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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 2008-08-20 to 2008-09-04
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:
2008
Report Date:
2008

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 21th August 2007 / Signed on 15th October 2007)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid
Details on test material:
- Physical state: solid
- Stability under test conditions: at least 7 days when kept at room temperature.
- Storage condition of test material: In the refrigerator (2 - 8 °C), protected from light
- Molecular formula : C16 H28 O
- Molecular weight : 236.4 g/mol

Method

Target gene:
Histidine gene for S. thyphimurium 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 Sprague-Dawley rats 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: 15, 50, 150, 500, 1500 and 5000 μg/plate for all S. strains; 50, 150, 500, 1500 and 5000 μg/plate for E. coli.
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 insoluble in water and dimethylsulphoxide at 50 mg/mL but was fully soluble in acetone at the same concentration in solubility checks performed in-house.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
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
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: 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 EXAMINATIONS:
- Other: Observations of precipitate of the test substance

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 2006 & 2007).
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 mL.
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 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 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: the test material is not classified as a VOC.
- Water solubility: the test material was solubilised in acetone to improve solubility.
- Precipitation: A stringy, oily precipitate was observed at and above 1500 µg/plate, this did not prevent the scoring of revertants colonies.
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES: the test material induced lowered revertant colony counts at the upper dose levels to TA100 but was non-toxic to WP2uvrA-. The test material 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: 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 no visible reduction in the growth of the bacterial background lawn at any dose level.

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

74

81

77

77

94

77

78

94

75

56

35

-

64

64

70

66

85

72

62

63

65

36

20

+

WP2

uvrA-

35

25

34

26

31

26

36

46

22

26

25

-

30

35

31

34

28

25

34

32

41

26

22

Applicant's summary and conclusion

Conclusions:
ST 10 C 08 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 to ST 10 C 08 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 ranged between 15 and 5000 µg/plate, depending on bacterial strain type. 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.

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.

ST 10 C 08 caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A stringy, oily precipitate, was observed at and above 1500 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, ST 10 C 08 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.