Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames, positive in Salmonella typhimurium TA1535 with and without metabolic activation

CA, positive in CHO cells without metabolic activation

CA, negative in CHO cells with metabolic activation

Key, MLA TK +/-, positive in mammalian cell lines with and without metabolic activation

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:
1986
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
10, 33, 100, 333, 1000, 3333, 6666 µg/plate
Vehicle / solvent:
Dimethyl Sulfoxide
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 2-Aminoanthracene, 4-Nitro-O-Phenylenediamine
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
333 µg/well
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
not specified
Conclusions:
The test item is positive in an bacteria reverse mutation assay with and without metabolic activation in the strain Salmonella typhimurium TA1535.
Executive summary:

The test item has been tested for mutagenic potential in bacteria in an bacteria reverse mutation assay (Ames test) according to international accepted protocolls. The following concentrations were tested: 10, 33, 100, 333, 1000, 3333, 6666 µg/plate with and without metabolic activation (S9 -mix) on S. typhimurium TA 1535, TA 1537, TA 98 and TA 100.

The test item was positive in S. typhimurium TA 1535 at 333 µg/plate 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
Study period:
1987
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
according to international standards
GLP compliance:
no
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
Test chemicals were supplied under code by the National Toxicology Program chemical repository (Radian Corp., Austin, TX).
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
Cloned Chinese hamster ovary cells (CHO-W-B1) were cultured in McCoy's 5a medium with 10% fetal calf serum, L-glutamine, and antibiotics.
Metabolic activation:
with and without
Metabolic activation system:
The S9 mix consisted of 15 µL/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
Test concentrations with justification for top dose:
16 - 160 µg/mL
Vehicle / solvent:
and were dissolved immediately before use in water, dimethyl sulfoxide (DMSO), ethanol, or acetone, in that order of preference.- Vehicle(s)/solvent(s) used: no data
- Justification for choice of solvent/vehicle: water, dimethyl sulfoxide (DMSO), ethanol, or acetone, in that order of preference.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
triethylenemelamine
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
Cells were exposed to the test chemical for 2 hr in the presence of S9 or throughout the incubation period without S9.
Cells were collected by mitotic shake-off. Slides were stained with Giemsa and coded, and 100 cells were scored from each of the three highest dose groups having sufficient metaphases for analysis.
All types of aberrations were recorded separately, but for data analysis they were grouped into categories of "simple" (breaks and terminal deletions), "complex" (exchanges and rearrangements), "other" (includes pulverized chromosomes), and "total." Gaps and endoreduplications were recorded but were not included in the totals. We did not score aberrations in polyploid cells but used metaphases with 19-23 chromosomes (the modal number being 21).
100 cells/dose were tested
Statistics:
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 (as opposed to Poisson) was used, and the test was that described by Margolin et al [1983, pp 714-715]. The P values were adjusted by Dunnett's method 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.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
There was a concentration-related increase in aberrations without S9; the increase in aberrations with S9 was not statistically significant.
Conclusions:
There was a concentration-related increase in aberrations without S9; the increase in aberrations with S9 was not statistically significant.
Executive summary:

The registered substance was tested in a Chromosome Aberration test in CHO cells. It was tested in the concentration range 16 to 160 µg/mL. There was a concentration-related increase in aberrations without S9; the increase in aberrations with S9 was not statistically significant.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
GLP compliance:
not specified
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:
TK gene
Species / strain / cell type:
mouse lymphoma L5178Y cells
Remarks:
TK+/— 3.7.2C
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system: S9 mix
- source of S9 : Aroclor 1254-induced male Sprague-Dawley rats.
- method of preparation of S9 mix : S9 mix was prepared according to the procedure of Clive et al. [1979].
Test concentrations with justification for top dose:
Cells at a concentration of 6x 10E5/mL (6x 10E6 cells total) were exposed for 4 hr to a range of concentrations from 0.0005 to 100 µL/mL.
The doses of chemical selected for testing were within the range yielding approximately 0-90% cytotoxicity.
Vehicle / solvent:
DMSO
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
other: ethylmethanesulfonate (without S9)
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 12 x 10E6 cells
- Test substance added in medium; in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 h at 37°C

