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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a GLP study conducted to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line, the test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells with or without metabolic activation. Therefore, the test item was considered to be non mutagenic in mammalian cells.

Link to relevant study records

Referenceopen allclose all

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.
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
Target gene:
Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.
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.
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.
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'.

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.

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.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The experimental phase of this study was performed between 15 February 2012 and 02 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 the relevant results.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
See Overall Remarks, Attachments
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine for Salmonella.
Tryptophan for E.Coli
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable.
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Not applicable.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/beta­naphthoflavone induced rat liver, S9
Test concentrations with justification for top dose:
Preliminary Toxicity Test; 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.
Expt 1: 50, 150, 500, 1500 and 5000 µg/plate.
Expt 2: All strains (with and without S9-mix): 150, 500, 1000, 1500, 2000, 3000 and 5000 µg/plate.
Expt 3: 500, 1000, 1500, 3000, 4000 and 5000 µg/plate.

Vehicle / solvent:
Vehicle: tetrahydrofuran
Justification for choice of solvent/vehicle: The test item was insoluble in sterile distilled water, dimethyl sulphoxide, dimethyl formamide and acetonitrile at 50 mg/ml and acetone at 100 mg/ml but was fully soluble in tetrahydrofuran at 200 mg/ml in solubility checks performed in house. Tetrahydrofuran was therefore selected as the vehicle.
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA100
Negative solvent / vehicle controls:
yes
Remarks:
THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 1 µg/plate
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1535
Negative solvent / vehicle controls:
yes
Remarks:
THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 2 µg/plate
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1537
Negative solvent / vehicle controls:
yes
Remarks:
THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 2 µg/plate
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of WP2uvrA
Negative solvent / vehicle controls:
yes
Remarks:
THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 10 µg/plate
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA98
Negative solvent / vehicle controls:
yes
Remarks:
THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
With S9 mix Migrated to IUCLID6: Benzo(a)pyrene: 5 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA98
Negative solvent / vehicle controls:
yes
Remarks:
THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9 mix Migrated to IUCLID6: 4-Nitroquinoline-1-oxide: 0.2 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1537
Negative solvent / vehicle controls:
yes
Remarks:
THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9 mix Migrated to IUCLID6: 9-Aminoacridine: 80 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA100
Negative solvent / vehicle controls:
yes
Remarks:
THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 3 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1535
Negative solvent / vehicle controls:
yes
Remarks:
THF
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 5 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of WP2uvrA
Negative solvent / vehicle controls:
yes
Remarks:
THF
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 2 µg/plate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) - Experiments 1 and 3

DURATION
- Preincubation period for bacterial strains: 10h
- Exposure duration: 48 - 72 hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable

NUMBER OF REPLICATIONS: Triplicate plating.

DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.

METHODS OF APPLICATION: in agar (pre-incubation) – Experiment 2
- Pre-incubation period for bacterial strains: 10hrs
- Exposure duration: 48-72hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (in incubation with a selective agent): 20 minutes at 37 degrees C

NUMBER OF REPLICATIONS: Triplicate plating.

DETERMINATION OF CYTOTOXICITY
-Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.
Evaluation criteria:
Acceptance Criteria:
The reverse mutation assay may be considered valid if the following criteria are met:
All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000). All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls. Combined historical negative and solvent control ranges for 2009 and 2010 are presented in Appendix 3. All tester strain cultures should be in the range of 0.9 to 9 x 109 bacteria per ml. Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation. The historical ranges of the positive controls for 2009 and 2010 are presented in Appendix 3. There should be a minimum of four non-toxic test item dose levels. There should be no evidence of excessive contamination.

Evaluation Criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby (1979)).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al (1989)).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Statistics:
Standard Deviation
Dunnetts Linear Regression Analysis
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
See Executive Summary
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
weakly mutagenic to one strain of bacteria (TA98 in the absence of S9-mix)
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
See Executive Summary
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECFIC CONFOUNDING FACTORS:
- Precipitation:
A greasy test item precipitate was observed at and above 1500 µg/plate, this observation did not prevent the scoring of revertant colonies.

