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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Non mutagenic

Link to relevant study records

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Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From May 02 to August 30, 1995
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The test was conducted by means of Read Across approach. The reliability of the source study report is 1. Further information was attached at section 13
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
not specified
Qualifier:
according to
Guideline:
EPA OTS 798.5300 (Detection of Gene Mutations in Somatic Cells in Culture)
Deviations:
not specified
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
not specified
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
Mutagenic effects are detected by the appearance of cells resistant to 6-TG
Species / strain / cell type:
other: CHINESE HAMSTER CELLS V79, clone 65/3
Details on mammalian cell type (if applicable):
Origin: Dr. D. Wild, Freiburg, Germany.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Post mitochondrial supernatant (S9 fraction) from Aroclor 1254 induced rat liver
Test concentrations with justification for top dose:
Cytotoxicity test
Range with metabolic activation:
0.24 to 500.0 ug/ml
Range without metabolic activation:
0.24 to 500.0 ug/ml
Mutagenicity test
Original experiment:
Range with metabolic activation:
18.52 to 500.0 ug/ml
Range without metabolic activation:
11.11 to 300.0 ug/ml
Confirmatory experiment:
Range with metabolic activation:
62.5 to 500.0 ug/ml
Range without metabolic activation:
37.5 to 300.0 ug/ml
Vehicle / solvent:
Dimethylsulfoxide (DMSO)
Untreated negative controls:
yes
Remarks:
vehicle control
Negative solvent / vehicle controls:
yes
Remarks:
Dimethylsulfoxide (DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-Nitrosodimethylamine
Remarks:
With metabolic activation
Untreated negative controls:
yes
Remarks:
vehicle control
Negative solvent / vehicle controls:
yes
Remarks:
Dimethylsulfoxide (DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Ethylmethansulfonate
Remarks:
Without metabolic activation
Details on test system and experimental conditions:
Preliminary cytotoxicity test
A cytotoxicity test was performed on V79 cells as a preliminary test to determine the highest concentration of the test substance to be applied in the mutagenicity assay. For each concentration and the untreated controls, 2.5x10s V79 cells were seeded in 5 ml growth medium into a 25 cm2 tissue culture flask and incubated overnight. The cultures were exposed to the test substance for five hours in the presence and for 21 hours in the absence of a metabolic activation system. In the two parts of the experiment, 12 concentrations of the test substance and two vehicle (DMSO) controls were tested. The highest concentration was determined in a preliminary solubility test. Lower concentrations were prepared by serial dilution by a factor of 0.5. The treatment was terminated by washing the cultures with phosphate buffered saline (PBS). Compound-induced cytotoxicity was estimated by cloning efficiency immediately after treatment. The cultures were counted and diluted so that 100 cells were seeded per 9.6 cm2 in 3 ml of growth medium. After seven to eight days of growth the cultures were fixed and stained with Giemsa and the surviving colonies determined with the aid of an electronic colony counter (Artek Counter®, Fisher Scientific) or by the naked eye. The sensitivity of the colony counter was adjusted to detect clones of about twenty or more cells. The concentration to be selected as the highest for the mutagenicity assay was the one causing about 50-90% reduction of viable cells in comparison with the mean of the two negative controls or corresponds to the substance's solubility limit (precipitates in the culture).
Mutagenicity test
Depending on the toxicity of the test compound 2.5-5.0x10ʌ6 cells of passage 25 (original experiment) and passage 25 (confirmatory experiment) were plated in 30 ml growth medium into 175 cm2 flasks and incubated overnight. The growth medium was replaced for five hours by 27 ml treatment medium and 3.0 ml S9 activation mixture, or for 21 hours by 30 ml treatment medium alone. In each assay, cultures were treated in duplicate with four test chemical concentrations, a positive and a negative (DMSO) control. In the non-activated part of the experiment, the positive control was the ultimate mutagen Ethylmethansulphonate (EMS) at a concentration of 0.3 ul/ml. In the part with metabolic activation the positive control was the promutagen N-Nitrosodimethylamine (DMN) at a concentration of 1.0 ul/ml. The treatment was terminated by washing the cell layer extensively with PBS. After washing, the cells were suspended by trypsinisation, pelleted, resuspended in fresh growth medium and counted with a haemocytometer or electronic coulter counter (Coulter Counter®, Model ZM), diluted with fresh growth medium and replated into flasks at 2x10ʌ6 cells. The cultures were incubated at 37°C for seven to eight days during which the cells could recover and divide to express the mutant phenotype. The cultures were subcultered after the second or third day transferring 2x10ʌ6 cells to a fresh flask to maintain exponential growth during the expression phase.
In parallel cytotoxicity of the compound was estimated from the cloning efficiency immediately after treatment. The counted cell suspension of each concentration level was further diluted so that 100 cells were seeded per 9.6 cm2 in 2.5 ml of growth medium and incubated at 37 °C. The number of colonies which developed within seven to eight days in these cultures reflected the viability at the end of the treatment (survival values).
At the end of the expression period the cultures were trypsinised, pelleted, resuspended in fresh growth medium and counted with a haemocytometer or electronic coulter counter (Coulter Counter®, Model ZM). The cell suspension of each culture was diluted with fresh growth medium and an aliquot replated into four flasks (75 cm2 growth area) each containing 2x10ʌ6 cells for the mutant selection. The high-density cultures were subjected to the mutant selection procedure by supplementing the growth medium with 8 ug/ml 6-thioguanine (6-TG). Only cells mutated at the hprt locus could survive the 6-thioguanine treatment. The number of colonies formed in these flasks during the following days reflected the overall number of mutations induced by the treatment with the test substance or the mutagen (positive control). After seven to eight days incubation at 37 °C, the cultures were fixed and stained with Giemsa. The mutant clones were counted with the naked eye.
In parallel the viability at the end of the expression period was estimated from the cloning efficiency. The remaining cell suspensions from the various expression cultures were further diluted such that 100 cells were seeded per 9.6 cm2 in 2.5 ml of growth medium and were incubated at 37 °C. The number of colonies which developed within these low-density cultures reflected the viability at the end of the expression period (viability values).
Evaluation criteria:
Assay acceptance criteria
• The results of the experiments should not be influenced by a technical error, contamination or a recognized artifact.
• From each experiment, at least three concentrations of the test substance, one positive and one solvent control should be evaluated.
• The mutant frequency of the solvent controls (spontaneous mutant frequency) should not exceed 35xl0-6.
• The positive control should fulfil the criteria for a mutagenic substance.
• The highest concentration of the test substance applied in the mutagenicity test should either reduce the viable cells by about 50-90% or correspond to the test substance's solubility limit (precipitates in the culture). In case of non-toxic freely soluble compounds the highest tested concentration
will be 5 mg/ml. In special cases the highest concentration can be determined by the sponsor.
Assay evaluation criteria
All mutant frequencies are normalized to a virtual cloning efficiency of 100 % at the end of the expression period. If the cloning efficiency of the viability cultures is lower than 15 %, the corresponding mutant frequency is usually not calculated, owing to the high statistical insignificance of the
result. For every concentration a mean mutant factor, which is defined as the ratio of the mean mutant frequencies of the treated cultures with the mean mutant frequencies of the solvent control cultures, will be calculated.
Statistics:
Assessment of statistical significance of mutation frequency
Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines.  
Species / strain:
other: CHINESE HAMSTER CELLS V79, clone 65/3
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

