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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From August 11 to 25, 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD test guideline No. 471 without any deviation.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

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
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Programme (inspected on December 02, 2002 /signed on February 13, 2003)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical state: Colorless to slightly yellow liquid
- Storage condition of test material: Cold and dark storage place
Specific details on test material used for the study:
Storage condition of test material: Stored at approximately 4 °C in the dark under nitrogen.

Method

Target gene:
Histidine and tryptophan for S. typhimurium and E. coli, respectively.
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:
10 % S9 mix: S9 from liver of male Sprague-Dawley rats orally received three consecutive daily doses of phenobarbitone/β-naphthoflavone (80/100 mg/kg bw/day) prior to S9 preparation on Day 4
Test concentrations with justification for top dose:
Preliminary toxicity study: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in TA100 and WP2uvrA-, with and without S9-mix using the direct plate incorporation method.

Mutation test (direct plate incorporation method):

- Experiment-1(range-finding test)
Salmonella strains (with and without S9-mix): 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
E.coli strain WP2uvrA- (with and without S9-mix): 50, 150, 500, 1500 and 5000 µg/plate

- Experiment-2 (main test)
Salmonella strains (with and without S9-mix): 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
E.coli strain WP2uvrA- (with and without S9-mix): 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: Test material was immiscible at 50 mg/mL in water, but miscible at 50 mg/mL in DMSO. Therefore, DMSO was selected as vehicle.
- Preparation of test materials: The test material was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer on the day of each experiment.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
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
Positive control substance:
benzo(a)pyrene
other: 2-aminoanthracene
Remarks:
With S9-mix.
Details on test system and experimental conditions:
SOURCE OF TEST SYSTEM: Salmonella typhimurium strains were obtained from the University of California at Berkeley whilst Escherichia coli strain WP2uvrA- was obtained from the British Industrial Biological Research Association.

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: Approximately 48 h at 37 °C.

NUMBER OF REPLICATIONS:
- Preliminary toxicity study: One plate/dose
- Mutation study: 3 plates/dose

DETERMINATION OF CYTOTOXICITY
- Method: Evaluation of the toxicity was performed on the basis of growth of the bacterial background lawn.
Evaluation criteria:
- There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS (Kirkland, 1989) can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
- A test material 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 a definitive judgement about the test material activity. Results of this type will be reported as equivocal.
Statistics:
Statistical methods, as recommended by the UKEMS (Kirkland, 1989) can be used as an aid to evaluation. However, statistical significance will not be the only determining factor for a positive response.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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: No data
- Water solubility: immiscible in water.
- Precipitation: A slight, oily precipitate was observed at 5000 µg/plate, but this did not prevent the scoring of revertant colonies.
- Other confounding effects: None

PRELIMINARY TOXICITY STUDY: Test material exhibited toxicity from 500 µg/plate to TA100 (without S9-mix) and was non-toxic to WP2uvrA- strain.

COMPARISON WITH HISTORICAL CONTROL DATA: The comparison was made with the historical control ranges for 2003 and 2004 of the corresponding testing laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The test material caused a visible reduction in the growth of the bacterial background lawn to all of the Salmonella strains, both with and without S9-mix, initially from 1500 µg/plate. No toxicity was observed to E.coli strain, WP2uvrA-. These results were not indicative of toxicity sufficiently severe enough to prevent the test material being tested up to the maximum recommended dose level of 5000 µg/plate.

Any other information on results incl. tables

Table 7.6.1/2: Preliminary toxicity test results

Metabolic activation

Strain

Dose (µg/plate)

0

0.15

0.5

1.5

5

15

50

150

500

1500

5000

-

TA100

79

76

86

93

104

87

78

88

73*

52*

0*P

+

TA100

95

81

93

93

80

81

98

78

85

40*

19*P

-

WP2uvrA-

19

25

26

30

20

30

23

24

26

22

20P

+

WP2uvrA-

34

24

22

27

30

24

43

31

15

22

18P

Key: *- Partial absence of bacterial background lawn

         P- Precipitate

See the attached document for information on tables of results – mutagenicity test

Applicant's summary and conclusion

Conclusions:
Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium (strains TA 1535, TA 1537, TA 98 and TA 100) and E. coli WP2 uvr A- according to the criteria of the Annex VI of the of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.
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 TA1535, TA1537, TA98 and TA100 and E.coli strain WP2 uvrA- were exposed to test material diluted in DMSO, both in the presence and absence of metabolic activation system (10% liver S9 in standard co-factors), using the plate incorporation method. The dose range for the range-finding test was determined in a preliminary toxicity assay and was 50 to 5000 and 5 to 5000 μg/plate for WP2uvrA-and the Salmonella strains respectively (with and without S9-mix). The experiment was repeated on a separate day using the same extended dose range as the range-finding test. Negative, vehicle (dimethyl sulphoxide) and positive control groups were also included in mutagenicity tests.

The vehicle 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. The test material caused a visible reduction in the growth of the bacterial background lawn to all of the Salmonella strains, with and without S9-mix, initially from 1500 µg/plate, although less toxicity was observed to TA 98. However, no toxicity was observed to E.coli WP2uvrA- strain. These results were not indicative of toxicity sufficiently severe enough to prevent the test material being tested up to the maximum recommended dose level of 5000 µg/plate. A slight, oily precipitate was observed at 5000 µg/plate but this did not prevent the scoring of revertant colonies. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation.

Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium strains TA1535, TA1537, TA98 and TA100, and E.coli WP2uvrA- according to the criteria of the Annex VI of the of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

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