(3E)-1,7,7-trimethyl-3-(4-methylbenzylidene)bicyclo[2.2.1]heptan-2-one

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item did not induce gene mutations or chromosomal aberrations in the V79 Chinese hamster cell line nor gene mutations in S. typhimurium bacterial cells in the presence and absence of metabolic activation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3 March 1986 to 25 April 1986

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)

Version / remarks:

1983

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

1982

Deviations:

not specified

GLP compliance:

yes

Type of assay:

in vitro mammalian cell transformation assay

Target gene:

Chinese hamster cell line V79

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Laboratory stock
- Suitability of cells: The V79 cell line has been used for many years in in vitro experiments with success. The high proliferation rate and plating efficiency of untreated cells are necessary for the appropriate performance of the study.
- Cell cycle length, doubling time or proliferation index: Doubling time 12 to 16 hours in stock cultures.
- Methods for maintenance in cell culture: Thawed stock cultures were propagated at 37°C in plastic flasks. Seeding was done with 100000 to 300000 cells per flask in 5 mL of MEM-medium supplemented with 10% fetal calf serum. Cells were subcultured twice a week.
- Modal number of chromosomes: 22

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9

Test concentrations with justification for top dose:

The highest concentration should reduce the mitotic index to about 50 % as compared to the solvent control.
7 hour preparation time: 10 μg/mL (without S9) and 36 μg/mL (with S9)
18 hour preparation time: 1, 5 and 11 μg/mL (without S9) and 3, 15 and 36 μg/mL (with S9)
28 hour preparation time: 11 μg/mL (without S9) and 36 μg/mL (with S9)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol (1%)
- Justification for choice of solvent/vehicle: Ethanol is relatively non-toxic in the concentrations applied.

Untreated negative controls:

yes

Remarks:

untreated cultures

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ethyl methanesulfonate (without metabolic activation), cyclophosphamide (with metabolic activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension
- Cell density at seeding: Two days old logarithmically growing stock cultures more than 50% confluent were trypsinised and a single cell suspension was prepared. The cells were seeded into four parallel plastic tissue culture flasks at a concentration of 300000 cells per flask (7 hour interval) and 150000 cells per flask (18 and 28 hour intervals).

DURATION
- Preincubation period: 48 hours
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 5, 15.5 and 25.5 hours after the start of treatment
- Fixation time: 2.0 (7 hour interval) or 2.5 (18 and 28 hour intervals) hours from addition of colcemid

STAIN: Aceto-orcein

NUMBER OF REPLICATIONS: 4

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Cells were trypsinised and suspended in a hypotonic solution (37.5 mM KCl) for 10 minutes at 37°C. After centrifugation (800 to 1000 rpm) the cell pellets were fixed in three steps with glacial acetic acid/ methanol 1 + 3 and stored overnight at 4°C. The next day the cells were spread on wet and ice cold slides and stained.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: 100 metaphases/flask were scored per test concentration.

DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index
- Any supplementary information relevant to cytotoxicity: Mitotic index was determined in 1000 cells in each replicate.

Rationale for test conditions:

The test item concentrations were determined in a preliminary experiment using the mitotic index and plating efficiencies as an indicator for toxicity response.

Evaluation criteria:

Gaps, breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural aberrations.
1. The test substance is classified as mutagenic if it induces with one of the concentrations tested a significantly increased aberration rate as compared with the negative control. The significance is obvious either by an enhancement of the rate clearly over the control range or it is proven by adequate biometry.
2. The test substance is classified as mutagenic if there is a concentration related increase in the aberration rates as compared with the solvent controls.
3. The test substance is considered negative when 1 and 2 do not apply.

Statistics:

No statistical analysis performed as there is no indication of a dose-response relationship.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: See Table 2.

The sensitivity of the test system was demonstrated by the clearly enhanced structural aberration rates found in the groups treated with the positive control substances.

