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Toxicological information

Genetic toxicity: in vitro

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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:
From October 31 to December 07, 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

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 Program (inspected on August 21, 2007 / Signed on October 15, 2007)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Remarks:
paste / solid block
Details on test material:
- Physical state: solid, pale yellow to yellow block, or pasty
- Stability under test conditions: no data
- Storage condition of test material: in the dark, preferably at about 4-10°C and under nitrogen.

Method

Target gene:
Histidine and tryptophan.
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
10% S9: S9-mix from the livers of male Wistar rats treated with phenobarbitone/β-naphthoflavone (80/100 mg/kg bw/day by oral route).
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 in TA100 or WP2uvrA strain with and without S9-mix using direct plate incorporation method.
- Experiment-1 (Range-finding test): 15, 50, 150, 500, 1500 and 5000 µg/plate in all strains with and without S9-mix using direct plate incorporation method.
- Experiment-2 (Main test): 50, 150, 500, 1500 and 5000 µg/plate in all strains with and without S9-mix using direct plate incorporation method.
Limiting factor was the maximum recommended dose.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide
- Justification for choice of solvent/vehicle: The test material formed a doseable suspension in sterile distilled water at 50 mg/mL but was fully soluble in dimethyl sulphoxide at the same concentration in solubility checks performed in-house. Dimethyl sulphoxide was therefore selected as the vehicle.
- Preparation of test materials: 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:
dimethyl sulphoxide
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:
dimethyl sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 2-Aminoanthracene (2AA)
Remarks:
With S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: not applicable
- Exposure duration: Plates were incubated for approximately 48 h at 37 °C.
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable

SELECTION AGENT (mutation assays): not applicable

NUMBER OF REPLICATIONS: Single plate/dose for preliminary toxicity study; triplicate plates per dose level for mutation study (Experiment 1 and 2).


NUMBER OF CELLS EVALUATED: not applicable

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

OTHERS:
After incubation, the plates were assessed for numbers of revertant colonies using a Domino colony counter.
Rationale for test conditions:
Experiment 1 & 2 - Maximum concentration was 5000 μg/plate (the maximum recommended dose level).
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 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 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 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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
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: No expected
- Water solubility: The test material was solubilised in dimethyl sulphoxide to improve solubility.
- Precipitation: No
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES: Test material exhibited a weak toxic response for TA100 in the absence of S9 and was non-toxic for TA100 in the presence of S9 and WP2uvrA in the presence and absence of S9.

COMPARISON WITH HISTORICAL CONTROL DATA: All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and positive controls. The comparison was made with the historical control ranges for 2006 and 2007 of the corresponding Testing Laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY: The test material caused a visible reduction in the growth of the bacterial background lawns of several of the Salmonella tester strains at 5000 µg/plate predominantly in the absence of S9.

OTHERS:
- The test material formulation and S9-mix used in this experiment were both shown to be sterile.

Any other information on results incl. tables

Table 7.6.1/2: Test results: Preliminary toxicity test

Metabolic activation

Strain

Dose (µg/plate)

0

0.15

0.5

1.5

5

15

50

150

500

1500

5000

-

TA100

115

125

127

102

108

121

100

111

120

126

67*

+

93

80

79

77

74

76

86

64

76

73

52

-

WP2

uvrA-

22

25

12

18

19

22

15

13

11

20

17

+

22

23

28

20

18

16

20

26

19

20

13

Key: * = Partial absence of bacterial background lawn.

Applicant's summary and conclusion

Conclusions:
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 WP2 uvrA- 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, Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and Escherichia coli strain WP2 uvrA- were exposed to test material diluted in dimethyl sulphoxide 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 ranged between 15 and 5000 µg/plate. The experiment was repeated on a separate day using a modified dose range, 50 to 5000 µg/plate based on the results of the range-finding test. 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 lawns of several of the Salmonella tester strains at 5000 µg/plate predominantly in the absence of S9. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence 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 material, 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 WP2 uvrA- 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.