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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
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 28 February 2012 and 23 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 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
Principles of method if other than guideline:
Add any principals of methods if other than guidelines: Or delete this section and leave blank.
GLP compliance:
yes
Remarks:
Date of GLP inspection: 19 - 21 July 2011 Date of GLP signature: 31 August 2011
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
other: white paste
Details on test material:
Identification: Bis(2,4-dichlorobenzoyl) peroxide (CAS# 133-14-2), paste, 50% in silicone
oil
Supplier: Akzo Nobel Polymer Chemicals, b.v.
Product name: Perkadox PD-50S-PS
Chemical name: Di(2,4-dichlorobenzoyl) peroxide
EC name: Bis(2,4-dichlorobenzoyl) peroxide
Product number: 66190
Batch number: 1012011395
CAS number: 133-14-2
Date of receipt: 08 November 2011
Expiry Date: 01 January 2013
Appearance: White Paste
Purity/Concentration: Bis(2,4-dichlorobenzoyl) peroxide (CAS# 133-14-2), paste (49-51%). The paste typically contains silicone oil (polydimethylsiloxanes, CAS # 63148-62-9) at 49 - 51%, but the typical average concentrations do not constitute product specifications.
The integrity of supplied data relating to the identity, purity and stability of the test item is the responsibility of the Sponsor.
Stored at approximately -20oC; used/formulated at ambient temperature <30oC.

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 (acetone) 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 batch A0277203 at 2 µg/ml was used as the positive control in the presence of metabolic activation.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used:
Solvent (Acetone) treatment groups were used as the vehicle controls.

- Justification for choice of solvent/vehicle:
Suitable for dosing at the required concentration.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Solvent (Acetone) 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 (Acetone) 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:
This 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 use of cultured mammalian cells for mutation studies may give a measure of the intrinsic response of the mammalian genome and its maintenance process to mutagens. Such techniques have been used for many years with widely different cell types and loci. The thymidine kinase heterozygote system, TK +/- to TK -/-, was described by Clive et al., (1972) and is based upon the L5178Y mouse lymphoma cell line established by Fischer (1958). This system has been extensively validated (Clive et al., 1979; Amacher et al, 1980; Jotz and Mitchell, 1981).

The method used 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. The technique used was a fluctuation assay using microtitre plates and trifluorothymidine as the selective agent and is based on that described by Cole and Arlett (1984). Two distinct types of mutant colonies can be recognised, i.e. large and small. Large colonies grow at a normal rate and represent events within the gene (base-pair substitutions or deletions) whilst small colonies represent large genetic changes involving chromosome 11b (indicative of clastogenic activity).
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
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 2.44 to 625 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. A cloudy precipitate of the test item was observed at and above 78.13 µg/ml and a greasy oily precipitate was observed at and above 156.25 µg/ml at the end of the exposure period in all three of the exposure groups. In addition, an increase in intensity was associated with an increase in dose concentration. Based on %RSG values observed, the maximum dose levels in the subsequent Mutagenicity Test were limited by test item-induced toxicity.

Mutagenicity Test

A summary of the results from the test is presented in attached Table 1.

Experiment 1

The results of the microtitre plate counts and their analysis are presented in attached Tables 2 to 7.

There was once again evidence of marked dose-related toxicity following exposure to the test item in the absence of metabolic activation, as indicated by the RTG and %RSG values (Table 3). In the presence of metabolic activation, the marked toxicity observed in the preliminary toxicity test was not reproduced and only modest levels of toxicity were achieved (Table 6). This was considered to be due to the very steep toxicity curve of the test item and inter-experimental variation. There was no evidence of any significant dose related reductions in viability (%V) in any of the dose levels, therefore indicating that no residual toxicity had occurred in either the absence or presence of metabolic activation. Based on the %RSG and RTG values observed, it was considered that optimum levels of toxicity had been achieved in the absence of metabolic activation. Whilst optimum levels of toxicity were not achieved in the presence of metabolic activation, it was considered that with no evidence of a response in Experiment 1, or Experiment 2 where optimum levels of toxicity were achieved in the presence of metabolic activation using a similar but slightly higher dose range, a repeat of this exposure group was not required. 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 in either the absence or presence of metabolic activation (Tables 3 and 6). 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 attached Tables 8 to 13.

As was seen in the preliminary toxicity test, 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 significant dose related reductions in viability (%V) in the presence of metabolic activation, therefore indicating that residual toxicity had occurred. However, it should be noted that the greatest reduction was observed at a dose level that had been excluded from the statistical analysis due to excessive levels of toxicity. It should also be noted that heterogeneity (poor correlation between A and B cultures), and a slight increase in mutant frequency, was observed at this dose level. However, this was considered to be due to the very high levels of toxicity and not a true genotoxic response. Based on the %RSG and / or 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 35 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. The toxicity observed at 60 µg/ml in the presence of metabolic activation exceeded the upper acceptable limit of 90%, therefore, this dose was excluded from the statistical analysis. 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 slight effect on the toxicity of the test item. It should also be noted that the lowering of the S9 concentration to 1% in this second experiment resulted in much 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 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 either the absence or presence of metabolic activation (Tables 9 and 12). Precipitate of test item was not observed at any of the dose levels. It was considered that the result obtained in the presence of metabolic activation demonstrated that a repeat of the 4-hour exposure group from Experiment 1, where optimum toxicity was not achieved, was not required and the test item had been adequately tested.

The numbers of small and large colonies and their analysis are presented in Tables 10 and 13.
Remarks on result:
other: strain/cell type: Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Please see Attached "Tables 1 to 13"

Due to the nature and quantity of tables it was not possible to insert them in this section.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
other: 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 up to ten 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 0.63 to 40 µg/ml in the absence of metabolic activation, and 2.5 to 80 µg/ml in the presence of metabolic activation for Experiment 1. In Experiment 2 the dose range was 5 to 50 µg/ml in the absence of metabolic activation, and 10 to 120 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. Precipitate of test item was not 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.