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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 2006-07-01 to 2006-09-01
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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

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 (inspection date: 2005-08-30)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical state: clear colourless liquid
- Storage condition of test material: ca. 4°C under nitrogen in the dark

Method

Target gene:
Histidine gene for Salmonella 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 (S9 fraction from Sprague-Dawley rats pre-treated with phenobarbitone/B-naphtoflavone)
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.
Range-finding test: 50, 150, 500, 1500 and 5000 μg/plate.
Main test: 50, 150, 500, 1500 and 5000 μg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The test material was immiscible in sterile distilled water and dimethyl sulphoxide at 50 mg/mL but was fully miscible in acetone at the same concentration in solubility checks performed in-house.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Remarks:
See Table 7.6.1/1
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:
Acetone
True negative controls:
no
Positive controls:
yes
Remarks:
See Table 7.6.1/1
Positive control substance:
benzo(a)pyrene
other: 2-Aminoanthracene
Remarks:
With S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- 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 was considered valid if the following criteria were met:
1. All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (according to historical control 2004 & 2005).
2. The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor etc.
3. All tester strain cultures should be in the approximate range of 1 to 9.9 billion bacteria per mLl.
4. Each mean positive control value should be at least two times the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
5. There should be a minimum of four non-toxic test material dose levels.
6. There should be no evidence of excessive loss of plates due to contamination.
Evaluation criteria:
Dose-related increase in revertant frequency over the dose range tested and/or reproducible at one or more concentrations in at least one bacterial strain with or without metabolic activation.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Statistics:
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.

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:
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: Test material was solubilised in acetone to improve solubility
- Precipitation: a precipitate (oily in appearance) was observed at 5000 µg/plate, this did not prevent the scoring of revertant colonies.
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES: The test material was non-toxic to the bacterial background lawns of the strains of bacteria used (TA100 and WP2uvrA-). The test material formulation and S9-mix used in this experiment were both shown to be sterile.

COMPARISON WITH HISTORICAL CONTROL DATA: 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-tnix and the sensitivity of the bacterial strains.

ADDITIONAL INFORMATION ON CYTOTOXICITY: The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level, although substantial decreases in revertant colony frequency were observed to all of the Salmonella strains dosed in the presence of S9 at 5000 µg/plate, The test material was, therefore, tested up to the maximum recommended dose level of 5000 ug/plate
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Preliminary test:

The numbers of revertant colonies for the toxicity assay were:

With or without metabolic activation

Strain

Dose (µg/plate)

0

0.15

0.5

1.5

5

15

50

150

500

1500

5000

-

TA100

103

91

108

110

93

99

85

88

70

87

98P

+

TA100

110

110

92

89

96

93

97

89

90

111

46P

-

WP2uvrA-

20

13

21

14

23

18

25

15

21

21

20P

+

WP2uvrA-

29

25

26

30

29

26

30

30

23

26

20P

Mutation test:

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. See "Tables of results" in "Attached background material"

Applicant's summary and conclusion

Conclusions:
The test material is not mutagenic with and without metabolic activation in S. thyphimurium strains TA1535, TA1537 TA98 & TA100, and E.coli WP2 uvrA-.
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 acetone 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 15 to 5000 µg/plate. The experiment was repeated on a separate day using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test material formulations.

The vehicle (acetone) 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 no visible reduction in the growth of the bacterial background lawn although substantial decreases in revertant colony frequency were observed to all of the Salmonella strains dosed in the presence of S9 at 5000 µg/plate. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A precipitate (oily in appearance) was observed at 5000 µg/plate, 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, the test material is not mutagenic with and without metabolic activation in S. thyphimurium strains TA1535, TA1537 TA98 & TA100, and E.coli WP2 uvrA-.

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