In only one group of cells treated with the lowest concentration of the test item, a higher number of cells with structural chromosome aberrations as compared to the range of the negative controls was found in the presence of S9 mix at preparation interval 18 h. In all other groups treated with the test item, aberration rates within or near the control range were found. This single higher value alone is not regarded as being sufficient to prove mutagenicity.

Table 1. Mutagenicity data and mitotic index

Preparation time	Treatment group	Total cells analysed	Number of aberrant cells including gaps	% of aberrant cells including gaps	Number of aberrant cells excluding gaps	% of aberrant cells excluding gaps	% of aberrant cell exchanges	% mitotic index (absolute)	% mitotic index (relative)
18 hours	Untreated control -S9	400	9	2.25	5	1.25	0.25	6.8	100.0
18 hours	Untreated control +S9	400	24	6.00	7	1.75	0.00	6.0	100.0
18 hours	Solvent control -S9	400	17	4.25	4	1.00	0.00	4.8	100.0
18 hours	Solvent control +S9	400	14	3.50	9	2.25	0.25	5.4	100.0
18 hours	Positive control -S9	200	60	60.00	52	52.00	45.00	7.7	113.2
18 hours	Positive control +S9	200	69	69.00	65	65.00	39.00	4.3	71.6
18 hours	1 μg/mL test item -S9	400	23	5.75	10	2.50	0.00	4.6	95.6
18 hours	3 μg/mL test item +S9	400	37	9.25	19	4.75	0.00	4.5	83.6
18 hours	5 μg/mL test item -S9	400	19	4.75	5	1.25	0.25	5.6	115.6
18 hours	15 μg/mL test item +S9	400	13	3.25	4	1.00	0.50	2.9	54.2
18 hours	11 μg/mL test item -S9	400	17	4.25	11	2.75	0.50	3.2	65.8
18 hours	36 μg/mL test item +S9	400	21	5.25	5	1.25	0.00	7.5	139.8
7 hours	Solvent control -S9	400	16	4.00	4	1.00	0.00	5.9	100.0
7 hours	Solvent control +S9	400	10	2.50	1	0.25	0.00	5.9	100.0
7 hours	10 μg/mL test item -S9	400	14	3.50	8	2.00	0.00	2.8	47.7
7 hours	36 μg/mL test item +S9	400	28	7.00	13	3.25	0.00	5.2	86.9
28 hours	Solvent control -S9	400	17	4.25	6	1.50	0.25	4.4	100.0
28 hours	Solvent control +S9	400	10	2.50	2	0.50	0.00	1.9	43.1
28 hours	11 μg/mL test item -S9	400	11	2.75	6	1.50	0.5	5.7	100.0
28 hours	36 μg/mL test item +S9	400	25	6.25	10	2.50	0.00	5.7	99.1

Table 2. Preliminary study results

Treatment group	Number of seeded cells per flask	Mean number of found cells per flask	% PE (absolute)	% PE (relative)
Untreated control -S9	496	300.0	60.5	100.0
Untreated control +S9	498	435.0	87.3	100.0
Solvent control -S9	496	311.5	62.8	100.0
Solvent control +S9	498	387.0	77.7	100.0
10.0 μg/mL test item -S9	496	224.0	45.2	71.9
11.0 μg/mL test item -S9	496	155.5	31.4	49.9
12.0 μg/mL test item -S9	496	20.0	4.0	6.4
13.0 μg/mL test item -S9	496	17.0	3.4	5.5
14.0 μg/mL test item -S9	496	0.0	0.0	0.0
15.0 μg/mL test item -S9	496	0.0	0.0	0.0
15.0 μg/mL test item +S9	498	356.5	71.6	92.1
20.0 μg/mL test item +S9	498	377.5	75.8	97.5
25.0 μg/mL test item +S9	498	350.5	70.4	90.6
30.0 μg/mL test item +S9	498	342.0	68.7	88.4
35.0 μg/mL test item +S9	498	233.5	46.9	60.3
40.0 μg/mL test item +S9	498	2.0	0.4	0.5

Conclusions:

The test item was not mutagenic to V79 Chinese hamster cell line in vitro in the presence and absence of metabolic activation.

