Ethyl [2-(4-phenoxyphenoxy)ethyl]carbamate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames, +/- S9 negative, S. typhimurium TA 1535, TA 1537, TA 1538, 97, TA 98, TA 100 and 102, according to OECD TG 471, Gocke 1988

- In vitro chromosome aberration study, +/- S9 negative, Chinese Hamster Ovary (CHO) cells, according to OECD TG 473, Ogorek 1988

- In vitro gene mutation assay, +/- S9 negative, Chinese Hamster lung cells (V79), according to OECD TG 476, Strobel 1982

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:

05 Oct 1988 to 03 Nov 1988

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)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

his- (S. typhimurium)

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Species / strain / cell type:

S. typhimurium TA 1538

Species / strain / cell type:

S. typhimurium TA 97

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9 : Phenobarbital/β-Naphthoflavone induced rat liver S9
- Method of preparation of S9 mix : Male albino rats were killed by decapitation and the livers were removed aseptically. The livers of were chopped and mixed with homogenizer with addition of a volume of ice cold 0.15 M potassium chloride equivalent to 3 times the weight of the livers. The homogenate was pooled and centrifuged at 4°C for 10 minutes at 9000 g. The supernatant fluid was collected and dispensed in 2 mL cryotubes. The tubes were rapidly cooled and kept in the deep freezer at -70°C until use. At the end of the preparation about 1 mL of the S9 fraction was dispensed on complete medium plates and incubated at 37°C for 2 days. Absence of colony growth verified the sterility of the S9 fraction
- Concentration or volume of S9 mix and S9 in the final culture medium: The composition of the S9 mixture was as followed: Potassium chloride 0.165 M, magnesium chloride 0.04 M (both 0.2 mL per mix), sodium phosphate buffered saline 0.2 M, pH 7.4 (0.5 mL per mL mix), NADP (Boehringer, 3.2 mg per mL mix), glucose-6-phosphate (Boehringer, 1.53 mg per mL mix) and S9 fraction (0.1 mL per mL mix).
- Quality controls of S9: In order to test the activity of the S9 mix used in the experiments positive controls requiring metabolic activation were tested concurrently. 2--AAF and 2-Aminoanthr. were tested with and without metabolic activation to examine the activity of the S9-mix.

Test concentrations with justification for top dose:

Ames standard assay (experiment 1 and 2): 15.8, 50, 158, 500 and 1580 µg/plate
Liquid pre-incubation assay (experiment 3 and 5): 10, 31.6, 100, 316 and 1000 µg/plate

Vehicle / solvent:

- Solvent used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

sodium azide

mitomycin C

other: 2-Aminoanthracene, ICR 191

Details on test system and experimental conditions:

PRE- EXPERIMENT FOR TOXICITY
Toxicity of the test substance was determined in a preliminary toxicity assay by evaluating the growth on NB- and/or minimal- medium (determination of the growth of the background lawn and/or frequency of spontaneous revertants). Each test substance dose, as well as the appropriate solvent control, was evaluated in duplicate in strain TA 100.

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation) for experiment 1 and 2. A liquid pre-incubation assay was performed for experiment 3 and 5.

EXPERIMENTAL PERFORMANCE
Ames test:
Test tubes containing 2 mL of 0.6% agar medium autoclaved and a prewarmed water bath at 42°C to 45°C. Following solutions were added in order:
- 0.2 mL of the histidine/biotin mixture corresponding to 21 µg L-histidine and 24.4 µg biotin.
- 0.1 mL of the test substance at different concentrations. The negative control tubes contained 0.1 mL of the solvent and the positive control ones received 0.05 mL of the different reference substances which were thawed shortly before use.
- 0.1 mL of the overnight cultures of the Salmonella typhimurium strains.
- 0.5 mL of the S9 mixture where metabolic activation was needed. The S9 mixture was replaced by 0.5 mL sodium phosphate buffered saline 0.2 M, pH 7.4 in the treatment without metabolic activation.
The contents of the tubes were mixed and poured immediately ontotable 1 Vogel-Bonner minimal agar plates. Four replicate plates for the test substance and negative control or two replicate plates for the positive controls were incubated at 37°C, upside down, for 2 days.

Liquid preincubation assay:
In the modified procedure 0.1 mL of the test compound solutions and of the solvent or 0.05 mL of the reference substances thawed shortly before use, 0.5 mL of sodium phosphate buffered saline 0.2 M, pH 7.4 or 0.5 mL of the 89 mixture and 0.1 mL of the NB cultures of the bacterial tester strains were incubated for 30 minutes at 37°C under shaking. 2.2 mL soft-agar supplemented with 21 µg L-histidine and 24.4 µg biotin was added afterwards and the tubes were poured on Vogel—Bonner minimal agar plates. Four replicate plates for the test compound and negative control or two replicate plates for the positive controls were incubated at 37°C, upside down, for 2 days.

DATA REPORTING
The determination of the number of colonies growing on the plates was made by hand. The colonies of the positive controls were generally evaluated using a New Brunswick Inc Biotran III Automatic colony counter. No correction factor was determined. Representative plates were examined microscopically for microcolony growth and the absence of a confluent lawn of bacteria. A reduced background lawn was reported as BR and the absence of bacterial lawn as B0. The inhibition of the growth was attributed to toxic effects by the substance

Evaluation criteria:

There is as yet no generally accepted statistical treatment of Ames test data. In most tests the results are either clearly positive or clearly negative. A positive result is defined reproducible, dose- related increase in the number of his+ revertants. The increase should reach at least a doubling of the number of spontaneous revertants for Salmonella typhimurium strains TA 1535, TA 1537, TA 1538 and TA 98. For strains TA 97, TA 100 and TA 102 a 1.5- fold increase over control values might be indicative of a mutagenic effect provided the negative control values fall within the historical control data. Other investigators have set higher limits for a mutagenic response (factor 3 and 2 for the respective groups of strains). These rules of thumb have a questionable scientific foundation (Claxton et al. 1987) and biological relevance should always be taken into account. Negative result is defined as the absence of a reproducible increase in the number of his+ revertant colonies.