FOR GENE MUTATION:
The mutagenicity assay was performed according to the procedure described by Clive and Spector[1975). A total of 12 x 10E6 cells in duplicate cultures were exposed to the test chemical, positive control and solvent control for 4 hr at 37° ± 1°C, washed twice with growth medium, and maintained at 37° ± 1°C for 48 hr in log-phase growth to allow recovery and mutant expression. Cells in the cultures were adjusted to 0.3 x 10E6 mL at 24-hr intervals. They were then cloned (1 X 10E6 cells/plate for mutant selection and 200 cells/plate for viable count determinations) in soft-agar medium containing Fischer's medium, 20% horse serum, 2 mM sodium pyruvate, 0.02% pluronic F-68, and 0.23% granulated agar (BBL, Inc.). Resistance to trifluorothymidine (TFT) was determined by adding TFT (final concentration, 3 µg/mL) to the cloning medium for mutant selection. The 100 x stock solution of TFT in saline was stored at —70°C and was thawed immediately prior to use. Plates were incubated at 37°C ± 1°C in 5% CO2 in air for 10-12 days and then counted with an automatic colony counter (Biotran D. New Brunswick Scientific Co., Edison, NJ). Mutant frequencies were expressed as mutants per 10E4 surviving cells, and the results were interpreted in a manner similar to that described by Clive et al. [1979].


Statistics:
not reported
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
other: within the range of approximately 0- 90 % cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Conclusions:
The test item is positive in a mouse lymphoma TK + /- mutagenicity assay with and without metabolic activation.
Executive summary:

The test item has been assessed in a mouse lymphoma TK + /- mutagenicity assay with and without metabolic (S9 mix) activation according to international accepted protocols. It was concluded that the substance is positive with and without metabolic activation for mutagenicity in mammalian cell lines.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Genetic toxicity in vitro_bacteria

In the key study (Mortelmans, 1986) the test item has been tested for mutagenic potential in bacteria in an bacteria reverse mutation assay (Ames test) according to international accepted protocolls. The following concentrations were tested: 10, 33, 100, 333, 1000, 3333, 6666 µg/plate with and without metabolic activation (S9 -mix) on S. typhimurium TA 1535, TA 1537, TA 98 and TA 100.

The test item was positive in S. typhimurium TA 1535 at 333 µg/plate with and without metabolic activation.

In the supporting study the mutagenicity of the test item towards bacteria was assessed in a plate incorporation assay according to Ames et al [1975] with histidine auxotrophs Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537. In the Salmonella assay, the chemical was tested at dose levels up to and including 10.000 µg/plate without metabolic activation and with liver S9 preparations from Sprague-Dawley rats and Syrian golden hamsters. The test item was positive in strain TA1535 in the absence and presence of metabolic activation with both rat and hamster S9. The responses obtained were more variable in strain TA100. A concentration-related increase was observed with iso-amyl nitrite. Strain TA100 was negative with metabolic activation and postive without.

Chromosome aberration in CHO cells

The registered substance was tested in a Chromosome Aberration test in CHO cells. It was tested in the dose range 16 to 160 µg/mL. There was a concentration-related increase in aberrations without S9; the increase in aberrations with S9 was not statistically significant.

Sister Chromatid Exchange Assay in CHO cells

The registered substance was tested in a Sister Chromatid Exchange Assay in CHO cells. It was tested in the concentration range 16 to 160 µg/mL. The SCE tests were positive with and without metabolic activation, although the increase with S9 was statistically significant in only one of two tests.

Mouse Lymphoma TK +/- Mutagenicity Assay

The test item has been assessed in a mouse lymphoma TK + /- mutagenicity assay with and without metabolic (S9 mix) activation according to international accepted protocols. It was concluded that the substance is positive for mutagenicity with and without metabolic activation in mammalian cell lines.

Justification for classification or non-classification

According to the positive results in the in vitro genetic toxicity studies, the test item needs to be classified as Mutagen Cat. 2 (H341: Suspected of causing genetic defects). This classification is in line with the classification in the disseminated lead substance dossier, published on the ECHA homepage.