Cytotoxicity test

A preliminary range finding test was run assessing cytotoxicity. The test substance was tested at concentrations up to 500.0 µg/ml. Higher concentrations could not be applied due to solubility limitations in the culture vehicle. In the part with metabolic activation, at the highest concentration of 500.0 µg/ml an acute growth inhibiting effect of 40.2 % could be seen. Without metabolic activation treatment with the test substance proved growth inhibiting by 99.98 % at the concentration of 500.0 µg/ml. The next lower concentration of 250.0 µg/ml revealed an acute inhibition of growth of 70.1 %. Accordingly, 500.0 µg/ml with and 300.0 µg/ml without metabolic activation were chosen as highest concentrations for the first mutagenicity assay.

Mutagenicity test with metabolic activation

The original experiment was performed at the following concentrations: 18.52, 55.56, 166.67 and 500.0 µg/ml. No toxicity was found at any concentration. In the confirmatory experiment the concentrations applied were 62.5, 125.0, 250.0 and 500.0 µg/ml. The highest concentration revealed a mean acute growth inhibition of 33.7%. N-Nitrosodimethylamine (DMN, 1.0 µl/ml) was used as positive control. In both experiments comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no significant increase of the mutant frequencies as determined by the screening with 6-Thioguanine (6-TG).

Mutagenicity test without metabolic activation

The original experiment was performed at the following concentrations: 11.11, 33.33, 100.0 and 300.0 µg/ml. The mean growth inhibition values found at the highest concentration after treatment and expression were 82.0% and 19.5% respectively. In the confirmatory experiment the concentrations applied were 37.5, 75.0, 150.0 and 300.0 µg/ml. The highest concentration revealed a mean acute growth inhibitory effect of 69.3%. The mean growth inhibition after the expression period was 24.0%. Ethylmethansulfonate (EMS, 0.3 µl/ml) was used as positive control. In both experiments comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no significant increase of the mutant frequencies as determined by the screening with 6-TG.