Key result

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 97, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

STUDY RESULTS
- Some signs of toxic activity (reduced background growth) was seen in the preincubation assay. In strain TA 1538 the negative control (-S9) was rather low so that a doubling of the mutant frequency was found at one intermediate test concentrations. This was however, clearly due to low spontaneous value and cannot be taken as test substance related. Accordingly no increase was found in the preincubation assay.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: Precipitation on the plates was observed at the dose level of 1580 μg/plate
- Water solubility: The test compound was insoluble in water. Stock solutions in DMSO were prepared before each experiment. Upon addition of the DMSO solutions to the aqueous medium a milky suspension was visible at concentrations of 158 pg/plate and above. At 1.58 mg/plate small droplets remained in the soft agar during the two day incubation period. Therefore, it was decided to use 1.58 mg/plate as the highest dose in the standard assay and 1 mg/plate in the preincubation assay where during the liquid incubation period the test concentration is higher than in the plate assay due to the smaller volume.

Table 1. Salmonella mutagenicity test (Ames standard assay). Mean values and standard deviations.

Experiment no	1	1	1	1	1	1
Activation	(-)S9	(+)S9	(-)S9	(+)S9	(-)S9	(+)S9
Strain	TA 1535	TA 1535	TA 1537	TA 1537	TA 1538	TA 1538
Concentration in µg/plate
0.00	11 ± 2	12 ± 3	13 ± 3	13 ± 4	8 ± 1	22 ± 6
15.80	11 ± 2	12 ± 3	13 ± 3	13 ± 4	8 ±  1	22 ± 8
50.00	13 ± 5	8 ±  3	10 ± 6	13 ± 5	12 ± 5	21 ± 5
158.00	15 ± 6	7 ± 4	14 ± 6	9 ± 3	14 ± 3	24 ± 3
500.00	9 ± 2	8 ± 4	11 ± 1	8 ± 3	17 ± 8	28 ± 4
1580	10 ± 3	12 ± 2	8 ± 4	10 ± 3	13 ± 6	17 ± 2

Experiment no	2	2	2	2	2	2	2	2
Activation	(-)S9	(+)S9	(-)S9	(+)S9	(-)S9	(+)S9	(-)S9	(+)S9
Strain	TA 97	TA 97	TA 98	TA 98	TA 100	TA 100	TA 102	TA 102
Concentration in µg/plate
0.00	150 ± 14	178 ± 18	23 ± 5	29 ± 2	96 ± 9	95 ± 6	295 ± 21	283 ± 10
15.80	156 ± 5	180 ± 15	25 ± 3	33 ± 2	88 ± 8	103 ± 11	288 ± 29	298 ± 16
50.00	167 ± 8	166 ± 8	33 ± 4	29 ± 7	81 ± 11	100 ± 8	296 ± 32	273 ± 9
158.00	160 ± 9	178 ± 12	29 ± 2	26 ± 5	106 ± 11	95 ± 8	255 ± 32	292 ± 15
500.00	151 ± 8	165 ± 8	24 ± 8	23 ± 8	102 ± 8	92 ± 8	166 ± 8	236 ± 8
1580	153 ± 8	181 ± 8	32 ± 8	26 ± 8	97 ± 8	87 ± 8	114 ± 8	160 ± 8

Table 2. Salmonella mutagenicity test (Liquid preincubation assay). Mean values and standard deviations

Experiment no	3	3	3	3	3	3
Activation	(-)S9	(+)S9	(-)S9	(+)S9	(-)S9	(+)S9
Strain	TA 1535	TA 1535	TA 1537	TA 1537	TA 1538	TA 1538
Concentration in µg/plate
0.00	6 ± 3	10 ± 1	12 ± 2	11 ± 5	19 ± 7	24 ± 5
10.00	9 ± 3	10 ± 2	14 ± 1	12 ± 3	13 ± 3	26 ± 5
31.60	8 ± 2	10 ± 2	12 ± 5	14 ± 1	16 ± 1	24 ± 2
100.00	10 ± 2	8 ± 2	6 ± 3	13 ± 4	6 ± 2 t	25 ± 6
316.00	10 ± 2	9 ± 5	2 ± 1 t	12 ± 3	5 ± 3 t	29 ± 10
1000.00	9 ± 3	8 ± 1	2 ± 1 t	7 ± 2 t	4 ± 1	10 ± 3 t

.

Experiment no	5	5	5	5	5	5	5	5
Activation	(-)S9	(+)S9	(-)S9	(+)S9	(-)S9	(+)S9	(-)S9	(+)S9
Strain	TA 97	TA 97	TA 98	TA 98	TA 100	TA 100	TA 102	TA 102
Concentration in µg/plate
0.00	167 ± 7	± 214 16	± 20 6	26 ± 5	106 ± 12	105 ± 7	179 ±15	248 ±18
10.00	174 ± 10	± 203 23	± 21 3	25 ± 8	116 ± 6	106 ± 3	196 ±16	265 ±25
31.60	165 ± 7	226 ± 20	19 ±5	28 ± 8	116 ± 15	112 ± 9	177 ±8	247 ±22
100.00	165 ± 9	221 ± 12	14 ±5	26 ± 5	54 ± 10 t	106 ± 6	163 ± 15	239 ± 8
316.00	155 ± 6	221 ± 17	13 ±3	23 ± 5	40 ± 4 t	105 ± 12	108 ± 12	137 ± 16
1000.00	160 ± 6	233 ± 14	13 ±3 t	20 ± 3	42 ± 4 t	81 ± 10	111 ± 15	118 ± 7

t : Toxic effect

Conclusions:

The test substance is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Executive summary:

The test substance was examined for mutagenic activity in the standard plate incorporation as well as in the preincubation versions of the Ames test, performed equivalent to OECD TG 471 and following GLP principles. The following concentrations were used for the Ames standard assay: 15.8, 50, 158, 500 and 1580 µg/plate and the following concentrations for the liquid preincubation assay: 10, 31.6, 100, 316 and 1000 µg/plate. The test substance was dissolved in DMSO. The test substance was tested in all strains currently in general use Salmonella typhimurium TA 1535, TA 1537, TA 1538, 97, TA 98, TA 100 and 102 in presence and absence of a metabolic activation system prepared from the livers of phenobarbital/ß-naphthoflavone-induced rats. Responsiveness of the strains and activity of the metabolic enzymes were verified by including appropriate positive controls in each experiment. Appropriate reference mutagens (sodium azide, 2-aminoanthracene, ICR 191, 2-acetylaminofluorene and mitomycin C) were used as positive controls. Precipitation on the plates was observed at the dose level of 1580 μg/plate in the standard assay and 1000 µg/plate for the preincubation assay.

Results showed that the test substance did not cause an increase of the number of revertant colonies under these test conditions. Some signs of toxic activity (reduced background growth) was seen in the preincubation assay. In strain TA 1538 the negative control (-S9) was rather low so that a doubling of the mutant frequency was found at one intermediate test concentrations. This was however, clearly due to low spontaneous value and cannot be taken as test substance related. Accordingly no increase was found in the preincubation assay.

In conclusion, the test substance is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 Jan 1998 to 18 Jun 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

1983

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5375 (In Vitro Mammalian Chromosome Aberration)

Version / remarks:

1987

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1992

Qualifier:

according to guideline

Guideline:

other: MAFF Japan

Version / remarks:

1985

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

K1

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: The cell line CHO CCL 61 has been used for cytogenetic studies for several years. The stability of the genome of these cells is assessed regularly on the basis of the cytogenetic analysis of control cultures in the course of the cytogenetic studies. It is judged to be adequate for the particular
purpose of cytogenetic studies.

For cell lines:
- Absence of Mycoplasma contamination: The cell cultures were periodically checked for mycoplasma contamination
- Cell cycle length : 12-13 hours

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: The 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 + Penicillin/Streptomycin 100 units/mL/100 µg/mL in 75 cm^2 tissue culture (plastic) flasks. The cultures were incubated at 37°C in a humidified atmosphere containing 5% CO2. The cells were passaged twice weekly.

Cytokinesis block (if used):

Colcemide (0.4 µg/mL)

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9 : Rat liver post-mitochondrial supernatant (S9 fraction) was prepared in advance from male rats (Tif:RAI/SPF)
- Method of preparation of S9 mix: The animals were treated with Aroclor 1254 (500 mg/kg, i.p) 5 days prior sacrifice. The livers were homogenised with 3 volumes of 150 mM KCl. The homogenate was centrifuged at 9000x g for 15 minutes and the resulting supernatant (S9 fraction) was stored at approximately -80 °C for no longer than one year. The protein content of the S9 fraction was 34.72 mg/mL.
- Concentration or volume of S9 mix and S9 in the final culture medium: The S9 fraction was thawed before use, mixed with NADP, isocitric acid and Nutrient Mixture F-12 (S9 mixture, Lit. 7) and added to culture medium. The concentrations in the S9 mixture were as followed: rat liver S9 fraction 150 µL/mL, NADP 31.4 µmol/mL, isocitric acid (trisodium salt) 153 µmol/mL. Unused portions of S9 fraction and 89 mixture were discarded. The S9 mixture was immediately sterilised by filtration through a 0.2 µm filter. The activation mixture was added (1:10) to the medium resulting in a final concentration of the S9 fraction of 1.5% during the treatment

Test concentrations with justification for top dose:

Experiments without metabolic activation:
- 21 hours treatment time: 6.25, 12.50 and 25.00 µg/mL (original experiment) and 12.50, 18.75 and 25.00 µg/mL (confirmatory experiment)
- 45 hours treatment time: 9.38, 12.50 and 18.75 µg/mL

Experiments with metabolic activation:
- 3 hours treatment followed by 18 hours recovery period: 9.77, 19.53 and 39.06 µg/mL (original experiment) and 30.00, 40.00 and 60.00 µg/mL (confirmatory experiment)
- 3 hours treatment followed by 42 hours recovery period: 30.00, 40.00 and 60.00 µg/mL

Vehicle / solvent:

- Solvent used: DMSO

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:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Quadruplicate cultures were prepared for each group in each assay
- Number of independent experiments : Three experiments

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: A series of glass in quadruple culture dishes was seeded with Chinese hamster ovary cells (preparation/passage number:106/3, 106/7, 106/9, 108/6) at a density of at least 1x10^4 cells/mL (21 hours experiments) or 4x10^3 cells/mL (45 hours experiments)
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: The preincubation time before treatment was about 24 hours
- Exposure duration/duration of treatment: Three experiments without metabolic activation and three trials with metabolic activation (twice 21 hours and once 45 hours each).

