Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three studies are available which included a bacterial reverse mutation assay (Ames test), a chromosome aberration assay and a mouse lymphoma assay. The Ames test provided a negative result both with and without metabolic activation, and utilising relevant bacterial strains. The chromosome aberration assay using Chinese hamster ovary (CHO) cells, showed a significant increase in aberration frequency at the highest concentration tested with metabolic activation. An increase in endoreduplication was also observed in cultures with metabolic activation from 2250 µg/mL. The final in vitro test was a mouse lymphoma assay conducted using mouse L5178Y lymphoma cells. This test provided a positive response both with and without metabolic activation. The genotoxic potential of this substance was further assessed in vivo.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Data migrated from NONS with permission to refer granted by ECHA.
Qualifier:
according to
Guideline:
other: 92/69/EEC (Ames)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Acrolor-induced rat liver S9
Test concentrations with justification for top dose:
Concentration range in the main test (with metabolic activation): 10...5000 µg/plate
Concentration range in the main test (without metabolic activation): 10...5000 µg/plate
Vehicle / solvent:
Solvent: Dimethylsulfoxide
Species / strain:
other: As specified above
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>/= 5000 µg/plate
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
other: As specified above
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
> 5000 µg/plate
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
other: As specified above
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>/= 5000 µg/plate
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
Observations:
The solvent control plates gave counts in the expected ranges. All positive controls produced marked increases in the revertant counts.
Remarks on result:
other: Preliminary test
Conclusions:
Negative with and without metabolic activation.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Data migrated from NONS with permission to refer granted by ECHA.
Qualifier:
according to
Guideline:
other: 96/69/EEC (Metaphase analysis)
GLP compliance:
yes
Type of assay:
other: Chromosome aberration study
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
Acrolor-induced rat liver S9
Test concentrations with justification for top dose:
Concentration range in the main test (with metabolic activation): 1360...3620 µg/plate
Concentration range in the main test (without metabolic activation): 1350...3620 µg/plate
Vehicle / solvent:
Dimethylsulfoxide
Details on test system and experimental conditions:
Exposure period (with metabolic activation): 3 hours
Exposure period (without metabolic activation): 17 hours
Fixation time: 20 hours
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
4810 µg/mL
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
3620 µg/mL
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
Observations:
A significant increase in aberration frequency was observed only at the top dose with activation. There was also an increase in endoreduplication in cultures with activation from 2250 µg/mL. Control values were within the expected ranges.
Remarks on result:
other: Preliminary test
Conclusions:
Increased chromosome aberration frequencies were seen in this study only at very high test concentrations, all of which were above the 10 mM recommended in the OECD Test guidelines for this endpoint. At 'conventional' dose levels, there were no increases. The increased frequency of endoreduplication in cells with activation may indicate that the test substance has potential to inhibit cell cycle progression.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
other: in vitro mammalian gene cell mutation test with L5178Y mouse lymphoma cells
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: American Type Culture Collection, (ATCC, Manassas, USA)
- Storage: Stock cultures of the cells were stored in liquid Nitrogen (-196°C).

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
- Basic media: RPMI 1640 Hepes buffered medium containing penicillin/streptomycin, sodium pyruvate and L-Glutamin.
- Growth medium: Basic medium supplemented with heat-inactivated horse serum.
- Exposure medium: 3 Hour: Cells were exposed to the test substance in basic medium supplemented with 5% heat-inactivated horse serum. 24 hour exposure: Cells were exposed to the test substance in basic medium supplemented with 10% heat-inactivated horse serum.
- Selective medium: Consisted of growth medium supplemented with 10% heat-inactivated horse serum and TFT.
- Non-selective medium: Consisted of growth medium supplemented with 10% heat-inactivated horse serum.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Prepared from the livers of adult male Wistar rats (dosed with phenobarbitol and beta-naphthoflavone).
Test concentrations with justification for top dose:
Range finding: 35, 105, 352, 704, 1047 and 1810 µg/mL (with and without activation: 3 and 24 hour treatment)

First mutagenicity/cytoxicity test test: 10, 33, 100, 333, 666, 1000, 1325 and 1772 µg/mL (without activation)
First mutagenicity/cytotoxicity test: 100, 500, 600, 700, 900, 950, 975, and 1000 µg/mL (with activation)

Second mutagenicity/cytotoxicity test: 50, 250, 500, 750, 1000, 1100, 1250 and 1375 µg/mL (without activation)
Vehicle / solvent:
Dimethyl sulfoxide
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
Environmental conditions:
All incubations were carried out in a controlled environment in the dark at 35.8 to 37.6°C in a humid atmosphere of 65 to 95% containing ca. 5% CO2 (monitored continuously). Temporary deviations from the study-plan ranges occured, caused by opening and closing of the incubator door, with minimal time of the deviations. Based on laboratory historical control data, these deviations did not affect the integrity of the study.