STERILITY, VEHICLE AND POSITIVE CONTROL DATA:
- Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and S9 mix used in all experiments was shown to be sterile. The culture density for each bacterial strain was also checked and considered acceptable. These data are not given in the report.
Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test item, reference item and vehicle controls, both with and without metabolic activation, are presented in Table 2 and Table 3 for Experiment 1 and Table 4 and Table 5 for Experiment 2 and Table 6 for Experiment 3.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Results

The test item was non-toxic to the strains of bacteria used (TA100 and WP2uvrA). The test item formulation and S9-mix used in this experiment were both shown to be sterile.

The numbers of revertant colonies for the toxicity assay were:

 

 

With (+) or without (-)

S9-mix

Strain

Dose (µg/plate)

0

0.15

0.5

1.5

5

15

50

150

500

1500

5000

-

TA100

98

98

108

131

102

111

113

136

200

211P

209P

+

TA100

81

115

110

109

90

108

118

129

124

121P

139P

-

WP2uvrA

36

32

38

41

43

45

43

32

32

42P

42P

+

WP2uvrA

46

48

55

44

36

51

34

52

40

47P

51P

P        Precipitate

 

 

Mutation Test

Table 1: Spontaneous Mutation Rates (Concurrent Negative Control)

Experiment 1

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

97

 

15

 

26

 

17

 

12

 

88

(98)

14

(14)

42

(32)

20

(18)

13

(11)

110

 

12

 

27

 

18

 

9

 

 

Experiment 2

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

99

 

24

 

51

 

25

 

9

 

112

(108)

23

(24)

53

(52)

26

(25)

14

(11)

113

 

24

 

51

 

25

 

11

 

129

 

 

15

 

125

(124)

15

(16)

118

 

18

 

: Experimental procedure repeated at a later date (without S9-mix) due to technical issues in the original test

 

Experiment 3

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

TA98

119

 

24

 

22

 

103

(109)

21

(23)

29

(24)

104

 

23

 

20

 

 

 

FOR TABLES OF RESULTS FOR MUTATION TEST: Please see attached in overall remarks, attachments.

References:

Ames B N, McCann J and Yamasaki E (1975), Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsome mutagenicity test, Mutation Research, 31, 347-364.

Maron D M and Ames B N (1983), Revised Methods for the Salmonella mutagenicity test, Mutation Research, 113, 173 - 215.

Mortelmans K and Zeiger E (2000), The Ames Salmonella/microsome mutagenicity assay, Mutation Research, 455, 29-60.

Green M H L and Muriel W J (1976), Mutagen Testing Using TRP+ Reversion in Escherichia coli, Mutation Research, 38, 3-32.

De Serres F J and Shelby M D (1979), Recommendations on data production and analysis using the Salmonella/microsome mutagenicity assay, Environmental Mutagenesis, 1, 87-92.

Mahon G A T et al (1989) Analysis of data from microbial colony assays. In: Statistical Evaluation of Mutagenicity Test Data, UKEMS sub-committee on guidelines for mutagenicity testing, (Kirkland D J Ed.),Cambridge University PressReport, 26-65.

Maron D M, Katzenellenbogen J and Ames B N (1981), Compatibility of organic solvents with the Salmonella/Microsome Test, Mutation Research, 88, 343-350.

Conclusions:
Interpretation of results (migrated information):
positive without metabolic activation weakly mutagenic to one strain of bacteria (TA98 in the absence of S9-mix)

The test item, Bis(2,4-dichlorobenzoyl) peroxide (CAS# 133-14-2), paste, 50% in silicone oil was considered to be weakly mutagenic to one strain of bacteria (TA98 in the absence of S9-mix) under the conditions of this test.
Executive summary:

Introduction.  The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Methods. Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA were treated with the test item, Bis(2,4-dichlorobenzoyl) peroxide (CAS# 133-14-2), paste, 50% in silicone oil using both the Ames plate incorporation and pre-incubation methods at up to seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate in the first experiment. The experiment was repeated on separate days (pre-incubation method) using an amended dose range of 150 to 5000 µg/plate. Fresh cultures of the bacterial strains and fresh test item formulations were also employed. Additional and intermediate dose levels, as well as an expanded dose range, were selected in order to achieve four non-toxic dose levels, the toxic limit of the test item and to confirm a weak mutagenic response noted in the first experiment.

A third experiment was performed to attain reproducibility and a dose-response relationship after small but statistically significant increases in TA100, TA98 and TA1535 revertant colony frequency had been noted in the first experiment (plate incorporation methodology). The experiment employed a tightened test item dose range of 500, 1000, 1500, 3000, 4000 and 5000 µg/plate using the plate incorporation method.

Results. The vehicle (tetrahydrofuran) control plates gave counts of revertant colonies within the normal range. All of the positive controls used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

In the first experiment (plate incorporation method) the test item caused no visible reduction in the growth of the bacterial background lawns of any of the tester strains, either in the absence or presence of S9-mix. In the second experiment (pre-incubation method) the test item induced a weak toxic response to TA98 (in the absence of S9-mix only) with weakened bacterial background lawns noted from 3000 µg/plate. The test item was tested up to the maximum recommended dose level of 5000 µg/plate. A greasy test item precipitate was observed at and above 1500 µg/plate, this observation did not prevent the scoring of revertant colonies.

In the first experiment (plate incorporation method) small but statistically significant increases in the frequency of TA100, TA1535 and TA98 revertant colonies were observed at the upper dose levels of the test item in the absence of metabolic activation (S9-mix) with a modest dose-response relationship noted for TA100 and TA98. However, the test item induced a toxic response in the second experiment to TA98 (absence of S9-mix) after employing pre-incubation methodology (20 minutes at 37°C) and the responses noted in the first experiment were not reproduced.

Therefore, an additional (confirmatory) experiment was performed employing plate incorporation methodology (as Experiment 1) using those bacterial strains that showed a statistically significant increase (TA100, TA1535 and TA98 in the absence of S9-mix only). In this experiment, there were no meaningful increases in colony frequency noted for TA100 and TA1535 but there were dose-related and statistically significant increases in the frequency of TA98 revertant colonies at the upper dose levels of the test item (3000, 4000 and 5000 µg/plate). The individual numbers of revertant colonies at the statistically significant dose levels were generally above the upper limit of the in-house vehicle/untreated historical control range. Any excursions outside the maxima ranges, particularly when reproducibility is apparent (TA98 employing plate incorporation methodology), must be considered to be evidence of a potential biological response.

Conclusion. The test item, Bis(2,4-dichlorobenzoyl) peroxide (CAS# 133-14-2), paste, 50% in silicone oil was considered to be weakly mutagenic to one strain of bacteria (TA98 in the absence of S9-mix) under the conditions of this test.

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The experimental phases of the study were performed between 13 February 2012 and 27 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 the relevant results.
Qualifier:
according to
Guideline:
other: OECD Guidelines for Testing of Chemicals (2010) No 487: In Vitro Mammalian Cell Micronucleus Test.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
The work described was performed in compliance with UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)). Date of GLP inspection 19-21 July 2011. Date of Certificate 31 August 2011
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
Not applicable.
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a volunteer who had been previously screened for suitability. The volunteer had not been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral
infection.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone and beta-naphthoflavone induced rat liver, S9
Test concentrations with justification for top dose:
Preliminary toxicity Test
The dose range of test item used was 4.88 to 1250 µg/ml. The maximum recommended dose was 5000 µg/ml, however due to formulation difficulties the
maximum dose that could be achieved was 1250 µg/ml.
Micronucleus Test - Experiment 1
The dose levels of the controls and the test item are given in the table below:
Group Final concentration of Bis(2,4-dichlorobenzoyl) peroxide (CAS# 133-14-2), paste, 50% in silicone oil (µg/ml)
4-hour without S9 0*, 20, 40*, 80*, 120*, 160*, 200, 240, 320, MMC 0.2*
4-hour with S9 (2%) 0*, 40*, 80*, 160*, 240, 320, 400, 480, 640, CP 5*
Micronucleus Test - Experiment 2
The dose levels of the controls and the test item are given in the table below:
Group Final concentration of Bis(2,4-dichlorobenzoyl) peroxide (CAS# 133-14-2), paste, 50% in silicone oil (µg/ml)
20-hour without S9 0*, 10, 20, 40*, 80*, 120*, 160*, 200, 240, DC 0.075*
4-hour with S9 (1%) 0*, 10, 20*, 40*, 80*, 160*, 240, 320, CP 5*
* Dose levels selected for scoring of micronuclei in binucleate cells
DC = Demecolcine
CP = Cyclophosphamide

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: Acetone was selected as the solvent because the test material was readily soluble in it at the required
concentrations.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: cyclophosphamide
Remarks:
In the presence of S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
4-hour exposure in the absence of S9 Migrated to IUCLID6: (MMC)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: demecolcine
Remarks:
20-hour exposure in the absence of S9
Details on test system and experimental conditions:
METHOD OF APPLICATION:
in medium

DURATION
- Preincubation period:
48 hrs

- Exposure duration:
Experiment 1 - 4 hrs with and without S9. Experiment 2 - 20 hrs without S9, 4 hrs with S9.

- Expression time (cells in growth medium):
Not applicable

- Selection time (if incubation with a selection agent):
28 hrs incubation with Cytochalasin B after exposure, prior to harvest

- Fixation time (start of exposure up to fixation or harvest of cells):
32 hours for 4-hour exposures, 48 hours for 20-hour exposure


SELECTION AGENT (mutation assays):
No selection agent.

SPINDLE INHIBITOR (cytogenetic assays):
Not applicable

STAIN (for cytogenetic assays):
When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and coverslipped using mounting medium.


NUMBER OF REPLICATIONS:
Duplicate cultures


NUMBER OF CELLS EVALUATED:
1000 binucleate cells/culture


DETERMINATION OF CYTOTOXICITY
-A minimum of 500 cells per culture were scored for the incidence of mononucleate, bi-nucleate and multinucleate cells and the Cytokinesis Block Proliferation Index (CBPI) value expressed as a percentage of the vehicle controls. The CBPI indicates the number of cell cycles per cell during the period of exposure to Cytochalasin B.


Scoring of Micronuclei
The micronucleus frequency in 2000 binucleated cells was analysed per concentration (1000 binucleated cells per culture, two cultures per
concentration). Cells with 1, 2 or more micronuclei were recorded as such but the primary analysis was on the combined
data.


OTHER:
None.

Evaluation criteria:
A positive response was recorded when the p value calculated from the statistical analysis of the frequency of cells with micronuclei was less than 0.05 and there was a dose-related increase in the frequency of cells with micronuclei which was reproducible
Statistics:
The frequency of cells with micronuclei was compared, where necessary, with the concurrent vehicle control value using the Chi-squared Test on observed numbers of cells with micronuclei. Other statistical analysis may be used if appropriate, Hoffman et al (2003).
Species / strain:
lymphocytes: Human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Refer to information on results and attached tables.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no significant change in pH when the test material was dosed into media.
- Effects of osmolality: The osmalality did not increase by more than 50 mOsm.
- Evaporation from medium: Not applicable.
- Water solubility: Not applicable, test material dissolved in Acetone
- Precipitation: Refer to results section below

