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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test:

The test chemical did not induce mutation in the Salmonella typhimurium strains both in the presence and absence of S9 metabolic activation system and hence is not likely to be mutagenic under the conditions of this study.

Chromosomal Abberation test:

The test chemical did not induce chromosome aberrations in the mammalian cell line in the presence and absence of S9 metabolic activation system and hence it is not mutagenic in the chromosome aberration study performed.

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
Study period:
25 March 1987 to 11 April 1987
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
no strain to detect cross linking included
Principles of method if other than guideline:
Gene mutation toxicity study of the test chemical
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Metabolic activation:
with and without
Metabolic activation system:
S-9 from aroclor 1254 induced rat livers
Test concentrations with justification for top dose:
dose range finding: 5, 50, 500 and 5000 ug/plate
main assay (with independent repeat): 15, 50, 150, 500 and 1500 ug/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: no data
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
N-ethyl-N-nitro-N-nitrosoguanidine
other: aminoanthrocene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation)
- Cell density at seeding (if applicable):ca 1E07/mL

DURATION
- Preincubation period: NA
- Exposure duration: 72 hours at 37 °C

NUMBER OF REPLICATIONS: 3/concentration (2 independent assays)

DETERMINATION OF CYTOTOXICITY
- Method:reduced bacterial back ground lawn and precipitate.
Evaluation criteria:
A test item is considered as mutagenic if:
- a significant and dose-related increase in the number of revertants occurs with sufficient reproducibility
- the number of revertant colonies is at least twice as high
Statistics:
NA
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at and above 5000 ug/plate
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at and above 5000 ug/plate
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at and above 5000 ug/plate
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at and above 5000 ug/plate
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at and above 5000 ug/plate
Vehicle controls validity:
valid
Positive controls validity:
valid
Conclusions:
Test chemical failed to induce gene mutation in the S. typhimurium TA1535, TA1537, TA98, TA100 and TA1538 and hence is negative for mutation in vitro.
Executive summary:

Gene mutation assay was performed to evaluate the mutagenic nature of the test compound. Plate incorporation assay was performed using S. typhimurium TA1535, TA1537, TA98, TA100 and TA1538 in the presence and absence of S9 metabolic activation system. Positive control mutagens were run concurrently for each strain in each test. When S9 was used, aminoanthrocene was used as a positive control for S9 activity with all strains.In those cases where positive mutagen or S9 controls did not give the expected results, the data from that particular experiment were disregarded. The plates were observed for a dose dependent increase in the number of revertants/plate. Test chemical failed to induce gene mutation in the S. typhimurium TA1535, TA1537, TA98, TA100 and TA1538 and hence is negative for mutation in vitro.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
Experimental data from various test chemicals
Justification for type of information:
Data for the target chemical is summarized based on the various test chemicals.
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Qualifier:
equivalent or similar to
Guideline:
other: As mentioned below
Principles of method if other than guideline:
WoE for the target CAS is summarized based on data from various test chemicals.
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
5. No data
6. Not specified
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Remarks:
5
Details on mammalian cell type (if applicable):
- Type and identity of media: Mc- Coy’s 5a medium with 10% fetal calf serum, L-glutamine, and antibiotics
- Properly maintained: No data available
- Periodically checked for Mycoplasma contamination: No data available
- Periodically checked for karyotype stability: No data available
- Periodically "cleansed" against high spontaneous background: No data available
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Remarks:
6
Details on mammalian cell type (if applicable):
not specified
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
5. No data
6. not specified
Metabolic activation:
with and without
Metabolic activation system:
5. The S9 mix consisted of 15 pl/ml liver homogenate (from male Sprague-Dawley rats, induced with Aroclor 1254), 2.4 mg/ ml NADP, and 4.5 mg/ml isocitric acid in serum-free medium.
6. induced male Sprague Dawley rat liver S9
Test concentrations with justification for top dose:
5. Without S9: 160-1600 µg/ ml
With S9: 500-5000 µg/ ml
6. -S9;0,6.4,12.8 and 18.3 µg/mL (Long duration)
0,6.02,7.96 and 10.21 µg/mL (short duration)
+S9; 0,50.2,174.8and 100.3 µg/mL
Vehicle / solvent:
5. Water, dimethyl sulfoxide (DMSO), ethanol, or Acetone (in the order of preference)
6. Vehicle
- Vehicle(s)/solvent(s) used: Dimethyl Sulfoxide
- Justification for choice of solvent/vehicle: The test substance is soluble in DMSO.
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: Triethylenemelamine
Remarks:
5
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: -S9 mix; Triethylenemelamine +S9 mix; Cyclophosphamide
Remarks:
6
Details on test system and experimental conditions:
5. METHOD OF APPLICATION: in medium

DURATION
- Preincubation period:
- Exposure duration: With S9: 2 hrs
Without S9: Apprx. 8.5-9 hrs
- Expression time (cells in growth medium): 8.5-9 hrs
- Selection time (if incubation with a selection agent): after 18-26 hrs
- Fixation time (start of exposure up to fixation or harvest of cells): 8-12 hr after the beginning of treatment.

SELECTION AGENT (mutation assays): Geimsa stain

NUMBER OF CELLS EVALUATED: 100 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
6. Details on test system and conditions
METHOD OF APPLICATION: In medium
DURATION
- Fixation time (start of exposure up to fixation or harvest of cells):
-S9; 11 hours (Long duration)
10.5 hours(short duration)
+S9; 10.5 hours
Rationale for test conditions:
5. No data
6. No data
Evaluation criteria:
5. Chromosomal aberrations were noted; Cells were selected for scoring on the basis of good morphology and completeness of karyotype (21 ± 2 chromosomes).

Classes of aberrations included simple (breaks and terminal deletions), complex (rearrangements and translocations), and other (pulverized chromosomes). Gaps and endo-reduplications were recorded but were not included in the totals. Aberrations were not scored in polyploidy cells but metaphases with 19-23 chromosomes were used (the modal number being 21).
6. The mammalian cells were observed for chromosome aberration, Chromosome gaps and breaks.
Statistics:
5. For chromosome aberrations, linear regression analysis of the percentage of cells with aberrations vs the log-dose was used as the test for trend. To examine absolute increases over control levels at each dose, a binomial sampling assumption was used. The P values were adjusted to take into account the multiple dose comparisons. For data analysis, we used the “total” aberration category, and the criterion for a positive response was that the adjusted P value be < 0.05.
6. Yes, SD ± Mean was observed.
Species / strain:
Chinese hamster Ovary (CHO)
Remarks:
5
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
Chinese hamster Ovary (CHO)
Remarks:
6
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
5. RANGE-FINDING/SCREENING STUDIES: doses were chosen for the aberration test based on a preliminary test of cell survival 24 hr after treatment. Doses were based on observations of cell confluence and mitotic cell availability in the SCE test.
6. No data
Remarks on result:
other: No mutagenic effects were observed
Conclusions:
The test chemical did not induce chromosome aberrations in the mammalian cell line in the presence and absence of S9 metabolic activation system and hence it is not mutagenic in the chromosome aberration study performed.
Executive summary:

In different studies, the given test chemical has been investigated for the mutagenic nature. The studies are as mentioned below:

 

In vitro mammalian chromosome aberration test was performed to evaluate the mutagenic nature of the test chemical. Cloned Chinese hamster ovary cells (CHO-W-B1) were cultured in Mc-Coy’s 5a medium with 10% fetal calf serum, L-glutamine, and antibiotics. Tests were carried out with and without an in vitro metabolic activation system (S9 mix). In tests without metabolic activation, the test chemical was left in culture until colcemid addition, whereas with activation the test chemical was added along with S9 mix for only 2 hr at the beginning of the test period. The doses used for the study were 160-1600µg/mL without S9 and 500-5000µg/mL with S9. The test chemical did not induce chromosome aberrations in the Chinese hamster ovary cell line in the presence and absence of S9 metabolic activation system and hence it is not mutagenic in the chromosome aberration study performed.

 

In another study, the chromosomal aberration induction potential was determined by performing the gene mutation study of the test chemical. For this purpose in vitro mammalian chromosome aberration test was performed .The test material was exposed toChinese hamster ovary cells inthe presence and absence of metabolic activation S9. The concentration of test material used in the presence and absence of metabolic activation were mention below – S9; 0, 6.4, 12.8 and 18.3 µg/mL (Long duration); 0, 6.02, 7.96 and 10.21 µg/mL (short duration) and +S9; 0, 50.2, 174.8 and 100.3 µg/mL. No chromosome aberration, Chromosome gaps and breaks were observed in the presence or absence of metabolic activation. Therefore test chemical was considered to be non-mutagenic inChinese hamster ovary cells by in vitro mammalian chromosome aberration test. Hence the substance cannot be classified as non -mutagenic in vitro.

 

Thus, based on the above summarized studies on test chemical, it can be concluded that the given test chemical did not induce chromosome aberrations in the mammalian cell line in the presence and absence of S9 metabolic activation system and hence it is not mutagenic in the chromosome aberration study performed.

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

Additional information

Gene mutation in vitro:

Data available from various sources was reviewed to determine the mutagenic nature of the given test chemical. The studies are as mentioned below:

Ames assay:

Gene mutation assay was performed to evaluate the mutagenic nature of the test compound. Plate incorporation assay was performed usingS. typhimuriumTA1535, TA1537, TA98, TA100 and TA1538 in the presence and absence of S9 metabolic activation system. Positive control mutagens were run concurrently for each strain in each test. When S9 was used, aminoanthrocene was used as a positive control for S9 activity with all strains.In those cases where positive mutagen or S9 controls did not give the expected results, the data from that particular experiment were disregarded. The plates were observed for a dose dependent increase in the number of revertants/plate. Test chemical failed to induce gene mutation in theS. typhimuriumTA1535, TA1537, TA98, TA100 and TA1538 and hence is negative for mutation in vitro.

In another study, Salmonella/microsome test in the absence of exogenous metabolic activation and in the presence of liver S-9 from Aroclor-induced male Sprague-Dawley rats and Syrian hamsters was performed to evaluate the mutagenic nature of the test chemical usingS. typhimuriumtest strains TA1535, TA97, TA98 and TA100. The study was performed as per the preincubation assay.The test compound was used at a dosage level of 0, 100, 333, 1000, 3333, 6666 or 10000 µg/plate in the preincubation assay of 48 hrs.Test chemical failed to induce gene mutation in theS. typhimuriumtest strains TA1535, TA97, TA98 and TA100 and hence is negative for mutation in vitro.

In another study, Gene mutation assay was performed to evaluate the mutagenic nature of the test chemical. Plate incorporation assay was performed usingS. typhimuriumTA1535, TA1537, TA1538, TA98 and TA100 in the presence and absence of S9 metabolic activation system. Positive control mutagens were run concurrently for each strain in each test. When S9 was used, aflatoxin B 1 (AFB1) was used as a positive control for S9 activity with strain TA100.In those cases where positive mutagen or S9 controls did not give the expected results, the data from that particular experiment were disregarded. The plates were observed for a dose dependent increase in the number of revertants/plate. Test chemical failed to induce gene mutation in theS. typhimuriumTA1535, TA1537, TA1538, TA98 and TA100 and hence is negative for mutation in vitro.

Chromosomal Abberstion test:

In different studies, the given test chemical has been investigated for the mutagenic nature. The studies are as mentioned below:

 

In vitro mammalian chromosome aberration test was performed to evaluate the mutagenic nature of the test chemical. Cloned Chinese hamster ovary cells (CHO-W-B1) were cultured in Mc-Coy’s 5a medium with 10% fetal calf serum, L-glutamine, and antibiotics. Tests were carried out with and without an in vitro metabolic activation system (S9 mix). In tests without metabolic activation, the test chemical was left in culture until colcemid addition, whereas with activation the test chemical was added along with S9 mix for only 2 hr at the beginning of the test period. The doses used for the study were 160-1600µg/mL without S9 and 500-5000µg/mL with S9. The test chemical did not induce chromosome aberrations in the Chinese hamster ovary cell line in the presence and absence of S9 metabolic activation system and hence it is not mutagenic in the chromosome aberration study performed.

 

In another study, the chromosomal aberration induction potential was determined by performing the gene mutation study of the test chemical. For this purpose in vitro mammalian chromosome aberration test was performed .The test material was exposed toChinese hamster ovary cells inthe presence and absence of metabolic activation S9. The concentration of test material used in the presence and absence of metabolic activation were mention below – S9; 0, 6.4, 12.8 and 18.3 µg/mL (Long duration); 0, 6.02, 7.96 and 10.21 µg/mL (short duration) and +S9; 0, 50.2, 174.8 and 100.3 µg/mL. No chromosome aberration, Chromosome gaps and breaks were observed in the presence or absence of metabolic activation. Therefore test chemical was considered to be non-mutagenic inChinese hamster ovary cells by in vitro mammalian chromosome aberration test. Hence the substance cannot be classified as non -mutagenic in vitro.

 

Thus, based on the above summarized studies on test chemical, it can be concluded that the given test chemical did not induce chromosome aberrations in the mammalian cell line in the presence and absence of S9 metabolic activation system and hence it is not mutagenic in the chromosome aberration study performed.

Justification for classification or non-classification

Based on the data available and applying weight of evidence approach, the given test chemical does not exhibit gene mutation in vitro by Ames assay and In vitro mammalian chromosome aberration study. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.