Executive summary:

The potential of the test item to induce chromosomal aberrations was determined in vitro with cells of the V79 Chinese hamster cell line according to OECD Guideline 473. Chinese hamster lung fibroblasts V79 were treated with the test item at concentrations of 1, 5, 10 and 11 µg/mL without S9-activation and 3, 15 and 36 µg/mL with S9-activation. Cells were incubated with the test item for 4 hours, followed by a 5, 15.5 or 25.5 -hour incubation period in growth medium. Colcemid was added as a fixing agent and the cells were incubated for a further 2.0 or 2.5 hours. The study included negative, solvent and positive (ethyl methanesulfonate and cyclophosphamide) controls . Mutagenicity was evaluated by determining the number of gaps, breaks, fragments, deletions, exchanges and chromosomal disintegrations in 400 metaphases per test concentration. Cytotoxicity was evaluated by determination of mitotic index in 1000 cells in each replicate. The test item was not mutagenic to V79 Chinese hamster cell line in the presence and absence of metabolic activation. This study is considered reliable without restriction (Klimisch 1).

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not applicable

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Remarks:

and TA 1538

Additional strain / cell type characteristics:

other: histidine mutation, uvrB deletion, rfa deletion, R-factor presence

Metabolic activation:

with and without

Metabolic activation system:

Liver postmitochondrial fraction (S-9 from male rats induced with Aroclor 1254)

Test concentrations with justification for top dose:

100, 500, 1000, 5000, 10000 and 15000 μg/plate
Toxic effect sought in the highest concentration.

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

methylmethanesulfonate

other: 2-aminoanthracene, daunomycin, 1-ethyl-2-nitro-3-nitrosoguanidine, N-methyl-N-nitro-N-nitrosoguanidine; 4-nitro-o-phenylene diamine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 8 for the solvent control, 4 for the test item

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Colonies were stained with triphenyltetrazolium if counting of bacteria colonies is affected by precipitation.

OTHER:
- Plate composition: 0.1 mL bacteria suspension, 0.1 mL test solution, 0.5 mL Na-phosphate buffer (in trials without metabolic activation), 0.5 mL S-9 mix (in trials with metabolic activation), 2.0 mL soft agar solution

Statistics:

Standard deviations were calculated.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Solubility: Toxic effect was not achieved at the highest dose due to low solubility of the test substance in the test medium.
- Precipitation: The compound precipitated from the test medium at the dose levels of 5000, 10000 and 15000 μg/plate.
- Definition of acceptable cells for analysis: Where counting of bacterial colonies was not possible because of precipitation of the test substance, the colonies were stained with triphenyltetrazolim chloride before evaluation of the plates. This staining enables the colonies to be distinguished from the precipitate and can then be counted as usual.

HISTORICAL CONTROL DATA
- Positive historical control data: Results were acceptable and within the normal range
- Negative (solvent/vehicle) historical control data: Results were acceptable and within the normal range.

Table 1. First trial for mutagenic effects without metabolic activation

Test item concentration (μg/plate)	Revertant colonies per plate (± standard deviation)
	Base-pair substitution			Frame-shift
	TA 100	TA 1535	TA 98	TA 1537	TA 1538
Solvent control (ethanol)	62 ± 8	10 ± 3	31 ± 7	5 ± 1	8 ± 3
100	57 ± 6	10 ± 3	28 ± 8	5 ± 1	7 ± 4
500	65 ± 10	7 ± 3	30 ± 6	6 ± 1	9 ± 5
1000	44 ± 9	8 ± 2	27 ± 7	5 ± 1	7 ± 2
5000	50 ± 8	5 ± 1	30 ± 9	3 ± 1	8 ± 3
10000	73 ± 13	6 ± 0	34 ± 6	4 ± 1	6 ±1
15000	75 ± 10	5 ± 4	36 ± 2	5 ± 0	7 ± 2