Preliminary Toxicity Test
The dose range for the Preliminary Toxicity Test was 4.88 to 1250 µg/ml. The maximum recommended dose was 5000 µg/ml, however due to formulation difficulties the maximum dose that could be achieved was 1250 µg/ml.
A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure period, at and above 312.5 µg/ml, which
became aggregated at 1250 µg/ml in all three exposure groups. Greasy/oily precipitate was also noted at and above 312.5 µg/ml in the parallel
blood-free cultures of the 4-hour with S9 exposure group and the 20-hour exposure group. Haemolysis was seen in the blood cultures at the end of the exposure period at and above 9.77 and 19.53 µg/ml in the 4 hour exposure groups in the absence and presence of S9 respectively. In the
20-hour exposure group haemolysis was seen at and above 78.13 µg/ml in the blood cultures at the end of the exposure period.
Microscopic assessment of the slides prepared from the exposed cultures showed that binucleate cells were present at up to 156.25 µg/ml in the
4-hour exposure group in the absence of S9 and the 20-hour exposure group in the absence of S9. In the 4-hour exposure group in the presence
of S9 bi-nucleate cells were present up to 312.5 µg/ml. The CBPI data are presented in the attached Table 1. The test item induced marked evidence
of toxicity in all three of the exposure groups.
The selection of the maximum dose level for the exposure groups of Experiment 1 and Experiment 2 was based on toxicity and was 320 and
640 µg/ml for the 4-hour exposure groups in the absence and presence of S9 respectively in Experiment 1. In Experiment 2, 240 µg/ml was selected as the maximum dose for the 20-hour exposure group and 320 µg/ml was selected as the maximum dose for the 4-hour exposure group in the
presence of S9.

Micronucleus Test - Experiment 1
The dose levels of the controls and the test item are given in the table below:
Group Final concentration of Bis(2,4-dichlorobenzoyl) peroxide (CAS# 133-14-2), paste, 50% in silicone oil (µg/ml)
4-hour without S9 0*, 20, 40*, 80*, 120*, 160*, 200, 240, 320, MMC 0.2*
4-hour with S9 (2%) 0*, 40*, 80*, 160*, 240, 320, 400, 480, 640, CP 5*
No precipitate was observed at the end of the exposure period. Haemolysis was observed at the end of exposure at and above 40 µg/ml in the
absence of S9 and at and above 80 µg/ml in the presence of S9.
The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were
binucleate cells suitable for scoring at 200 µg/ml in the absence of S9 and 320 µg/ml in the presence of S9, although due to the obvious toxicity at
this dose level it was not selected for CBPI analysis.
The CBPI and micronucleus data are given in the attached Table 2 and Table 3 for the without and with S9 groups. They confirm the qualitative
observations in that a dose-related inhibition of CBPI was observed in both exposure groups. A plateau of toxicity was seen in the 4-hour exposure group in the absence of S9 between 120 and 160 µg/ml where approximately 50% inhibition of cell proliferation was achieved. In the presence of S9 a similar plateau was seen at 160 µg/ml and 240 µg/ml where there was a 49% and 56% inhibition of cell proliferation respectively. The maximum dose level selected for analysis of binucleate cells was 160 µg/ml for both exposure groups where near optimum toxicity was achieved.
The vehicle control cultures had frequencies of cells with micronuclei within the expected range. The positive control items induced statistically
significant increases in the frequency of cells with micronuclei. The metabolic activation system was therefore shown to be functional and the test
method itself was operating as expected.
The test item did not induce any statistically significant increases in the frequency of cells with micronuclei, either in the absence or presence of
metabolic activation.
Micronucleus Test - Experiment 2
The dose levels of the controls and the test item are given in the table below:
Group Final concentration of Bis(2,4-dichlorobenzoyl) peroxide (CAS# 133-14-2), paste, 50% in silicone oil (µg/ml)
20-hour without S9 0*, 10, 20, 40*, 80*, 120*, 160*, 200, 240, DC 0.075*
4-hour with S9 (1%) 0*, 10, 20*, 40*, 80*, 160*, 240, 320, CP 5*
* Dose levels selected for scoring of micronuclei in binucleate cells
DC = Demecolcine
CP = Cyclophosphamide

