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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
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 (incl. certificate)
Remarks:
The Department of Health of the Government of the United Kingdom
Type of assay:
other: mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline

Method

Target gene:
Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Type and identity of media:
RPMI 1640
Properly maintained:
yes
Periodically checked for Mycoplasma contamination:
yes
Periodically checked for karyotype stability:
no
Periodically "cleansed" against high spontaneous background:
yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and beta-naphthoflavone induced rat liver, S9
Test concentrations with justification for top dose:
The maximum dose levels used in the Mutagenicity Test was the 10 mM dose limit.
Vehicle and positive controls were used in parallel with the test item. Solvent (R0 media) treatment groups were used as the vehicle controls. Ethylmethanesulphonate (EMS) Sigma batch BCBG1395V at 400 µg/mL and 150 µg/mL for Experiment 1 and Experiment 2, respectively, was used as the positive control in the absence of metabolic activation. Cyclophosphamide (CP) Acros batch A0302605 at 2 µg/mL was used as the positive control in the presence of metabolic activation.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used:
Solvent (R0 medium) treatment groups were used as the vehicle controls.
- Justification for choice of solvent/vehicle:
soluble
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Solvent (R0 medium) treatment groups were used as the vehicle controls.
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: cyclophosphamide
Remarks:
With metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Solvent (R0 medium) treatment groups were used as the vehicle controls.
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ethylmethanesulphonate
Remarks:
Without metabolic activation
Details on test system and experimental conditions:
The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No. 476 "In Vitro Mammalian Cell Gene Mutation Tests", Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and be acceptable to the Japanese METI/MHLW guidelines for testing of new chemical substances.
Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at six dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test item at six dose levels using a 4-hour exposure group in the presence of metabolic activation (1% S9) and a 24-hour exposure group in the absence of metabolic activation.
The dose range of test item was selected following the results of a preliminary toxicity test and was 23.44 to 750 µg/mL in absence and presence of metabolic activation in Experiment 1. In Experiment 2 the dose range was again 23.44 to 750 µg/mL in the absence and presence of metabolic activation.
The maximum dose levels used in the Mutagenicity Test was the 10 mM dose limit. No precipitate of the test item was observed at any dose level. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.
Evaluation criteria:
Please see ''Any other information on materials and methods incl. tables'' section
Statistics:
Please see ''Any other information on materials and methods incl. tables'' section

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
non-mutagenic
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
modest toxicity 24 hour only
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Preliminary Toxicity Test
The dose range of the test item used in the preliminary toxicity test was 2.93 to 750 µg/mL.
In both of the 4 hour (+/- S9) exposure groups there was no evidence of dose-related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls. In the 24-hour exposure group there was evidence of modest reductions in %RSG when compared to the vehicle control values. No precipitate of the test item was observed at any dose level. The maximum dose level used in the subsequent Mutagenicity Test was the maximum recommended 10 mM dose limit.
Experiment 1
The results of the microtitre plate counts and their analysis are presented in Tables 2 to 7.
There was no evidence of dose-related toxicity following exposure to the test item in both the absence and presence of metabolic activation, as indicated by the RTG and %RSG values (Tables 3 and 6). There was no evidence of a marked reduction in viability (%V), therefore indicating that residual toxicity had not occurred. Acceptable levels of toxicity were seen with both positive control substances (Table 3 and Table 6).
Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 x 10-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (Tables 3 and 6).
The test item did not induce and statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell in the absence or presence of metabolic activation. No Precipitate of the test item was observed at any dose level.
The numbers of small and large colonies and their analysis are presented in Tables 4 and 7.
Experiment 2
The results of the microtitre plate counts and their analysis are presented in Tables 8 to 13.
As was seen previously there was no evidence of toxicity following exposure to the test item in the presence of metabolic activation and there was evidence of moderate dose-related toxicity following exposure to the test item in the absence of metabolic activation as indicated by the RTG and %RSG values (Tables 9 and 12). There was no evidence of any reductions in viability (%V) in either the absence or presence of metabolic activation, therefore indicating that residual toxicity had not occurred. Acceptable levels of toxicity were seen with both positive control substances (Tables 9 and 12).
The 24-hour exposure without metabolic activation demonstrated that the extended time point had a marked effect on the toxicity of the test item.
Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 x 10-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (Tables 9 and 12).
The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell in either the absence or presence of metabolic activation. No precipitate of the test item was observed at any dose level.
The numbers of small and large colonies and their analysis are presented in Tables 10 and 13.

Any other information on results incl. tables

Please see Attached ''Tables 1 -13'' Due to the quantity and the size of the tables it was not possible to insert them into this section.

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative