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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 March 16 to April 14, 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted according to OECD 471 Guideline without any deviation.
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:
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:
other: liquid
Details on test material:
- Physical state: Pale yellow liquid
- Storage condition of test material: Approximately 4 °C in the dark under nitrogen

Method

Target gene:
Histidine and tryptophan gene
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 (direct plate incorporation method): 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in TA 100 and WP2 uvr A strains, with or without S9-mix.

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): 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.

OTHER:
- Plates were assessed for numbers of revertant colonies using a Domino colony counter.
Evaluation criteria:
The test material may be considered to be positive in this test system if the following criteria are met:
The test material should have induced a reproducible, dose-related and statistically (Dunnett' s method of linear regression) significant increase in the revertant count in at least one strain of bacteria.
Statistics:
Dunnett' s method of linear regression

Results and discussion

Test results
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
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: Not expected
- Water solubility: Test item was immiscible in sterile distilled water at 50 mg/mL, but was fully miscible in DMSO at same concentration.
- Precipitation: A light (oily in appearance) precipitate was observed at and above 1500 µg/plate, this did not prevent the scoring of revertant colonies.
- Other confounding effects: None

PRELIMINARY TOXICITY STUDY: The test material exhibited toxicity to TA100 at and above 1500 µg/plate as a weakened bacterial background lawn in both the absence and presence of S9. No toxicity was noted to WP2uvrA-.

COMPARISON WITH HISTORICAL CONTROL DATA: All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the untreated controls.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- The test material caused a visible reduction in the growth of the bacterial background lawn and/or a substantial decrease in revertant colony frequency to all of the Salmonella strains, initially at 1500 µg/plate both in the presence and absence of S9. No toxicity was noted to E.coli strain WP2uvrA- at any test material dose level.

OTHERS:
- Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).
- Test material formulation and S9-mix used in this experiment were both shown to be sterile.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 7.6.1/2: Preliminary toxicity Test

S9 mix

Strain

Dose (µg/plate)

0

0.15

0.5

1.5

5

15

50

150

500

1500

5000

- S9

TA 100

133

145

143

144

141

163

154

133

130

146 *P

0 *P

+ S9

TA 100

129

134

104

105

115

124

110

117

111

72 *P

94 *P

- S9

WP2 uvr A

24

27

36

26

25

30

34

24

25

23 P

21 P

+ S9

WP2 uvr A

35

31

24

31

27

25

27

29

22

21 P

24 P

 

*: 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:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The 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 Regulation (EC) No 1272/2008 (CLP).
Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and Escherichia coli WP2 uvr A were exposed to the test material diluted in DMSO at the following concentrations using the plate incorporation method. The dose range for the range-finding test was determined in a preliminary toxicity assay and was 5 to 5000 and 50 to 5000 µg/plate for the Salmonella strains and E.coli strain WP2uvrA- respectively. The experiment was repeated on a separate day using a slightly amended dose range (15 to 5000 and 50 to 5000 µg/plate for the Salmonella strains and E.coli strain WP2uvrA- respectively), fresh cultures of the bacterial strains and fresh test material formulations. Experiment was carried out in triplicate, both with and without metabolic activation (10 % liver S9 in standard co-factors). Negative, vehicle (DMSO) 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 and/or a substantial decrease in revertant colony frequency to all of the Salmonella strains, initially at 1500 µg/plate both in the presence and absence of S9. However no toxicity was noted to E.coli strain WP2uvrA- at any test material dose level. 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 light (oily in appearance) 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, the test material is not mutagenic with and without metabolic activation to S. typhimurium (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 Regulation (EC) No. 1272/2008 (CLP).

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