Conclusions:
Non mutagenic
Executive summary:

Method

The test substance was tested for its mutagenic effect by the gene mutation test with chines hamster cells V79, according to OECD Guideline 476.

The test system allows the detection of base-pair substitutions, frameshift mutations and deletions induced by the test substance or by its metabolites. Mutagenic effects are manifested by the appearance of cells resistant to 6-TG and can be quantified by comparison of the numbers of 6-TG resistant colonies in the treated and control cultures. To ensure that any mutagenic effect of metabolites of the test substance found in mammals is also detected, an experiment is performed, in which the metabolic turnover of the test material is simulated in vitro by the addition of an activation mixture to the cell cultures containing rat-liver post mitochondrial supernatant (S9 fraction) and cofactors.

Observation

The original experiment was performed at the following concentrations: 11.11, 33.33, 100.0 and 300.0 µg/ml. The mean growth inhibition values found at the highest concentration after treatment and expression were 82.0 % and 19.5 % respectively. In the confirmatory experiment the concentrations applied were 37.5, 75.0, 150.0 and 300.0 µg/ml. The highest concentration revealed a mean acute growth inhibitory effect of 69.3 %. The mean growth inhibition after the expression period was 24.0 %. Ethylmethansulfonate (EMS, 0.3 µl/ml) was used as positive control.

Results

Non mutagenic

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2015
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The test was conducted by means of Read Across approach. The reliability of the source study report is 1. Further information was attached at section 13
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Principles of method if other than guideline:
None
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable.
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
Exponentially growing CHO-K1 cells were seeded in complete medium (McCoy's 5A medium containing 10% fetal bovine serum, 1.5 mM L-glutamine, 100 units/mL penicillin, 100 μg/mL streptomycin and 2.5 μg/mL Amphotericin B) for each treatment condition at a target of 5 x 105 cells/culture. The cultures were incubated under standard conditions (37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2 in air) for 16-24 hours.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
The S9 liver microsomal fraction
Test concentrations with justification for top dose:
dose levels 2.5 to 300 μg/mL in the non-activated 4-hour exposure group, and at doses 5 to 200 μg/mL in the S9-activated 20-hour exposure group
Vehicle / solvent:
Dimethyl formamide (DMF)
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:
Chromosome Aberration Assays
Seven to nineteen dose levels were tested using duplicate cultures at appropriate dose intervals based on the toxicity profile of the test substance. Precipitation of test substance dosing solution in the treatment medium was determined using unaided eye at the beginning and conclusion of treatment. The highest dose level evaluated for chromosome aberrations was either based on cytotoxicity (cell growth inhibition relative to the vehicle control) or visible precipitate at the conclusion of the treatment period. Two or three additional dose levels were included in the evaluation.

Treatment of Target Cells (Preliminary Toxicity Test and Chromosome Aberration Assay)
The pH at the highest test substance concentration was measured prior to dosing using a pH meter or test strips. Treatment was carried out by re-feeding the cultures with 5 mL complete medium for the non-activated exposure or 5 mL S9 mix (4 mL culture medium + 1 mL of S9 cofactor pool) for the S9-activated exposure, to which was added 50 μL of test substance dosing solution or vehicle alone. Untreated controls were re-fed with 5 mL complete medium for the non-activated exposure or 5 mL S9 mix (4 mL culture medium + 1 mL of S9 cofactor pool) for the S9-activated exposure. In the definitive assay, positive control cultures were resuspended in either 5 mL of complete medium for the non-activated studies, or 5 mL of the S9 reaction mixture (4 mL serum free medium + 1 mL of S9 cofactor pool), to which was added 50 μL of positive control in solvent.
After the 4 hour treatment period in the non-activated and the S9-activated studies, the treatment medium were aspirated, the cells were washed with calcium and magnesium free phosphate buffered saline (CMF-PBS), re-fed with complete medium, and returned to the incubator under standard conditions.
For the chromosomal aberration assay only, two hours prior to cell harvest, cultures with visible precipitate were washed with CMF-PBS to avoid precipitate interference with cell counts, and then Colcemid® was added to all cultures at a final concentration of 0.1 μg/mL. Thus the treatment time for the precipitating dose levels was 18 hours instead of 20 hours.