FOR CHROMOSOME ABERRATION:
- Spindle inhibitor: indicate the identity of mitotic spindle inhibitor used (e.g., colchicine), its concentration and, duration and period of cell exposure. 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, v/v).
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): Whenever possible two hundred well spread metaphase gures with 17 to 21 centromeres from two cultures (100 metaphases per replicate culture) in the vehicle control and in the treated groups were scored. At least fifty metaphases were scored in the positive controls (25 per replicate culture).
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): Slides were air-dried and stained with orcein.

The slides were examined for the following structural aberrations:
- Chromatid and chromosome deletions (including breaks, deletions and fragments), chromatid exchanges (including triradials quadriradials, endfusions, acentric rings), chromosome exchanges (including dicentrics, polycentrics, centric and acentric rings),
- Multiple aberrations: metaphases containing more than 10 aberrations of different types or more than S aberrations of one particular type (excluding gaps), unspecic aberrations: gaps (chromatid- and chromosome),
In addition the frequency of polyploid metaphases (multiples of ‘2n’, > 30 centromers, including endoreduplication gures) was recorded. Using a computerized coordinate reading system attached to the vernier scale on the microscope stage, the coordinates of all metaphases were recorded.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: Mitotic index (MI)

Evaluation criteria:

Criteria for a positive response
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 negative control range) and differs statistically significant from the respective value of the negative control. A concentration- related response should be demonstrable.

Criteria for a negative response
Under the standard conditions, 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 specic aberrations in all treatment groups is less than or equal to 6.0 (based on negative control range) and does not differ statistically significant from the respective value of the negative control.

Exceptions
At the limits of the criteria for a positive or for a negative response or if the criteria for a positive response are only partially fulfilled or if effects are obtained at high concentrations or in the toxic range of the test substance only, the Study Director will decide by experience about the interpretation of the results.

Assay acceptance criteria
The results of the experiments should not be influenced by a technical contamination or a recognized artifact. The quality of the slides should allow, at least to a large the chromosomes to be easily identifiable. In the negative controls the percentage of metaphases showing specific chromosomal aberrations should be less than 6.0 (based on historical negative control range). The results of the positive control experiments should meet the criteria for a positive response. The highest concentration to which cells were exposed in the mutagenicity test
should exert sufficient toxicity (suppression of mitotic activity by 50% or more), represent the limit of solubility of the test substance, or be at least 5 mg/mL.

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-squared test. The nonsignificant 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-squared test for trend was performed modelling all cells in a given experiment as independent (Lit. 8). That is, the individual cell is taken as the experimental unit. Consequently the power of the test is substantially increased, resulting in a rather safe judgement of the observed effects.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- No precipitation was observed in this study

Gene mutation tests in mammalian cells:
- Toxicity: Higher concentrations were not scored due to cytotoxicity. The highest concentration of 25.00 µg/mL selected for chromosome analysis in the first experiment of the original study (without metabolic activation, 21 hours treatment) caused 49.2% suppression of mitotic activity. At the next higher concentration of 50.00 µg/mL mitotic activity was nearly completely suppressed due to toxicity. The highest concentration selected for chromosome analysis (39.06 µg/mL in the second experiment of the original study (with metabolic activation, 3 hours treatment/18 hours recovery) caused 6.2% suppression of mitotic activity. The next higher concentration of 78.13 µg/mL suppressed mitotic activity by 89.3% and was not scored because of excessive toxicity. In the first experiment of the confirmatory study (without metabolic activation, 21 hours treatment) the highest concentration selected for chromosome analysis (25.00 µg/mL caused 74.7% suppression of mitotic activity. The next higher concentration (37.50 µg/mL suppressed mitotic activity by 91.6% and was not scored due to strong toxicity. In the second confirmatory experiment (with metabolic activation, 3 hours treatment/18 hours recovery) the highest concentration selected for chromosome analysis (60.00 µg/mL caused 66.8% suppression of mitotic activity. The next higher concentration (80.00 µg/mL suppressed mitotic activity by 90.4%. The highest concentration selected for chromosome analysis (18.75 µg/mL in the third experiment of the confirmatory study (without metabolic activation, 45 hours treatment) caused 64.2% suppression of mitotic activity. The next higher concentration (25.00 µg/mL suppressed mitotic activity by 92.6%. In the fourth confirmatory experiment (with metabolic activation, 3 hours treatment/42 hours recovery) the highest concentration selected for chromosome analysis (60.00 µg/mL caused 62.1% suppression of mitotic activity. At the next higher concentration (80.00 µg/mL not enough cells for scoring were present on the slides due to toxicity.

- Controls: The treatment of the cultures with mitomycin-C and cyclophosphamide was followed by a high incidence of specific chromosomal aberrations in the experiments one and two of the original study (74.0% and 72.0%, respectively) and in the experiments one and two of the confirmatory study (68.0% and 56.0%, respectively).

In both experiments performed without and with metabolic activation no statistically significant increase in the number of metaphases containing specific chromosomal aberrations was observed for the test substance (see tables in 'Any other information on results incl. tables').