Table 2. First trial for mutagenic effects with metabolic activation

Test item concentration (μg/plate)	Revertant colonies per plate (± standard deviation)
	Base-pair substitution			Frame-shift
	TA 100	TA 1535	TA 98	TA 1537	TA 1538
Solvent control	74± 10	13± 6	52± 9	9± 3	20± 4
100	55± 15	11±3	39± 5	7± 4	17± 3
500	70± 15	9± 2	41± 8	7± 3	14± 4
1000	71± 9	10± 3	50± 13	7± 2	14± 6
5000	61± 5	11± 0	48± 11	8± 3	12± 2
10000	68± 3	12± 3	50± 2	7± 2	18± 6
15000	76± 6	14± 5	42± 8	6± 1	18± 5

Table 3. Second (repeat) trial for mutagenic effects without metabolic activation

Test item concentration (μg/plate)	Revertant colonies per plate (± standard deviation)
	Base-pair substitution			Frame-shift
	TA 100	TA 1535	TA 98	TA 1537	TA 1538
Solvent control	96±10	11± 2	22± 7	7± 2	6± 1
100	87± 13	6±2	25± 3	6± 3	5± 2
500	106± 26	9± 4	20± 3	5 ± 2	5± 1
1000	106± 17	12± 5	21± 3	4± 3	3± 2
5000	97± 5	4± 2	31± 4	5± 1	4 ± 2
10000	107± 8	5± 4	9± 1	2± 2	2± 1
15000	106± 12	9± 1	18± 5	3± 2	5± 1

Table 4. Second (repeat) trial for mutagenic effects with metabolic activation

Test item concentration (μg/plate)	Revertant colonies per plate (± standard deviation)
	Base-pair substitution			Frame-shift
	TA 100	TA 1535	TA 98	TA 1537	TA 1538
Solvent control	97±17	16± 5	30± 8	5± 2	17± 2
100	119± 17	15±3	24± 3	4± 2	13± 2
500	112± 15	11± 1	40± 17	7± 3	10± 1
1000	128± 12	12± 2	24± 10	3 ± 1	15± 5
5000	125± 5	17± 5	25± 7	3± 2	11± 5
10000	124± 15	8± 3	41± 1	6± 4	11± 4
15000	122± 11	10± 3	28± 4	3± 1	5± 2

Conclusions:

The test substance did not show mutagenic activity in S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 up to 15000 μg/plate, with and without metabolic activation.

Executive summary:

The mutagenic potential of the test item was determined in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 using a plate incorporation method similar to OECD Guideline 471. The test substance was incubated with and without metabolic activation (rat liver S-9 mix) at nominal concentrations of 100, 500, 1000, 5000, 10000 and 15000 μg/plate. Two experiments were performed, each consisting of 4 test item replicates and 8 solvent control (ethanol) replicates. 2 -aminoanthracene, 9 -aminoacridine, daunomycin, 1 -ethyl-2 -nitro-3 -nitrosoguanidine, methyl methanesulfonate, N-methyl-N-nitro-N-nitrosoguanidine, 2 -nitrofluorene and 4 -nitro-1,2 -phenylenediamine were tested as the positive controls. The test substance did not show mutagenic activity in any S. typhimurium strain up to 15000 μg/plate, with and without metabolic activation. This study is reliable without restrictions (Klimisch 1).

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 July 2017 to 24 August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

Kanpoan No. 287 (Environmental Protection Agency), Eisei No. 127 (Ministry of Health & Welfare), Heisei 09/10/31 Kikyoku No. 2 (Ministry of International Trade & Industry)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian cell gene mutation test using the Hprt and xprt genes (migrated information)

Target gene:

Hprt and xprt

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Laboratory cell bank.
- Suitability of cells: The V79 cell line has been used successfully in in vitro experiments for many years, particularly due to the doubling time and cloning efficiency.
- Cell cycle length, doubling time or proliferation index: 12-16 h doubling time in stock cultures and more than 50% cloning efficiency of untreated cells.
- Methods for maintenance in cell culture if applicable: Storage in liquid nitrogen.
- Modal number of chromosomes: 22