Collection of Metaphase Cells (Preliminary Toxicity Test and Chromosome Aberration Assayd)
For the preliminary toxicity test and chromosome aberration assays, cells were collected 20 hours (± 30 minutes), 1.5 normal cell cycles, after initiation of treatment to ensure that the cells are analyzed in the first division metaphase. Just prior to harvest, the cell cultures was visually inspected for the degree of monolayer confluency relative to the vehicle control. The cells were trypsinized and counted and the cell viability was assessed using trypan blue dye exclusion.
The cell count was determined from a minimum of two cultures to determine the number of cells being treated (baseline). The data was presented as cell growth inhibition in the treatment group compared to vehicle control. Cell growth was determined by Relative Increase in Cell Counts (RICC) as a measure of cytotoxicity (Fellows and O'Donovan 2007; Lorge et al., 2008). The cell counts and percent viability were used to determine cell growth inhibition relative to the vehicle control (% cytotoxicity).
Evaluation criteria:
The test substance was considered to have induced a positive response if:
• at least one of the test concentrations exhibited a statistically significant increase when compared with the concurrent negative control (p ≤ 0.05), and
• the increase was concentration-related (p ≤ 0.05), and
• results were outside the 95% control limit of the historical negative control data.

The test substance was considered to have induced a clear negative response if none of the criteria for a positive response were met.
Statistics:
The percentage of cells in mitosis per 500 cells scored (mitotic index) was determined and recorded for each coded treatment group selected for scoring chromosomal aberrations. Slides were coded using random numbers by an individual not involved with the scoring process. Metaphase cells with 20 ± 2 centromeres were examined under oil immersion without prior knowledge of treatment groups. Whenever possible, a minimum of 300 metaphase spreads from each dose level (150 per duplicate culture) were examined and scored for chromatid-type and chromosome-type aberrations.

The number and types of aberrations (structural and numerical) found, the percentage of structurally damaged cells in the total population of cells examined (percent aberrant cells), the percentage of numerically damaged cells in the total population of cells examined, and the average number of structural aberrations per cell (mean aberrations per cell) were calculated and reported for each treatment group. Chromatid and isochromatid gaps are presented in the data but are not included in the total percentage of cells with one or more aberrations or in the average number of aberrations per cell.
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:
not examined
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Negative for the induction of structural and numerical chromosome aberrations.
Executive summary:

Method

The test substance was tested in the chromosome aberration assay using Chinese hamster ovary (CHO) cells in both the absence and presence of an Aroclor-induced rat liver S9 metabolic activation system according to OECD Guideline 473.

A preliminary toxicity test was performed to establish the dose range for the chromosome aberration assay. The chromosome aberration assay was used to evaluate the clastogenic potential of the test substance. In both phases, CHO cells were treated for 4 and 20 hours in the non-activated test system and for 4 hours in the S9-activated test system. All cells were harvested 20 hours after treatment initiation. Dose formulations were adjusted for the purity of the test substance (65%), using a correction factor of 1.54.

Water was used as the vehicle based on the solubility of the test substance and compatibility with the target cells. In a solubility test conducted at BioReliance, the test substance formed a workable suspension in water at a maximum concentration of approximately 10 mg/mL. Cyclophosphamide and mitomycin C were evaluated as the concurrent positive controls for treatments with and without S9, respectively.

In the preliminary toxicity assay, the doses tested ranged from 0.2 to 2000 μg/mL. Cytotoxicity (≥ 50 % reduction in cell growth index relative to the vehicle control) was observed at dose levels 20, 200, 500, and 2000 μg/mL in the non-activated 4-hour exposure group, at dose levels ≥ 60 μg/mL in the S9-activated 4-hour exposure group, and at dose levels ≥ 600 μg/mL in the non-activated 20-hour exposure group. Based on these findings, the doses chosen for the chromosome aberration assay ranged from 2.5 to 300 μg/mL for the non-activated 4-hour exposure group, from 5 to 200 μg/mL for the S9-activated 4-hour exposure group, and from 5 to 300 μg/mL for the non-activated 20-hour exposure group.

Results

In the initial chromosome aberration assay, 55 ± 5 % cytotoxicity (reduction in cell growth index relative to the vehicle control) was not observed at any dose level in the non-activated 4-hour exposure group. Cytotoxicity was observed at 200 μg/mL in the S9-activated 4-hour exposure group and at dose levels ≥ 200 μg/mL in the non-activated 20-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed at dose levels ≥ 75 μg/mL in all three treatment groups. The highest dose analyzed under each treatment condition exceeded the limit of solubility in treatment medium at the conclusion of the treatment period, which met the dose limit as recommended by testing guidelines for this assay.

The percentage of cells with structural or numerical aberrations in the non-activated 4 and 20-hour exposure groups was not significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher's Exact test).