Table 1. Values printed bold represented a statistically significant difference from the control

Without S9-mix
dose μg/ml	21 h continuous treatment
	MI frequency % of control	% cells with specific aberr
0	100	2.5
6.25.	96	3.5
12.5	90	1
25	51	3.5
pos. c.		74

 

With S9-mix
dose μg/ml	3 h treatment/ 18 hr recovery
	MI frequency % of control	% cells with specific aberr
0	100	2.0
9.77	66	2.5
19.5	50	2.0
39	94	3.0
pos. c.		72.0

Without S9-mix
dose μg/ml	21 h continuous treatment
	MI frequency % of control	% cells with specific aberr
0	100	4.0
12.5	72	4.5
18.75	77	3.5
25	25	3.5
pos. c.		68

 

With S9-mix
dose μg/ml	3 h treatment/ 18 hr recovery
	MI frequency % of control	% cells with specific aberr
0	100	2.5
30	86	4.0
40	77	4.0
60	33	5.5
pos. c.		56

 

Without S9-mix
dose μg/ml	45 h continuous treatment
	MI frequency % of control	% cells with specific aberr
0	100	2.5
9.38	74	3.5
12.5	37	4.0
18.75	36	3.5

 

With S9-mix
dose μg/ml	3 h treatment/ 42 hr recovery
	MI frequency % of control	% cells with specific aberr
0	100	3.0
30	107	4.0
40	98	1.0
60	38	5.0

 

Conclusions:

It is concluded that under the given experimental conditions, no evidence of clastogenic effects was obtained in Chinese hamster ovary cells in vitro with the test substance

Executive summary:

The objective of this in vitro assay was to evaluate the ability of the test substance to induce chromosomal aberrations in Chinese hamster ovary (CHO) cells with and without metabolic activation, according to GLP principles and OECD TG 473. The test substance was dissolved in DMSO. The following concentrations were used in experiments without metabolic activation: 6.25, 12.5 and 25.0 µg/mL (21 hours treatment time, original experiment) and 12.5, 18.75 and 25.0 µg/mL (21 hours treatment time, confirmatory experiment) and 9.38, 12.5 and 18.75 µg/mL (45 hours treatment time). Higher concentrations were not scored due to cytotoxicity. Mitomycin C (0.2 µg/mL) was used as a positive control in the 21 hours experiments. The following concentrations were used with metabolic activation and 3 hour treatment: 9.77, 19.53 and 39.06 µg/mL (original experiment, followed by 18 hours recovery period), 30, 40 and 60 µg/mL (confirmatory experiment, followed by 18 hours recovery period) and 30, 40 and 60 µg/mL (followed by 42 hours recovery period). Higher concentrations were not scored due to cytotoxicity. Cyclophosphamide (20.0 µg/mL) was used as a positive control in the 3 hours/18 hours experiments.

Results showed that in both experiments performed without and with metabolic activation no statistically significant increase in the number of metaphases containing specific chromosomal aberrations was observed. The incidence of aberrant cells was within the historical control range at all doses assessed. No precipitation was observed in this study.

It is concluded that under the given experimental conditions, no evidence of clastogenic effects was obtained in Chinese hamster ovary cells in vitro with the test substance.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 Mar 1982 to 12 May 1982

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:

1984

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

Hypoxanthine guanine phosphoribosyl transferase (HGPRT) locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Established cell line V79 clone 66 A/4

MEDIA USED
- Type and composition of media: HAM F10, 15% fetal bovine serum, penicillin, streptomycin, kanamycin.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9 : Male albino SPF mice were pre-treated with 500 mg Aroclor 1254 per kg body weight. Five days after treatment, the animals were killed by cervical dislocation.
- Method of preparation of S9 mix: The livers were removed, homogenised and centrifuged for 10 minutes at 5500 g and another 20 minutes at 900 g. The supernatant fraction(S-9 mix) was pooled and stored at -196 °C for about 5 months.
- Method of preparation of CM- S9 mix: The CM S-9 mix containing 10% S-9 fraction and 4 mM NADP, 5mM glucose-6- phosphate, 50 mM Na2HPO4, 30 mM KCL, 10 mM MgCl2 and 10 mM CaCl2 as cofactors was prepared according to Tan and Hsie (1981)

Test concentrations with justification for top dose:

Experiment without metabolic activation: 1, 5, 25 µg/mL of the test substance
Experiment with metabolic activation: 25, 50, 100 µg/mL of the test substance

Vehicle / solvent:

- Solvent used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF TREATMENT/EXPOSURE:
- Cell density at seeding: 10^6 cells plated in culture flask and incubated.
- Test substance added in medium (Hank's balanced salt solution)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 24 hour
- Exposure duration/duration of treatment: Exposition time was either 16 hours when using no metabolic activation or 5 hours when using CM S-9 mix as metabolic activation system.

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): Cells were incubated in growth medium for five days and subcultured after 2-3 days to keep them in logarithmic growth phase.
- Selection time (if incubation with a selective agent): To select for 8-azaguanine resistant cells, 10^5 cells were plated per dish (Ø50 mm) and incubated in growth medium substituted with 20 µg 8-azaguanine per mL as selective agent.
- Fixation time (start of exposure up to fixation or harvest of cells): Because of low stability of 8-azaguanine in solution it was resubstituted every 2 to 3 days during the first week of incubation. Fixation and staining of 8-azaguanine resistant cells was carried out two weeks after starting the selection.

OTHER:
- Survival of cells were determined at the beginning as well as the end of the phenotypic expression phase because of recovering of the cells during the period of time. Four dishes per dose were used. Clones were scored macroscopically.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

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
- No precipitation was observed

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements: Cytotoxicity observed at dose level: 25 μg/ml (-S9) and 50 μg/ml and above (+S9)
- Sensitivity of the test system was shown by using the well-known mutagens in both presence and absence of a metabolic activation system.

Conclusions:

It is concluded that under these experimental conditions described in the report, the test substance did not induce point mutations in mammalian cells in vitro.

Executive summary:

The test substance was evaluated for induction of point mutations at the hypoxanthine guanine phosphoribosyl transferase (HGPRT) locus in the established cell line V49, derived from Chinese hamster lung cells according to OECD TG 476 and GLP principles. The cells were treated with 1,5 and 25 µg/mL for 16 hours without metabolic activation and 25, 50 and 100 µg/mL of the test substance with metabolic activation for five hours using a post-mitochondrial supernatant fraction (S-9 mix) of mouse liver homogenate. No precipitation was observed.