No precipitate of the test item was seen at the end of the exposure period in either exposure group. Haemolysis was noted at the end of the exposure period at and above 80 µg/ml in the 20-hour exposure group and at and above 20 µg/ml in the 4-hour exposure group
The qualitative assessment of the slides determined that there were binucleate cells suitable for scoring up to 160 µg/ml in both exposure groups.
The CBPI and micronucleus data are given in the attached Table 4 and Table 5 for the without and with S9 exposure groups. They confirm the
qualitative observations in that a dose-related inhibition of CBPI was observed in both exposure groups. In the 20-hour exposure group there was a plateau of toxicity with 44% and 43% inhibition of cell proliferation at 120 and 160 µg/ml respectively. In the 4-hour exposure group in the presence of S9 a dose-related inhibition of CBPI was observed and 39% and 68% inhibition of cell proliferation was achieved at 80 and 160 µg/ml respectively.
The increased toxicity of the test item seen in the 4-hour exposure group with S9 in Experiment 2 compared to Experiment 1 was considered to be
due to the reduced S9 concentration having less of a protective effect to the cells. The maximum dose level selected for binucleate cell analysis was
160 µg/ml for both exposure groups and was the maximum dose level with binucleate cells suitable for scoring.
The vehicle control cultures had frequencies of cells with micronuclei within the expected range. The positive control items induced statistically significant increases in the frequency of cells with micronuclei. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.
The test item did not induce any statistically significant increases in the frequency of cells with micronuclei, either in the absence or presence of
metabolic activation.
Remarks on result:
other: strain/cell type:
Remarks:
Migrated from field 'Test system'.

For the tables of results mentioned above, please refer to the attached background material section for the following tables:

Table 1 CBPI - Preliminary Toxicity Test

Table 2 CBPI and Micronucleus Data – Experiment 1- 4-hour exposure without S9                

Table 3 CBPI and Micronucleus Data – Experiment 1 - 4-hour exposure with S9 (2%)

Table 4 CBPI and Micronucleus Data – Experiment 2 - 20-hour exposure without S9

Table 5 CBPI and Micronucleus Data – Experiment 2 - 4-hour exposure with S9 (1%)

Conclusions:
Interpretation of results (migrated information):
negative

The test item did not induce a statistically significant increase in the frequency of cells with micronuclei in either the absence or presence of a metabolising system, in either of two separate experiments. The test item was therefore considered to be non-clastogenic and non aneugenic to human lymphocytes in vitro.
Executive summary:

Introduction. 

This report describes the results of an in vitro study for the detection of the clastogenic and aneugenic potential of the test item on the nuclei of normal human lymphocytes The test method was designed to be compatible with the following:

OECD Guidelines for Testing of Chemicals (2010) No 487:In Vitro Mammalian Cell Micronucleus Test.

Methods. 

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells at up to four dose levels, together with vehicle and positive controls. Four exposure conditions were used for the study. Experiment 1 used a 4 hour exposure in the presence and absence of a standard metabolising system (S9, at a 2% final concentration). Experiment 2, used a repetition of the 4 hour exposure with S9 (at a 1% final concentration), whilst in the absence of metabolic activation the exposure time was increased to 20 hours.

The dose levels used in the main experiments were selected using data from the preliminary toxicity test and were as follows:

Group

Final concentration of the test item(µg/ml)

4-hour without S9

20, 40, 80, 120, 160, 200, 240, 320

4-hour with S9 (2%)

40, 80, 160, 240, 320, 400, 480, 640

20-hour without S9

10, 20, 40, 80, 120, 160, 200, 240

4-hour with S9 (2%)

10, 20, 40, 80, 160, 240, 320

Results. 

All vehicle (solvent) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes.

The positive control items induced statistically significant increases in the frequency of cells with micronuclei, indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test item was toxic but did not induce any statistically significant increases in the frequency of cells with micronuclei, in either of the two experiments, using a dose range that included a dose level that induced approximately 50% reduction in CBPI or greater.

Conclusion. 

The test item was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Additional information from genetic toxicity in vitro:

1.    Mammalian cell gene mutation: Mouse Lymphoma Assay (Flanders, 2012)

A 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 Guideline 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.

Study Conclusion:The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells with or without metabolic activation and is therefore considered to be non mutagenic under the conditions of the test.

2.    Bacterial gene mutation: Bacterial reverse mutation assay by Ames test (Thompson, 2012)

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing by the Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guideline No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonized guidelines.