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Cells were cultured in MEM (minimal essential medium) containing Hank’s salts supplemented with 10% foetal bovine serum (FBS), neomycin (5 μg/mL) and amphotericin B (1%). During treatment no FBS was added to the medium. All incubations were done at 37°C with 1.5% carbon dioxide (CO2) in humidified air.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

With metabolic activation: 0.75, 1.5, 3, 6, 8, 10, 12, 16 and 20 μg/mL
Without metabolic activation: 0.78, 1.6, 3.1, 6.25, 12.5, 25, 50 and 100 μg/mL
The top dose was chosen to have a relevant cytotoxic effect, indicated by a relative cloning efficiency of 50% or below.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (1.0% v/v)
- Justification for choice of solvent/vehicle: The solvent was chosen for its solubility properties and its relative non-toxicity to the cell cultures.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding: 2.0×10^6 cells per test vessel

DURATION
- Preincubation period: 24 hours
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 8 days

STAIN (for cytogenetic assays): 10% methylene blue in 0.01% KOH solution

NUMBER OF REPLICATIONS: 5 (+ 2 for determination of cytotoxicity)

NUMBER OF CELLS EVALUATED: Stained colonies with more than 50 cells were counted.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
- Any supplementary information relevant to cytotoxicity: Cloning efficiency was determined after exposure to the test item (survival) and after the expression time (viability).

Rationale for test conditions:

Two parallel cultures were used throughout the assay. In order to establish a concentration response effect of the test item at least four concentration levels are tested. To overcome problems with possible deviations in toxicity the main experiment was started with more than four concentrations.

Evaluation criteria:

A test item is classified as clearly mutagenic if, in any of the experimental conditions examined, all of the following criteria are met:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is dose-related when evaluated with an appropriate trend test,
c) any of the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).

A test item is classified as clearly non-mutagenic if, in all experimental conditions examined, all of the following criteria are met:
a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no concentration-related increase when evaluated with an appropriate trend test,
c) all results are inside the distribution of the historical negative control data (based 95% control limits).

In cases when the response is neither clearly negative nor clearly positive as described above, or in order to judge the biological relevance of a result, the data was evaluated by expert judgement or further investigations.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. A trend is judged as significant whenever the p-value (probability value) is below 0.05 when compared to the solvent control groups. A t-Test was performed to evaluate an isolated increase of the mutation frequency at a test point exceeding the 95% confidence interval. Again a t-test is judged as significant if the p-value (probability value) is below 0.05. Both biological and statistical significance were considered together.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No relevant shift of pH of the medium even at the maximum concentration of the test item.
- Effects of osmolality: No relevant shift of osmolarity of the medium even at the maximum concentration of the test item.
- Precipitation: Precipitation was observed at the end of the treatment in the three highest test concentation without metabolic activation (12, 16 and 20 μg/mL) and the two highest test concentration with metabolic activation (50 and 100 μg/mL). Mutation rate was not assessed for these precipitating test concentrations (except at 50 μg/mL, mutation rate was assessed).
- Other confounding effects: Severe cytotoxicity was observed in the four highest test concentation without metabolic activation (10, 12, 16 and 20 μg/mL), therefore mutation rate was not assessed for these cytotoxic test concentrations. The data at 8.0 μg/mL without metabolic activation were rejected as severe cytotoxicity was observed, therefore the mutant frequency was not taken into the consideration of the evaluation of this assay.