Dose range finding phase:
In order to obtain suitable doses, cytotoxicty data was initially obtained by treating cells for 3 or 24 hours with a number of test substance concentrations. Cell cultures were exposed for 3 hours in the presence of S9-mix and for 3 and 24 hours in the absence of S9-mix. After a series of standard techniques the cells were counted with a coulter particle counter. For determining cytotoxicity, the surviving cells were subcultured and counted. The suspension growth expressed as the reduction in cell growth after approximately 24 and 48 hours cell growth, compared to the cell growth of the solvent control, was used to determine an appropriate dose range for the mutagenicity study.

Mutagenicity test:
In the initial experiment cells were exposed for 3 hours in the absence and presence of S9-mix. In the second experiment cells were exposed for 24 hours in the absence of S9-mix. Standard exposure techniques were utilised as per the relevant guideline after which cells were counted using a coulter particle counter.

Expression period:
For expression of the mutant phenotype, the remaining cells were cultured for two days after the treatment period. Two days after the end of the treatment with the test substance the cells were plated for determination of cloning efficiency (CEDay 2) and and the mutation frequency (MF).

Determination of mutation frequency:
Eight doses of the substance were selected for the mutation assay in the absence and presence of S9-mix. Standard guideline processes were used to prepare plates which were scored and used to determine the CEday2 and MF.
Rationale for test conditions:
Standard guideline procedures were followed with final concentrations based on the results of a dose range finding study.
Evaluation criteria:
Determination of mutant colonies:
The colonies were divided into small and large colonies with small colonies suffering extensive genetic damage and large colonies were less severly affected. Smaller colonies can be associated with the induction of chromosomal aberrations whilst larger colonies appear to result from mutants with single gene mutations affecting the TK gene.

Calculation of the survival or viability:
The cloning efficiency was determined by dividing the number of empty wells by the total number of wells. The relative survival (RS) in each treatment group was determined by comparing cloning efficiencies in treatment and control cultures. The relative total growth (RTG) was also calculated as the product of the cumulative relative suspension growth (RSG) and the relative cloning efficiency for each culture.

Calculation of mutation frequency:
This was expressed as the number of mutants per 10^6 cells.

Acceptability of the assay:
A mutation assay is considered acceptable if the following criteria are met:
1) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120%.
2) The spontaneous mutation frequency in the solvent control is > 50x10^-6 and < 170x10^-6.
3) The growth rate over the 2-day expression period for the negative controls should be between 8 and 32.
4) The mutation frequency of MMS should not be below 500x10^-6 and for CP not below 700 x 10^-6.
Statistics:
The experimental results were not subject to statistical analysis.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The test substance did not precipitate in the exposure medium up to 1722 µg/mL (0.01 M). Therefore this concentration was used as the highest test substance concentration. After the 3 hour treatment period, precipitation of the substance was observed at dose levels of 1300 µg/mL and above in the presence of S9-mix only.

The pH and osmolarity of a concentration of 1810 µg/mL were 7.03 and 0.414 Osm/kg respectively (compared to 7.25 and 0.425 Osm/Kg) in the solvent control.

The substance induced up to 13 and 6.4 fold dose related increases in the mutation frequency at the TK locus in the absence and presence of S9-mix respectively.

The substance induced up to 11 and 5.2 fold increase in the MF of the smaller colonies, compared with solvent controls in the absence and presence of S9-mix respectively. The MF of large colonies was increased up to 9.6 and 6.2 fold compared with solvent controls in the absence and presence of S9 -mix respectively. These increases in small and large colonies indicate that the substance has the potential to induce both chromosomal aberrations and single cell gene mutations.

Conclusions:
Based on the results obtained, it is concluded that the substance has the potential to be mutagenic in the mouse lymphoma L5178Y test system under the conditions used on study.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Three studies are available assessing the genotoxicity of the substance in vivo. This included two mouse micronucleus assays (both providing negative results) and a rat UDS assay, which also provided a negative result for genotoxicity.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Data migrated from NONS with permission to refer granted by ECHA.
Qualifier:
according to
Guideline:
other: 96/69/EEC (Micronucleus)
GLP compliance:
yes
Type of assay:
other: Mouse micronucleus assay
Species:
mouse
Strain:
CD-1
Sex:
male/female
Route of administration:
oral: unspecified
Vehicle:
Corn oil
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
2 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Males and females: 10 per group (5 sacrificed at 24 hours and 5 at 48 hours after exposure)
Control animals:
yes, concurrent vehicle
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Animals that received 2000 mg/kg were observed as having decreased fecal output (females) and weakness in 3 males at this dosage
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
There were no increases in the frequency of micronuclei in any dose group. In females that received 2000 mg/kg, there was a significant trend for bone marrow depression.

