Formaldehyde, reaction products with phenol heptyl derivs. and 1,3,4-thiadiazolidine-2,5-dithione

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The experimental phases of the study were performed between 23 February 2012 and 18 April 2012.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of therelevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction product of 1,3,4-thiadiazolidine-2,5-dithione, formaldehyde and phenol, heptyl derivs.

EC Number:

939-460-0

Cas Number:

1471311-26-8

Molecular formula:

Too complex

IUPAC Name:

Reaction product of 1,3,4-thiadiazolidine-2,5-dithione, formaldehyde and phenol, heptyl derivs.

Details on test material:

Sponsor's identification:OS48604M
Description :Amber coloured liquid
Purity :Mixture
Batch number :OS48604M
Date received :13 January 2012
Expiry date :31 December 2013
Storage conditions:Room temperature in the dark

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 were limited by test item-induced toxicity.
Vehicle and positive controls were used in parallel with the test item. Solvent (DMSO) treatment groups were used as the vehicle controls. Ethylmethanesulphonate (EMS), Sigma batches BCBC4573V and 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 batches A0277203 and A0302605 at 2 µg/ml for Experiment 1 and Experiment 2, respectively, was used as the positive control in the absence of metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
Solvent (DMSO) treatment groups were used as the vehicle controls.
- Justification for choice of solvent/vehicle:
Suitable for dosing at required concentration

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Solvent (DMSO) treatment groups were used as the vehicle controls.

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

EXAMPLE: Solvent (DMSO) treatment groups were used as the vehicle controls.

True negative controls:

no

Positive controls:

yes

Positive control substance:

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 eight 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 eight 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 for Experiment 1 was 2.5 to 80 µg/ml in both the absence and presence of metabolic activation. In Experiment 2 the dose range was 1.25 to 80 µg/ml in the absence of metabolic activation, and 2.5 to 100 µg/ml in the presence of metabolic activation.
The maximum dose levels used in the Mutagenicity Test were limited by test item-induced toxicity. No precipitate of test item was observed at any of the dose levels in the Mutagenicity Test. 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 incl. tables'' section

Statistics:

Please see ''Any other information on materials 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

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 19.53 to 5000 µg/ml
In all three of the exposure groups there was evidence of marked dose-related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls. The steep nature of the toxicity curve was taken to indicate that achieving optimum toxicity would be difficult. Immediately on dosing, precipitate of the test item was observed at and above 78.13 µg/ml in the 4-hour exposure group in the absence of metabolic activation, at and above 156.25 µg/ml in the 24-hour exposure group in the absence of metabolic activation and the 4-hour exposure group in the presence of metabolic activation. Greasy / oily aggregated precipitate was observed at and above 2500 µg/ml in all three of the exposure groups. In addition, an increase in intensity was associated with an increase in dose concentration. Based on the %RSG values observed, the maximum dose levels in the subsequent Mutagenicity Test were limited by test item induced toxicity.
Experiment 1
The results of the microtitre plate counts and their analysis are presented in Tables 2 to 7.
There was once again evidence of marked 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). The levels of toxicity observed were similar to those of the preliminary toxicity test. There was evidence of very modest reductions in viability (%V) in both the absence and presence of metabolic activation, therefore indicating that residual toxicity had occurred. However, it should be noted that the reduction observed in the absence of metabolic activation was at a dose level that had been excluded from the statistical analysis due to the toxicity exceeding the upper acceptable limit of 90%. Based on the %RSG and RTG values observed, it was considered that optimum levels of toxicity had been achieved in the presence of metabolic activation and near optimum levels in the absence of metabolic activation. Whilst optimum levels of toxicity were not achieved in the absence of metabolic activation, a dose level that marginally exceeded the usual upper limit of acceptable toxicity was plated for viability and 5-TFT resistance as sufficient cells were available at the time of plating. Acceptable levels of toxicity were seen with both positive control substances (Tables 3 and 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 any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell at any of the dose levels in the presence of metabolic activation (Table 6). A very modest but statistically significant dose related (linear trend) increase in mutant frequency was observed in the absence of metabolic activation (Table 3). The greatest increases in mutant frequency were only observed at dose levels that approached or exceeded the upper limit of acceptable toxicity. However, the GEF was not exceeded at any of the dose levels, including the dose level that marginally exceeded the usual upper limit of acceptable toxicity, there was no evidence of any increases in absolute numbers of mutant colonies, and statically significant increases were not observed at any of the individual dose levels. The mutant frequency value observed at 60 µg/ml marginally exceeded the upper limit for vehicle controls; however, there was no evidence of a shift towards small colony formation that would have indicated a clastogenic response. It was considered that the response observed in the absence of metabolic activation was artefactual and due to cytotoxicity and not a true genotoxic response and were, therefore, of no toxicological significance. Precipitate of test item was not observed at any of the dose levels.
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 evidence of marked 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 9 and 12). There was evidence of a very modest reduction in viability (%V) in the presence of metabolic activation, therefore indicating that residual toxicity had occurred. Based on the %RSG and RTG values observed, it was considered that optimum levels of toxicity had been achieved in both the absence and presence of metabolic activation. The excessive toxicity observed at and above 40 µg/ml in the absence of metabolic activation, and at and above 80 µg/ml in the presence of metabolic activation, resulted in these dose levels not being plated for viability or 5-TFT resistance. 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 modest effect on the toxicity of the test item. It should be noted that the lowering of the S9 concentration to 1% in this second experiment resulted in slightly greater levels of toxicity being observed when compared to 4-hour exposure groups in the presence of 2% metabolic activation in the Preliminary Toxicity Test and Experiment 1.
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 induced very modest but statistically significant dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell in both the absence and presence of metabolic activation (Tables 9 and 12). The greatest increases in mutant frequency were only observed at dose levels that approached the upper limit of acceptable toxicity. However, the GEF was not exceeded at any of the dose levels, there was no evidence of any marked increases in absolute numbers of mutant colonies, and statically significant increases were not observed at any of the individual dose levels. The mutant frequency value observed at 20 µg/ml in the absence of metabolic activation marginally exceeded the upper limit for vehicle controls, however, there was no evidence of a shift towards small colony formation that would have indicated a clastogenic response. It was considered that the responses observed in both the absence and presence of metabolic activation were artefactual and due to cytotoxicity and not a true genotoxic response and were, therefore, of no toxicological significance. Precipitate of test item was not observed at any of the dose levels.
The numbers of small and large colonies and their analysis are presented in Tables 10 and 13

Remarks on result:

other: strain/cell type:

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Please see attached Tables 1 -13

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative Non-mutagenic

The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non mutagenic under the conditions of the test.

Executive summary:

Introduction. 

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.

Methods. 

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 eight 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 eight 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 for Experiment 1 was 2.5 to 80 µg/ml in both the absence and presence of metabolic activation. In Experiment 2 the dose range was 1.25 to 80 µg/ml in the absence of metabolic activation, and 2.5 to 100 µg/ml in the presence of metabolic activation.

Results. 

The maximum dose levels used in the Mutagenicity Test were limited by test item-induced toxicity. No precipitate of test item was observed at any of the dose levels in the Mutagenicity Test. 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.

The test item did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment.

Conclusion. 

The test item was considered to be non-mutagenic to L5178Y cells under the conditions of the test.