Results showed that the test substance did not induce 8-azaguanine resistant cells without and with metabolic activation. Concentration of 50 µg/mL and above were cytotoxic when treating V79 cells with metabolic activation and 25 µg/mL without metabolic activation. Sensitivity of the test system was shown using ethyl methanesulfonate and 2-acet-aminofluorene (200 µg/mL) as reference substances.

It is concluded that under these experimental conditions described in the report, the test substance did not induce point mutations in mammalian cells in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

- In vivo micronucleus test in mice, negative, according to OECD TG 474, Ogorek 1996

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:

13 Sep 1996 to 29 Nov 1996

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:

1983

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1992

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1987

Qualifier:

according to guideline

Guideline:

other: MITI Japan

Version / remarks:

1987

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

Tif:MAGf

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 7 to 8 weeks
- Weight at study initiation: 23-28 g for females and 27-38 g for males.
- Housing: The animals were housed 5/cage (reserve animals: 3/cage) and marked with color pens.
- Diet: Pelleted standard diet, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.5
- Humidity (%): 41-48
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 13 Sep 1996 to 29 Nov 1996

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: Arachis oil
- Amount of vehicle: 10 mL/kg body weight
- Justification for choice of vehicle: Another in vivo study performed with the test substance has revealed arachis oil to be the best suited vehicle, yielding an applicable suspension at the dose level of 5000 mg/kg

Duration of treatment / exposure:

Negative control + high dose: 16, 24 and 48 hours
Intermediate dose + low dose + positive control: 24 hours

Frequency of treatment:

Once

Post exposure period:

16, 24 or 48 hours

Dose / conc.:

1 250 mg/kg bw/day (actual dose received)

Remarks:

Low dose

Dose / conc.:

2 500 mg/kg bw/day (actual dose received)

Remarks:

Intermediate dose

Dose / conc.:

5 000 mg/kg bw/day (actual dose received)

Remarks:

High dose

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Route of administration: Oral by stomach tube, 10 mL/kg body weight
- Doses: 64 mg/kg dissolved in bidistilled water

Tissues and cell types examined:

Subsequently femoral bone marrow cells were prepared and polychromatic erythrocytes were scored for micronuclei

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
A tolerability test was not performed, since LD50- data obtained in another in vivo study performed (p.o.) on mouse with the test substance indicated a maximum tolerated dose (MTD) > 5000 mg/kg. Signs of toxicity were recorded hourly for the first few hours after application and once the following days. Body weight were recorded daily.

TREATMENT AND SAMPLING TIMES: The animals were sacrificed by CO2 gas. Bone marrow was harvested in fetal calf serum from the shafts of both femurs, centrifuged and resuspended in fetal calf serum. Smears prepared from this suspension were stained with May-Grünwald/Giemsa solution and mounted. See table 1 in ''any other information on material and methods''.

DETAILS OF SLIDE PREPARATION:
Micronuclei are uniform, darkly stained, more or less round bodies in the cytoplasm of erythrocytes. Inclusions which are reflective, improperly shaped or stained, or which are not in the focal plain of the cell are judged to be artifacts and are not scored as micronuclei. Cells containing more than one micronucleus are only counted once. Prior to analysis the slides were coded. The slides of five animals/sex/dose, showing good between mature and polychromatic erythrocytes, were scored by a laboratory technician. The incidence of micronucleated polychromatic erythrocytes (MNPCE) among at least 2000 polychromatic erythrocytes (PCB), and the ratio of PCB to normochormatic erythrocytes (NCE) among a total of at least 1000 erythrocytes was determined for each slide.

OTHER:
Analytical chemistry: To confirm that the animals were actually exposed to the intended doses of the test substance and to confirm the stability of the in vehicle used substance in the vehicle used, determinations of the applied concentrations of the test substance in the vehicle used performed by the analytical unit. These determinations were performed with the 16 h and 24/48 h samples of the test substance prepared for application in the micronucleus test, representing the high dose (5000 mg/kg) and with the 24h sample prepared which represents the low dose (1250 mg/kg).

Evaluation criteria:

The results of the experiments were evaluated with respect to the mean number pf PCEs with micronuclei. The groups compared differed by treatment, sampling time and sec of the animals. The data from females and males were pooled for evaluation. In case of significant sex differences data have to be evaluated for each sex separately in addition.

Statistics:

The significance of differences was assessed by the Chi-Squared-Contingency-Test (F=1, p<0.05).

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

At all sampling times (16, 24 and 48 hours) there was no statistically significant increase in the number of micronucleated polychromatic erythrocytes in the animals treated with the respective doses of the test substance as compared with the negative control animals. The percentages of micronucleated PCEs found in the negative controls were within the range of the negative control (0.05 ± 0.04). In the positive control (24 hours) the percentage of micronucleated cells within polychromatic erythrocytes was clearly increased. The mean percentage of micronucleated PCEs was 1.85. In comparison with the negative control (0. 06) this value15 highly significant (p<0.05) and within the range of the historical positive control (1.40± 0.80).