The percentage of cells with structural aberrations in the S9-activated 4-hour exposure group was statistically significantly increased (3.0%) relative to vehicle control at 75 μg/mL (p ≤ 0.01, Fisher's Exact test). However, the Cochran-Armitage test was negative for a dose response (p > 0.05). The percentage of cells with numerical aberrations in the S9-activated 4-hour exposure group was not significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher's Exact test).

In order to confirm the positive response observed, the chromosome aberration assay was repeated in the S9-activated 4-hour exposure group at dose levels ranging from 25 to 100 μg/mL. In the repeat assay, 55 ± 5% cytotoxicity (reduction in cell growth index relative to the vehicle control) was not observed at any dose level in the non-activated 4-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed at dose levels ≥ 55 μg/mL. The highest dose analyzed exceeded the limit of solubility in treatment medium at the conclusion of the treatment period, which met the dose limit as recommended by testing guidelines for this assay.

In the repeat assay, the percentage of cells with structural aberrations was not significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher's Exact test). The percentage of cells with numerical aberrations in the S9-activated 4-hour exposure group was statistically significantly increased (6.0% and 5.7%) relative to vehicle control at dose levels 25 and 50 μg/mL, respectively (p ≤ 0.05, Fisher's Exact test). However, the Cochran-Armitage test was negative for a dose response (p > 0.05). In addition, the percentage of cells with numerical aberrations was within the historical control range of 0.0% to 9.5% and also within the 95% control limit of historical data. Therefore, the statistically significant induction was considered to have no biological relevance.

All vehicle control values were within historical ranges, and the positive controls induced significant increases in the percent of aberrant metaphases (p ≤ 0.01). Thus, all criteria for a valid study were met.

Conclusion

Under the conditions of the assay described in this report, the test substance was considered to be negative for the induction of structural and numerical chromosome aberrations in the non-activated and S9-activated test systems in the in vitro mammalian chromosome aberration test using CHO cells.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

No studies on the "Genetic toxicity" are available for the substance in itself.

Nevertheless studies have been conducted with an analogue molecule (Similar Substance 01). Further information are reported in the Read Across justification attached to section 13.

In the HPRT study the test substance was tested for its mutagenic effect by the gene mutation test with chines hamster cells V79, according to OECD Guideline 476.

The test system allows the detection of base-pair substitutions, frameshift mutations and deletions induced by the test substance or by its metabolites. Mutagenic effects are manifested by the appearance of cells resistant to 6-TG and can be quantified by comparison of the numbers of 6-TG resistant colonies in the treated and control cultures. To ensure that any mutagenic effect of metabolites of the test substance found in mammals is also detected, an experiment is performed, in which the metabolic turnover of the test material is simulated in vitro by the addition of an activation mixture to the cell cultures containing rat-liver post mitochondrial supernatant (S9 fraction) and cofactors.

The original experiment was performed at the following concentrations: 11.11, 33.33, 100.0 and 300.0 µg/ml. The mean growth inhibition values found at the highest concentration after treatment and expression were 82.0 % and 19.5 % respectively. In the confirmatory experiment the concentrations applied were 37.5, 75.0, 150.0 and 300.0 µg/ml. The highest concentration revealed a mean acute growth inhibitory effect of 69.3%. The mean growth inhibition after the expression period was 24.0%. Ethylmethansulfonate (EMS, 0.3 µl/ml) was used as positive control.

The results of this experiment showed no genotoxicity on the chines hamster cells V79.

 

The Chromosomal Aberration study was performed to investigate the potential of the test substance to induce gene mutations according to OECD 473 and in accordance with GLP.

Under the conditions of the assay described in this report, the test substance was considered to be negative for the induction of structural and numerical chromosome aberrations in the non-activated and S9-activated test systems in the in vitro mammalian chromosome aberration test using CHO cells.

Justification for classification or non-classification

GERM CELL MUTAGENICITY

This hazard class is primarily concerned with substances that may cause mutations in the germ cells of humans that can be transmitted to the progeny. However, the results from mutagenicity or genotoxicity tests in vitro and in mammalian somatic and germ cells in vivo are also considered in classifying substances and mixtures within this hazard class.

Category 1: Substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans. Substances known to induce heritable mutations in the germ cells of humans.

Categoty 2: Substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.

Classification for heritable effects in human germ cells is made on the basis of well conducted, sufficiently validated tests as In vitro mutagenicity tests such as these indicated in 3.5.2.3.8:

- in vitro mammalian chromosome aberration test;

- in vitro mammalian cell gene mutation test;

- bacterial reverse mutation tests

Therefore the test substance is Not classified.