Registration Dossier

Administrative data

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:
Between 26 September 2012 and 21 October 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to OECD test guideline No. 471 and in compliance with GLP.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

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 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Monitoring Programme (inspection date: 10 July 2012/ signed on 30 November 2012)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Details on test material:
Description: clear colourless liquid
Specific details on test material used for the study:
- Storage conditions: room temperature in the dark
- Expiration date of the lot/batch: June 2013

Method

Target gene:
Histidine gene for S. typhimurium and tryptophan gene for E.coli
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix from the livers of rats induced with phenobarbitone/B-naphthoflavone
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.
Experiment 1: 50, 150, 500, 1500 and 5000 μg/plate .
Experiment 2: 15, 50, 150, 500, 1500 and 5000 μg/plate.
Justification: The maximum concentration was 5000 μg/plate (the maximum recommended dose level).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water at 50 mg/ml but was fully miscible in dimethyl sulphoxide at 50 mg/ml in solubility checks performed in-house.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
See Table 7.2.1/1
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
See Table 7.2.1/1
Positive control substance:
benzo(a)pyrene
other: 2-Aminoanthracene
Remarks:
With S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: Experiment 1 in agar (plate incorporation), Experiment 2 preincubation

DURATION
- Preincubation period (Exp 2): 20 minutes at 37°C.
- Exposure duration: ca. 48 hours at 37°C.

NUMBER OF REPLICATIONS: triplicate plates per dose level

DETERMINATION OF CYTOTOXICITY
- Method: growth assessment of the bacterial background lawn

OTHER: ACCEPTANCE CRITERIA: The reverse mutation assay may be considered valid if the following criteria are met:
1. 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).
2. All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (according to the historical control for 2010 & 2011).
3. All tester strain cultures should be in the approximate range of 0.9 to 9.9 billion bacteria per mL.
4. 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 (according to historical positive controls for 2010 & 2011).
5. There should be a minimum of four non-toxic test material dose levels.
6. There should be no evidence of excessive contamination.
Rationale for test conditions:
Experiment 1 - Maximum concentration was 5000 μg/plate (the maximum recommended dose level).
Experiment 2 - Maximum concentration was 5000 μg/plate (the maximum recommended dose level).
Up to six test item dose levels per bacterial strain were selected in the second mutation test in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test item.
Evaluation criteria:
There are several criteria for determining a positive result. Any, one or all of the following may be used to determine the overall result of the study, weighing the results in terms of their biological significance. The criteria are listed in order of priority:
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.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgement about test item activity. Results of this type will be reported as equivocal.
Statistics:
As recommended by UKEMS (Mahnon et al (1989).

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
At 5000 µg/plate using the plate incorporation method and from 1500 µg/plate using the pre-incubation method
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: not applicable
- Effects of osmolality: not applicable
- Evaporation from medium: the test material is not classified as a VOC.
- Water solubility: the test material was solubilised in DMSO to improve solubility.
- Precipitation: None observed.
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES: 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. See table 7.6.1/2.

COMPARISON WITH HISTORICAL CONTROL DATA:
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable.
All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

ADDITIONAL INFORMATION ON CYTOTOXICITY: In the range-finding test (plate incorporation method) the test item caused no toxicity in terms of weakened bacterial background lawns. However, decreases in revertant colony frequency were noted for several of the Salmonella strains at 5000 μg/plate. In the main test (pre-incubation method) the test item induced toxicity to all of the Salmonella strains
as weakened bacterial background lawns and/or decreases in revertant colony frequency, initially from 1500 μg/plate in the absence and presence of S9-mix. No toxicity was noted to Escherichia coli strain WP2uvrA. These results were not indicative of toxicity sufficiently severe enough to prevent the test item being tested up to the maximum recommended dose level of 5000 μg/plate.
Remarks on result:
other:
Remarks:
Table of results are in "Attached background documents"

Any other information on results incl. tables

Table 7.6.1/2: Test results: Preliminary toxicity test

Metabolic activation

Strain

Dose (µ/plate)

0

0.15

0.5

1.5

5

15

50

150

500

1500

5000

+

TA100

96

95

86

112

102

100

97

95

85

85

78

-

105

134

147

120

115

120

150

113

116

61

78

+

WP2

uvrA-

25

27

21

26

29

27

31

33

31

21

21

-

33

43

30

35

28

25

32

38

31

31

19

Applicant's summary and conclusion

Conclusions:
Under the test conditions, test item is not considered as mutagenic in S. typhimurium (TA1535, TA1537, TA98 and TA100) and E. coli WP2 uvrA strains.
Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and E.coli strain WP2 uvrA were exposed the test material diluted in DMSO both in the presence and absence of metabolic activation system (10% liver S9 in standard co-factors) using both the Ames plate incorporation and pre-incubation methods at up to six dose levels, in triplicate. The dose range for the range-finding test was determined in a preliminary toxicity assay and was 50 to 5000 μg/plate. The

experiment was repeated on a separate day (pre-incubation method) using an amended dose range (15 to 5000 μg/plate), fresh cultures of the bacterial strains and fresh test item formulations. An additional dose level and an expanded dose range were selected in the main test in order to achieve four non-toxic dose levels and the toxic limit of the test item.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals 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 range-finding test (plate incorporation method) the test item caused no toxicity in terms of weakened bacterial background lawns. However, decreases in revertant colony frequency were noted for several of the Salmonella strains at 5000 μg/plate. In the main test (pre-incubation method) the test item induced toxicity to all of the Salmonella strains as weakened bacterial background lawns and/or decreases in revertant colony frequency, initially from 1500 μg/plate in the absence and presence of S9-mix. In the range-finding test (plate incorporation method) the test item caused no toxicity in terms of weakened bacterial background lawns. However, decreases in revertant colony frequency were noted for several of the Salmonella strains at 5000 μg/plate. In the main test (pre-incubation method) the test item induced toxicity to all of the Salmonella strains as weakened bacterial background lawns and/or decreases in revertant colony frequency, initially from 1500 μg/plate in the absence and presence of S9-mix.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method

 

Under the test conditions, test item is not considered as mutagenic in S. typhimurium (TA1535, TA1537, TA98 and TA100) and E. coli WP2 uvrA strains.

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.