Negative and positive (cyclophosphamide) control groups gave the expected responses.
Conclusions:
There were no increases in the frequency of micronuclei in any dose group. In females that received 2000 mg/kg, there was a significant trend for bone marrow depression.
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
other: Mouse micronucleus assay
Species:
mouse
Strain:
CD-1
Details on species / strain selection:
Crlj: CD1(ICR) strain
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: CRL Japan
- Age at study initiation: 7 weeks
- Weight at study initiation: 32.9 to 36.8 g (male DRF), 25.4 to 29.3 g (female DRF), 34 to 36.8 g (male micronucleus test).
- Assigned to test groups randomly: Yes
- Housing: Polycarbonate cages (group housed)
- Diet (e.g. ad libitum): Pelleted diet (adlib)
- Water (e.g. ad libitum): Provided adlib
- Acclimation period: At least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 50 +/- 20°C
- Air changes (per hr): > 10 changes per hour
- Photoperiod (hrs dark / hrs light): 12 h day (150 to 300 Lux)

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Olive oil
- Justification for choice of solvent/vehicle: Substance insoluble in water and 0.5% carboxymethyl cellulose.
- Concentration of test material in vehicle: 10 mL/Kg

Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance 4000 mg and 1000 mg were weighed for the tests of dose determination and micronucleus assay respectively. These were added to a volume of olive oil and adjusted to 20 mL (DRF) or 5 mL (micronucleus test). The lower doses were serially diluted from the concentration. Test solutions were prepared before use.
Duration of treatment / exposure:
DRF: 24 or 48 hours
Micronucleus test: 24 hours
Frequency of treatment:
Administered once
Post exposure period:
24 or 48 hours (DRF) and 24 hours (micronucleus test)
Dose / conc.:
125 mg/kg bw/day (nominal)
Remarks:
DRF
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
DRF
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
DRF
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
DRF
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Remarks:
DRF
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
Micronucleus assay
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Micronucleus assay
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Remarks:
Micronucleus assay
No. of animals per sex per dose:
2 males and 2 females per dose group in the DRF and 5 males per dose group in the micronucleus study.
Control animals:
yes, concurrent vehicle
Positive control(s):
Positive control: Mitomycin C (as prescribed in the test guideline)
Tissues and cell types examined:
Bone marrow cells
Details of tissue and slide preparation:
The animals were sacrificed by cervical dislocation at 24 or 48 hours after dosing in the DRF and after 24 hours in the micronucleus test. The right femurs were dissected and bone marrow cells were treated and prepared on to glass slides using standard procedures. The slides were subsequently stained as appropriate.
Evaluation criteria:
Positive: The number of MNPCE increased dose-dependantly or significantly increased at a single dose point compared with negative controls.
Negative: Not meeting positive criteria.
Statistics:
The incidences of MNPCE and the ratio of PCE to RBC of each test substance concentration group and positive control group were compared with that of the negative control group for significant differences using Wilcoxon's rank sun test.
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The induced frequency of the MNPCE in the positive control group was significantly higher with that of the negative control group. The PEC ratio to RBC did not differ compared with the negative control group. The results show that the study was conducted appropriately, and all values values were within relevant historical ranges.
Conclusions:
The experimental findings conclude that the test substance was negative for mutagenicity as the substance did not increase the incidence of MNPCE in the mouse femoral bone marrow.
Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.39 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells In Vivo)
Deviations:
no
GLP compliance:
yes
Type of assay:
unscheduled DNA synthesis
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: CRL Germany
- Age at study initiation: 8 weeks
- Weight at study initiation: 241 +/- 13 g
- Assigned to test groups randomly: Yes
- Fasting period before study: No
- Housing: Group housed in polycarbonate cages
- Diet (e.g. ad libitum): Standard pelleted animal diet (generally adlib although food was witheld to assist in perfusion)
- Water (e.g. ad libitum): Adlib
- Acclimation period: 5 to 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 3°C
- Humidity (%): 30 to 70% (max range was exceeded for a minimal time and based on historical control data this deviation was considered not to have affected study integrity).
- Air changes (per hr): Approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle

Route of administration:
oral: gavage
Vehicle:
Corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The substance was dissolved in corn oil and dosed within 1 hour of preparation.
Duration of treatment / exposure:
DRF: Two dose groups, one comprising 1 male and the other 3 males, received a single dose of 2000 or 1000 mg/kg. The study duration was 2 days (for the 2000 mg/kg group) or 2 to 3 days (for the 1000 mg/kg group).