RANGE-FINDING/SCREENING STUDIES: The first pre-experiment was performed in the presence and absence of metabolic activation for test item concentrations between 15.6 and 2000 μg/mL. A relevant cytotoxic effect (indicated by a relative cloning efficiency of 50% or below) was observed at 15.6 μg/mL and above without metabolic activation and at 125.0 μg/mL and above with metabolic activation. Due to strong toxic effects in the absence of S9 mix, this part of the pre-experiment was repeated with lower concentrations (0.78 μg/mL to 50 μg/mL). In the second pre-experiment, relevant cytotoxic effect was observed at 12.5 μg/mL and above without metabolic activation.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Number of mutant colonies per 10^6 cells, without metabolic activation: mean 190.3, range 53.9 to 872.3, standard deviation 88.4, 95% confidence limit not reported. Number of mutant colonies per 10^6 cells, with metabolic activation: mean 215.8, range 56.7 to 739.9, standard deviation 110.9, 95% confidence limit not reported.
- Negative (solvent) historical control data: Number of mutant colonies per 10^6 cells, without metabolic activation: mean 15.9, range 3.4 to 41.0, standard deviation 7.1, 95% confidence limit 1.7 to 30.2. Number of mutant colonies per 10^6 cells, with metabolic activation: mean 15.7, range 2.4 to 39.2, standard deviation 6.8, 95% confidence limit 2.0 to 29.4.

Table 1. Summary of results for culture 1

Treatment	S9 mix	Relative cloning efficiency (%)	Relative cell density (%)	Relative adjusted cloning efficiency (%)	Number of mutant colonies / 10^6 cells	95% confidence interval
Solvent control	-	100.0	100.0	100.0	21.8	1.7 - 30.2
Positive control (300.00 μg/mL EMS)	-	103.5	85.1	88.1	253.0	1.7 - 30.2
Test item (0.75 μg/mL)	-	101.7	108.3	110.1	14.8	1.7 - 30.2
Test item (1.50 μg/mL)	-	102.1	91.0	92.9	10.5	1.7 - 30.2
Test item (3.00 μg/mL)	-	102.4	106.4	109.0	21.4	1.7 - 30.2
Test item (6.00 μg/mL)	-	102.7	89.5	91.9	30.3	1.7 - 30.2
Test item (8.00 μg/mL) (X)	-	10.4	53.6	5.6	43.9	1.7 - 30.2
Test item (10.00 μg/mL)	-	7.0	28.0	2.0	-	1.7 - 30.2
Test item (12.00 μg/mL) (P)	-	-	2.9	##	##	1.7 - 30.2
Test item (16.00 μg/mL) (P)	-	##	##	##	##	1.7 - 30.2
Test item (20.00 μg/mL) (P)	-	##	##	##	##	1.7 - 30.2
Solvent control	+	100.0	100.0	100.0	7.2	2.0 - 29.4
Positive control (2.30 μg/mL DMBA)	+	81.0	98.0	79.4	124.3	2.0 - 29.4
Test item (0.78 μg/mL)	+	-	109.6	#	-	2.0 - 29.4
Test item (1.60 μg/mL)	+	94.1	124.2	116.9	-	2.0 - 29.4
Test item (3.10 μg/mL)	+	84.4	121.0	102.1	5.9	2.0 - 29.4
Test item (6.25 μg/mL)	+	93.1	119.8	111.5	14.6	2.0 - 29.4
Test item (12.50 μg/mL)	+	90.9	114.9	104.4	15.3	2.0 - 29.4
Test item (25.00 μg/mL)	+	88.0	114.7	100.9	28.6	2.0 - 29.4
Test item (50.00 μg/mL) (P)	+	75.0	98.4	73.8	15.8	2.0 - 29.4
Test item (100.00 μg/mL) (P)	+	-	8.1	##	##	2.0 - 29.4

EMS: Ethylmethane sulfonate

DMBA: 7,12 -dimethylbenz(a)anthracene

(P): Precipitation visible at the end of treatment

(X): Data not considered in evaluation due to severe cytotoxicity

#: Culture was not continued as a minimum of only four analysable concentrations are required