Table 1. Micronuclues test

Overall mean percentages of micronucleated PCEs
Treatment time	High dose (GD) 5000 mg/kg	Intermediate dose (ID) 2500 mg/kg	Low dose (LD) 1250 mg/kg	Positive control (CPA)	Negative control (vehicle
16 hours	0.07				0.08
24 hours	0.06	0.08	0.08	1.85	0.06
48 hours	0.09				0.06

Conclusions:

It is concluded that under the given experimental conditions, no evidence for clastogenic or aneugenic effects was obtained in mice treated with the test substance

Executive summary:

 

The test substance was investigated for clastogenic (and/or aneugenic) on mouse bone marrow cells in vivo according to OECD TG 474 and GLP principles. The test substance was administered once by oral gavage to groups of 5 male and 5 female Tif: MAGf (SPF) mice at doses of 5000, 2500 and 1250 mg/kg. Additional groups of animals were treated with the vehicle alone (arachis oil, 10mL/kg body weight) or with the positive control cyclophosphamide (64 mg/kg body weight). From the high dose group and from the negative control group animals were sacrificed 16, 24 and 48 hours thereafter. From the intermediate and the low dose group and from the positive control group animals sacrificed 24 hours after application. Subsequently femoral bone marrow cells were prepared and polychromatic erythrocytes were scored for micronuclei.

Results showed that none of the animals treated with the test material showed symptoms of toxicity. In all dosage groups assessed at the different periods post treatment, no statistically significant increase in the number of micronucleated polychromatic erythrocytes was observed when compared with the respective negative control group. In the positive control group the percentage of micronucleated cells with polychromatic erythrocytes was clearly increased.

It is concluded that under the given experimental conditions, no evidence for clastogenic or aneugenic effects was obtained in mice treated with the test substance

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

AMES, Gocke 1988 - Key

The test substance was examined for mutagenic activity in the standard plate incorporation as well as in the preincubation versions of the Ames test, performed equivalent to OECD TG 471 and following GLP principles. The following concentrations were used for the Ames standard assay: 15.8, 50, 158, 500 and 1580 µg/plate and the following concentrations for the liquid preincubation assay: 10, 31.6, 100, 316 and 1000 µg/plate. The test substance was dissolved in DMSO. The test substance was tested in all strains currently in general use Salmonella typhimurium TA 1535, TA 1537, TA 1538, 97, TA 98, TA 100 and 102 in presence and absence of a metabolic activation system prepared from the livers of phenobarbital/ß-naphthoflavone-induced rats. Responsiveness of the strains and activity of the metabolic enzymes were verified by including appropriate positive controls in each experiment. Appropriate reference mutagens (sodium azide, 2-aminoanthracene, ICR 191, 2-acetylaminofluorene and mitomycin C) were used as positive controls. Precipitation on the plates was observed at the dose level of 1580 μg/plate in the standard assay and 1000 µg/plate for the preincubation assay.

Results showed that the test substance did not cause an increase of the number of revertant colonies under these test conditions. Some signs of toxic activity (reduced background growth) was seen in the preincubation assay. In strain TA 1538 the negative control (-S9) was rather low so that a doubling of the mutant frequency was found at one intermediate test concentrations. This was however, clearly due to low spontaneous value and cannot be taken as test substance related. Accordingly no increase was found in the preincubation assay.

In conclusion, the test substance is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

In vitro gene mutation assay in Saccharomyces cerevisiae, Chételat 1982 - Supporting

The diploid yeast strain Saccharomyces cerevisiae D7 was used for the evaluation of DNA damage of the test substance according to OECD TG 480 and GLP principles. With strain D7, a broad spectrum of damages in the genetic material can be detected: mutation induction as well as induction of mitotic crossing over and gene conversions are covered by this test. Furthermore, S. cerevisiae D7 has the advantage of being a diploid eukaryotic organism. The following concentration were used: 0.017, 0.04, 0.17 and 0.40 mg/mL for 3 hours treatment. DMSO was used as solvent.

Results showed, no miotic crossing over at the ade 2 locus, no mitotic gene conversion at the rtp 5 locus and no reverse mutation at the ile 1 locus with and without metabolic activation. The positive control cyclophosphamide showed a positive response of S. cerevisiae D7 to this mutagen and the activity of the S9 mix. At concentrations of 0.715, 2.45 or 7.5 mg cyclophosphamide per mL incubation mix the number of convertants/10^5 survivors, the number of revertrants/10^6 survivors and the percentage of coloured colonies are higher dan the control values without cyclophosphamide or without S9 mix activation. No precipitation was observed and no cytotoxicity was observed up to and including the dose level of 0.40 mg/mL.

In conclusion, the test substance did not induce DNA damage under the described in vitro conditions.

In vitro chromosome aberration assay, Ogorek 1988 - Key

The objective of this in vitro assay was to evaluate the ability of the test substance to induce chromosomal aberrations in Chinese hamster ovary (CHO) cells with and without metabolic activation, according to GLP principles and OECD TG 473. The test substance was dissolved in DMSO. The following concentrations were used in experiments without metabolic activation: 6.25, 12.5 and 25.0 µg/mL (21 hours treatment time, original experiment) and 12.5, 18.75 and 25.0 µg/mL (21 hours treatment time, confirmatory experiment) and 9.38, 12.5 and 18.75 µg/mL (45 hours treatment time). Higher concentrations were not scored due to cytotoxicity. Mitomycin C (0.2 µg/mL) was used as a positive control in the 21 hours experiments. The following concentrations were used with metabolic activation and 3 hour treatment: 9.77, 19.53 and 39.06 µg/mL (original experiment, followed by 18 hours recovery period), 30, 40 and 60 µg/mL (confirmatory experiment, followed by 18 hours recovery period) and 30, 40 and 60 µg/mL (followed by 42 hours recovery period). Higher concentrations were not scored due to cytotoxicity. Cyclophosphamide (20.0 µg/mL) was used as a positive control in the 3 hours/18 hours experiments.

Results showed that in both experiments performed without and with metabolic activation no statistically significant increase in the number of metaphases containing specific chromosomal aberrations was observed. The incidence of aberrant cells was within the historical control range at all doses assessed. No precipitation was observed in this study.