Method and results:Salmonella typhimuriumstrains TA1535, TA1537, TA98, TA100 andEscherichia colistrain WP2uvrAwere treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to 7 dose levels, in triplicate, both with and without the addition of a metabolising system (10% rat liver S9 in standard co-factors).  A total of 3 experiments were performed.The range of revertant colonies in vehicle (tetrahydrofuran) control plates and all positive controls, both with or without metabolic activation showed that the sensitivity of the assay and the efficacy of the S9-mix were validated. In the first experiment (plate incorporation method) small but statistically significant increases in the frequency of TA100, TA1535 and TA98 revertant colonies were observed at the upper dose levels of the test item (minus S9-mix) with a modest dose-response relationship noted for TA100 and TA98. However, the test item induced a toxic response in the second experiment to TA98 (absence of S9-mix) after employing pre-incubation methodology (20 minutes at 37°C) and the responses noted in the first experiment were not reproduced. Therefore, an additional (confirmatory) experiment was performed employing plate incorporation methodology (as Experiment 1) using those bacterial strains that showed a statistically significant increase (TA100, TA1535 and TA98 (plus S9-mix only). In this experiment, there were no meaningful increases in colony frequency noted for TA100 and TA1535 but there were dose-related and statistically significant increases in the frequency of TA98 revertant colonies at the upper dose levels of the test item (3000, 4000 and 5000 µg/plate). The individual numbers of revertant colonies at the statistically significant dose levels were generally above the upper limit of the in-house vehicle/untreated historical control range. Any excursions outside the maxima ranges, particularly when reproducibility is apparent (TA98 employing plate incorporation methodology), must be considered to be evidence of a potential biological response.

Study Conclusion.The test item was considered to be weakly mutagenic to one strain of bacteria (TA98 in the absence of S9-mix) under the conditions of this test.

3.    Chromosome aberration: In vitro mammalian cell micronucleus test in human lymphocytes (Morris, 2012)

The test was an in vitro study for the detection of the clastogenic and aneugenic potential of the test item on the nuclei of normal human lymphocytes The test method was designed to be compatible with the OECD Guideline No. 487: In Vitro Mammalian Cell Micronucleus Test.

Study Conclusion:The test item did not induce a statistically significant increase in the frequency of cells with micronuclei in either the absence or presence of a metabolising system, in either of two separate experiments. The test item was therefore considered to be non-clastogenic and non aneugenic to human lymphocytes in vitro.

Overall discussion of the endpoint summary: Although the test item, Bis(2,4-dichlorobenzoyl) peroxide, paste, 50% in silicone oilwas weakly mutagenic to one strain of bacteria in Ames test (TA98 in the absence of S9 -mix), the test item was shown to be non-mutagenic in L5178Y cells (no toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells, with or without metabolic activation) as well as non-clastogenic and non-aneugenic to human lymphocytes in vitro (no significant increase in the frequency of cells with micronuclei, with or without metabolic activation). Both are mammalian cells. In accordance with Endpoint Specific Guidance Chapter R.7A, Figure R.7.7 -1 "Flow chart of the mutagenicity testing strategy", no further testing (ie. no in vivo testing) need be proposed in the event of a negative mouse lymphoma assay or hprt assay, regardless of whether or not the gene mutation test in bacteria is positive or negative. This, therefore, implies that when considering whether an in vivo gene mutation request is required for substances requiring Annex IX test proposals due to their volume bands, a negative mouse lymphoma assay is sufficient evidence to waive the need for an in vivo gene mutation test.


Justification for selection of genetic toxicity endpoint
The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells with or without metabolic activation and is therefore considered to be non mutagenic.

Justification for classification or non-classification

In a bacterial reverse mutation assay, the test substance was mutagenic to one starin of bacteria only, TA98 (-S9), in the plate incorporation method. The response was not reproducible in the preincubation method. In an in vitro mammalian cell micronucleus test in human lymphocytes (Morris, 2012), a chromosomal abberation study, the test substance is shown to be non-clastogenic and non-aneugenic. Lastly, the test item was not mutagenic to mammalian cells in an MLA test in vitro. Therefore, based on the Weight of Evidence, the substance is not expected to be mutagenic.

Therefore, the genetic toxicity data are considered conclusive but not sufficient for classification.