##: Culture was not continued due to severe cytotoxic effects

-: Not reported

Table 2. Summary of results for culture 2

Treatment	S9 mix	Relative cloning efficiency (%)	Relative cell density (%)	Relative adjusted cloning efficiency (%)	Number of mutant colonies / 10^6 cells	95% confidence interval
Solvent control	-	100.0	100.0	100.0	10.0	1.7 - 30.2
Positive control (300.00 μg/mL EMS)	-	98.3	71.1	69.8	234.3	1.7 - 30.2
Test item (0.75 μg/mL)	-	98.3	88.1	86.6	23.3	1.7 - 30.2
Test item (1.50 μg/mL)	-	104.0	62.4	64.8	15.6	1.7 - 30.2
Test item (3.00 μg/mL)	-	101.4	77.2	78.3	12.8	1.7 - 30.2
Test item (6.00 μg/mL)	-	100.7	82.2	83.4	11.0	1.7 - 30.2
Test item (8.00 μg/mL) (X)	-	11.4	42.4	4.8	41.3	1.7 - 30.2
Test item (10.00 μg/mL)	-	2.7	28.6	0.8	-	1.7 - 30.2
Test item (12.00 μg/mL) (P)	-	-	1.1	##	##	1.7 - 30.2
Test item (16.00 μg/mL) (P)	-	##	##	##	##	1.7 - 30.2
Test item (20.00 μg/mL) (P)	-	##	##	##	##	1.7 - 30.2
Solvent control	+	100.0	100.0	100.0	7.4	2.0 - 29.4
Positive control (2.30 μg/mL DMBA)	+	81.0	71.6	58.0	149.5	2.0 - 29.4
Test item (0.78 μg/mL)	+	#	#	#	#	2.0 - 29.4
Test item (1.60 μg/mL)	+	94.8	58.1	55.1	-	2.0 - 29.4
Test item (3.10 μg/mL)	+	99.3	69.2	68.7	16.0	2.0 - 29.4
Test item (6.25 μg/mL)	+	94.4	66.1	62.5	21.7	2.0 - 29.4
Test item (12.50 μg/mL)	+	49.5	70.0	34.6	24.4	2.0 - 29.4
Test item (25.00 μg/mL)	+	55.4	84.5	46.9	14.6	2.0 - 29.4
Test item (50.00 μg/mL) (P)	+	39.7	45.9	18.3	20.9	2.0 - 29.4
Test item (100.00 μg/mL) (P)	+	-	8.1	##	##	2.0 - 29.4

EMS: Ethylmethane sulfonate

DMBA: 7,12 -dimethylbenz(a)anthracene

(P): Precipitation visible at the end of treatment

(X): Data not considered in evaluation due to severe cytotoxicity

#: Culture was not continued as a minimum of only four analysable concentrations are required

##: Culture was not continued due to severe cytotoxic effects

-: Not reported

Conclusions:

The test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test item is considered to be non-mutagenic in the HPRT assay.

Executive summary:

The potential of the test item to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster was determined according to OECD Guideline 476, EU Method B.17, EPA OPPTS 870.5300 and Japanese guidelines. The study was conducted in medium at test item concentrations of 0.75, 1.5, 3, 6, 8, 10, 12, 16 and 20 μg/mL in the absence metabolic activation and 0.78, 1.6, 3.1, 6.25, 12.5, 25, 50 and 100 μg/mL in the presence of metabolic activation. A solvent control and positive controls were tested concurrently. Five replicates were assessed per test item treatment and control, and two parallel cultures were used throughout the assay. The treatment period was 4 hours with and without metabolic activation, with a 24 hour pre-exposure period and 7 day expression (post-exposure) period in medium. Cells were fixed and stained with 10% methylene blue in 0.01% KOH solution 8 days after treatment. Precipitation and / or severe cytotoxicity were observed in the four highest test concentation without metabolic activation (10, 12, 16 and 20 μg/mL) and at the highest test concentration with metabolic activation (100 μg/mL), therefore mutation frequency was not assessed for these concentrations. Severe cytotoxicity also occurred in the 8 μg/mL treatment level without metabolic activation, and this data was not taken into the consideration of the evaluation of this assay. No relevant and reproducible increase in mutant colony numbers per 10^6 cells was observed with and without metabolic activation. Therefore, the test item is considered to be non-mutagenic in the HPRT assay. This study is considered to be reliable without restrictions (Klimisch 1).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Three in vitro studies are available to determine the genetic toxicity of the test item.

