In comparison with the negative control (0.03 %) this value is highly significant (p <0.05).

Conclusions:

The test item was tested in an in vivo micronucleus test (MNT) according to the OECD TG 474 (1983) using Chinese hamster. No evidence of mutagenic effects was obtained in Chinese hamsters treatedby gavage with 5000 mg/kg bw test material.

Executive summary:

The test article was tested in an in vivo micronucleus test (MNT) according to the OECD TG 474 (1983) using Chinese hamster. The test item was administered by single gavage at a dose of 5000 mg/kg bw, which had been selected based on a preliminary tolerability test. The treated animals were sacrificed 16, 24 and 48 hours thereafter. From the bone marrow smears were made and evaluation of any mutagenic effect of the test item on polychromatic erythrocytes as manifested by micronuclei was done.

At all three sampling times, no statistically significant increase in the number of micronucleated polychromatic erythrocytes in comparison with the negative control animals could be evidenced. The respective positive control with cyclophosphamide (64 mg/kg bw) yielded an average of 2.0% polychromatic erythrocytes with micronuclei. This was significantly different from the negative vehicle controls (0.03%). It is concluded that no evidence of mutagenic effects was obtained in Chinese hamsters treated with the test article in the MNT.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Ames Test

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of bacterial strains Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA in a reverse mutation assay. The dose range was 33 μg - 5000 μg/plate in standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats). A weak bacteriotox effect (slight decrease in the number of his+ revertants) was observed only with tester strain TA 1535 with S9 mix at a concentration of 5000 μg/plate. In the preincaubation assay no bacteriotoxic effect (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants) was observed up to the highest concentration. Test substance precipitation was found from about 1000 μg/plate onward both with and without S9 mix. According to the results of the present study, the test substance did not lead to a relevant increase in the number of revertant colonies without S9 mix or after adding a metabolizing system in two experiments carried out independently of each other (standard plate test and preincubation assay). Besides, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria. In this study with and without S9 mix, the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain. In addition, the positive control substances with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data. In conclusion, under the experimental conditions chosen here, it is concluded that the test article is not a mutagenic test substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation.

Assessment of structural analogue substance (EC 406-040-9)

Ames test

In the presence and absence of rat liver S-9-microsomal activation system and at doses of 313-5'000 micrograms per plate, S-typhimurium strains TA 98, TA 100, TA 1535, and TA 1537 as well as E. coli strain WP2uvrA were tested for the induction of base-pair substitutions and frameshift mutations. The study was performed according to OECD testing guideline 471 and under GLP. No evidence of a mutagenic potential associated with the test substance was observed (Ciba-Geigy, 1989).

 

Chromosome Aberration

Chinese hamster ovary cells were treated in vitro at concentrations up to 1000 µg/ml with and without microsomal ac­tivation. The treat­ments lasted with microsomal activation for 3 hours and the expression periods were 15 hours and 39 hours (treatment periods not included). In the experiment without microsomal acti­vation the combined treatment and expression periods lasted for 18 and 42 hours. The analysis of the metaphases from tests with and without microsomal activation re­vealed no evidence for a clastogenic effect. The procedure followed OECD Guideline 473 and the principles of GLP (Ciba-Geigy, 1993)

In vivo micronucleus test

The substance was administered orally to groups of 24 female and 24 male animals each in the negative and in the 5'000 mg/kg dose group. The positive control group consisted of 8 female and 8 male animals. Treatment occurred in a single application. 16, 24 and 48 hours after application 8 female and 8 male animals per sampling time were sacrificed and bone-marrow smears were prepared. The procedure followed OECD Guideline 474 and the principles of GLP. No evidence of a mutagenic potential associated with the test substance was observed (Ciba-Geigy, 1989).

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for mutagenicity under Regulation (EC) No 1272/2008, as amended for the eighth time by Regulation (EU) No 2016/218.