It is concluded that under the given experimental conditions, no evidence of clastogenic effects was obtained in Chinese hamster ovary cells in vitro with the test substance.

In vitro chromosomal aberration assay, Hugentobler 1982 - Supporting

The test substance was evaluated for a potential induction of chromosomal aberrations in vitro in human peripheral blood lymphocytes in the presence and in the absence of metabolic activation according to GLP principles. For metaphase analysis without metabolic activation, the human lymphocytes cultures were treated with 0.4, 2.0 and 4.0 µg of the test substance per mL medium for 24 hours. For metaphase analysis with metabolic activation by an Aroclor 1254-inducd S-9 liver homogenate of albino rats, the human lymphocytes were treated with 1.0; 5.0 and 10 µg of the test substance per mL medium for 2 hours. The test substance was dissolved in DMSO.

Results showed that there was no increase in the amount of detectable numerical or structural chromosomal aberrations when compared with the solvent control treated cultures, neither in the presence nor absence of metabolic activation was observed. Higher concentrations of the test substance could not be test due its cytotoxicity. Sensitivity of the test systems could be demonstrated with the positive control compound bleomycin as a direct acting mutagen and cyclophosphamide as indirect acting mutagen. No precipitation was observed in this study.

In conclusion, the test substance did neither induce chromosomal aberrations in human lymphocytes in vitro at concentrations of 0.4; 2.0 and 4.0 µg/mL in the absence of metabolic activation, nor at concentrations of 1.0; 5.0 and 10 µg/mL in the presence of metabolic activation.

In vitro chromosome aberration assay, Dresp 1989 - Supporting

The test substance was assessed for its ability to induce chromosomal aberrations in human peripheral blood lymphocytes in vitro under GLP principles. In a previous experiment (Hugentobler 1982) cells had been treated with the test substance (dissolved in DMSO) at final concentrations of 0.4, 2.0 and 4.0 µg/mL for 24 h in the absence and of 1.0 and 5.0 and 10 µg/mL for 2 h in the presence of an Aroclor- induced rat S9 liver homogenate fraction. As this experiment was criticised for not evaluating dose levels which cause cytotoxic effects, the present supplementary study was carried out. A dose range from 25- 150 µg/mL was tested but only doses of 25 µg/mL in the absence and 25 and 50 µg/mL in the presence of a metabolic activation system respectively could be evaluated because the higher doses turned out to be toxic.

Results showed that exposure of the lymphocytes to the test substance for 2 h did not increase the rate of structural chromosome aberrations neither in the presence nor in the absence of a metabolic activating system. The use of the Bromodesoxyuridine (brdUrd) technique together with the fact that human peripheral blood lymphocytes present an only weakly synchronised cell population ascertains that cells analysed after such a experimental design represent a mixture of cells treated in G1, S- or G2- phase of the cell cycle. The sensitivity of the test system and the activity of the metabolic activation were demonstrated by using the direct acting mutagen bleomycin-sulfate and the promutagen cyclophosphamide as positive controls. Both substances increased significantly the rate of structural chromosomal aberrations. No precipitation was observed precipitation to this study.

In conclusion, under the described conditions, neither the test substance nor any of its metabolites formed by rat liver enzymes induced chromosomal damage in human peripheral lymphocytes at doses up to toxicity levels.

In vitro gene mutation assay, Strobel 1982 - Key

The test substance was evaluated for induction of point mutations at the hypoxanthine guanine phosphoribosyl transferase (HGPRT) locus in the established cell line V49, derived from Chinese hamster lung cells according to OECD TG 476 and GLP principles. The cells were treated with 1,5 and 25 µg/mL for 16 hours without metabolic activation and 25, 50 and 100 µg/mL of the test substance with metabolic activation for five hours using a post-mitochondrial supernatant fraction (S-9 mix) of mouse liver homogenate. No precipitation was observed.

Results showed that the test substance did not induce 8-azaguanine resistant cells without and with metabolic activation. Concentration of 50 µg/mL and above were cytotoxic when treating V79 cells with metabolic activation and 25 µg/mL without metabolic activation. Sensitivity of the test system was shown using ethyl methanesulfonate and 2-acet-aminofluorene (200 µg/mL) as reference substances.

It is concluded that under these experimental conditions described in the report, the test substance did not induce point mutations in mammalian cells in vitro.

In vivo micronucleus test, Ogorek 1996 - Key

The test substance was investigated for clastogenic (and/or aneugenic) on mouse bone marrow cells in vivo according to OECD TG 474 and GLP principles. The test substance was administered once by oral gavage to groups of 5 male and 5 female Tif: MAGf (SPF) mice at doses of 5000, 2500 and 1250 mg/kg. Additional groups of animals were treated with the vehicle alone (arachis oil, 10mL/kg body weight) or with the positive control cyclophosphamide (64 mg/kg body weight). From the high dose group and from the negative control group animals were sacrificed 16, 24 and 48 hours thereafter. From the intermediate and the low dose group and from the positive control group animals sacrificed 24 hours after application. Subsequently femoral bone marrow cells were prepared and polychromatic erythrocytes were scored for micronuclei.

Results showed that none of the animals treated with the test material showed symptoms of toxicity. In all dosage groups assessed at the different periods post treatment, no statistically significant increase in the number of micronucleated polychromatic erythrocytes was observed when compared with the respective negative control group. In the positive control group the percentage of micronucleated cells with polychromatic erythrocytes was clearly increased.

It is concluded that under the given experimental conditions, no evidence for clastogenic or aneugenic effects was obtained in mice treated with the test substance

Justification for classification or non-classification

Based on the available data classification for genetic toxicity is not warranted in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.