The mutagenic potential of the test item to bacteria was determined in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 using a plate incorporation method similar to OECD Guideline 471 (1980). The test substance was incubated with and without metabolic activation (rat liver S-9 mix) at nominal concentrations of 100, 500, 1000, 5000, 10000 and 15000 μg/plate. Two experiments were performed, each consisting of 4 test item replicates and 8 solvent control (ethanol) replicates. 2 -aminoanthracene, 9 -aminoacridine, daunomycin, 1 -ethyl-2 -nitro-3 -nitrosoguanidine, methyl methanesulfonate, N-methyl-N-nitro-N-nitrosoguanidine, 2 -nitrofluorene and 4 -nitro-1,2 -phenylenediamine were tested as the positive controls. The test substance did not show mutagenic activity in any S. typhimurium strain up to 15000 μg/plate, with and without metabolic activation. This study is reliable without restrictions (Klimisch 1).

The potential of the test item to induce chromosomal aberrations was determined in vitro with cells of the V79 Chinese hamster cell line according to OECD Guideline 473 (1986). Chinese hamster lung fibroblasts V79 were treated with the test item at concentrations of 1, 5, 10 and 11 µg/mL without S9-activation and 3, 15 and 36 µg/mL with S9-activation. Cells were incubated with the test item for 4 hours, followed by a 5, 15.5 or 25.5 -hour incubation period in growth medium. Colcemid was added as a fixing agent and the cells were incubated for a further 2.0 or 2.5 hours. The study included negative, solvent and positive (ethyl methanosulfonate and cyclophosphamide) controls. Mutagenicity was evaluated by determining the number of gaps, breaks, fragments, deletions, exchanges and chromosomal disintegrations in 400 metaphases per test concentration. Cytotoxicity was evaluated by determination of mitotic index in 1000 cells in each replicate. The test item was not mutagenic to V79 Chinese hamster cell line in the presence and absence of metabolic activation. This study is considered reliable without restrictions (Klimisch 1).

The potential of the test item to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster was determined according to OECD Guideline 476, EU Method B.17, EPA OPPTS 870.5300 and Japanese guidelines (2017). The study was conducted in medium at test item concentrations of 0.75, 1.5, 3, 6, 8, 10, 12, 16 and 20 μg/mL in the absence metabolic activation and 0.78, 1.6, 3.1, 6.25, 12.5, 25, 50 and 100 μg/mL in the presence of metabolic activation. A solvent control and positive controls were tested concurrently. Five replicates were assessed per test item treatment and control, and two parallel cultures were used throughout the assay. The treatment period was 4 hours with and without metabolic activation, with a 24 hour pre-exposure period and 7 day expression (post-exposure) period in medium. Cells were fixed and stained with 10% methylene blue in 0.01% KOH solution 8 days after treatment. Precipitation and / or severe cytotoxicity were observed in the four highest test concentation without metabolic activation (10, 12, 16 and 20 μg/mL) and at the highest test concentration with metabolic activation (100 μg/mL), therefore mutation frequency was not assessed for these concentrations. Severe cytotoxicity also occurred in the 8 μg/mL treatment level without metabolic activation, and this data was not taken into the consideration of the evaluation of this assay. No relevant and reproducible increase in mutant colony numbers per 10^6 cells was observed with and without metabolic activation. Therefore, the test item is considered to be non-mutagenic in the HPRT assay. This study is considered to be reliable without restrictions (Klimisch 1).

Justification for classification or non-classification

Three in vitro studies are available to evaluate the genotoxicity of the test item. All studies were considered reliable without restriction (Klimisch 1) and sufficient for classification. None of studies indicated any potential for mutagenicity, clastogenicity or cytogenicity. Consequently, in accordance with the CLP Regulation (EC) No. 1272/2008, the test item is not classified as a genetic toxicant.