Main phase: Two dose groups (500 or 1000 mg/kg) were sampled at various times ranging from 2 to 16 hours
Frequency of treatment:
Animals were dose once during the DRF and main phase
Post exposure period:
Varies from 2 days in the DRF and from 2 to 16 hours in the main phase
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
DRF
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Remarks:
DRF
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
Main phase
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Main phase
No. of animals per sex per dose:
DRF: See Duration of treatment/exposure.
Main phase: 6 per dose group sampled at varying times.
Control animals:
yes, concurrent vehicle
Positive control(s):
2-4 hour treatment: dimethylnitrosamine (DMN)
12-16 hour treatment: 2-acetylaminofluorene
Tissues and cell types examined:
Liver hepatocytes
Details of tissue and slide preparation:
Liver perfusion:
The liver was removed after perfusion of the animals using standard techniques as detailed in the guidelines. The cell suspensions obtained were incubated and all non-relevant cells removed via centrifugation, leaving only the hepatocytes.

Cell culturing:
Viability of the cells was determined using the trypan blue dye exclusion method. Four microscope slides were prepared by animal. All incubations were carried out in a controlled environment with a humidity of 80 to 100% containing ca. 5% CO2 in air in the dark at 37 +/- 1°C. Temporary deviations from the temperature occured as a result of opening and closing the incubator door. These deviations were minimal and based on laboratory historical data, did not affect the study integrity.

Labelling of cell cultures:
The cultures were washed using standard procedures and labelled using a tritiated thymidine solution. The resulting cultures were fixed with a methanol-acetix acid mixture.

Autoradiographic procedure:
After fixation of the cells the coverslips were mounted on microscope slides and stained with a H&E stain according to standard procedures. Four scorable coverslips per animal were subject to the autoradiographic procedure.

Scoring:
Slides were assessed for the presence of sufficient cells and examined for signs of overt cytotoxicity using a randomised blind procedure. Grain counts were determined over the nuclei and the nucleus equivalent areas over the cytoplasm manually on the coverslips. Standard criteria were employed to determine whether a cell was countable, and averages along with a standard deviation were calculated. Grain counts over nuclear areas (N) were compared to grain counts over the most heavily labelled adjacent cytoplasm area (C) of the same size as the corresponding nuclear area. The net nuclear area grain count (NNG) was calculated for each cell by subtracting cytoplasmic grain counts from nuclear grain counts. the background counts of a single area of the same size as the corresponding nuclear area was recorded per coverslip.
Evaluation criteria:
A test substance is considered positive if it yields greater than or equal to 5 net nuclear grain (NNG) counts per group average, and greater than or equal to 20% of the cells responding (i.e. with NNG values of 5 or more).

A test substance is considered negative it it does not induce a net nuclear grain (NNG) count >/= 5 and >/= 20% of cells responding.

Biological relevance of the data is also considered.
Statistics:
No statistical analysis is performed.
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid

Mortality/signs of toxicity:

All animals survived the sampling period. The control animals and those dosed at 500 mk/kg showed no signs after dosing.

At the 2 -4 hour sampling time, all animals treated at 1000 mg/kg presented as lethargic and showed ataxia.

At the 12 -16 hour sampling time, animals initially showed no signs of treatment. However, by 12 hours all animals had a rough coat and hunched posture, with two animals presenting as lethargic in those animals that received 1000 mg/kg.

Visibility of hepatocytes:

At the 2 -4 hour sampling point, viability was at least 71% with 89% observed in controls, indicating no direct liver toxicity.

At the 12 -16 hour sampling point, viability was at least 75% with 89% observed in controls, indicating no direct liver toxicity.

DNA repair assay:

In all slides assessed, sufficient cells of normal morphology were available. No positive responses were observed on study.

Acceptability:

All validity criteria as per the guideline were met and the study was considered acceptable.       

Conclusions:
The substance was not genotoxic in the DNA repair assay using hepatocytes obtained from male rat livers following in vivo exposure, as no induction of DNA repair was evident at the MTD.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

A total of six genotoxicity tests are available to assess the genotoxic potential of the substance including three in vitro and three in vivo tests. The positive in vitro tests included the mouse lymphoma and chromosome aberration assays. Although the chromosome aberration assay showed a positive response, this was only observed at high dose concentrations all of which were greater than the guideline required concentration. The mouse lymphoma test also showed a positive response indicating that the substance had the potential to induce both chromosomal aberrations and single cell gene mutations. The positive result observed in these tests was also not replicated in vivo based on the results of two independant micronucleus assays showing a negative response, and accordingly there is a low concern that this substance has the potential to induce chromosome aberrations (either structural or numerical). An in vivo UDS assay is also available providing a negative response which shows the substance did not induce DNA repair, and accordingly is unlikely to be genotoxic.

Overall the results of the two positive in vitro tests have been shown not to be replicated in vivo test systems, and accordingly the substance is considered not be be genotoxic with no further testing deemed appropriate.