Registration Dossier

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 June 25 to August 02, 2004
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:
2004
Report Date:
2004

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:
- Name of test material (as cited in study report): Dorinone beta
- Physical state: Pale yellow liquid
- Storage condition of test material: Approximately 4 ºC 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: 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 using the direct plate incorporation method.

Mutation Test (direct plate incorporation method):

Experiment 1 (Range-finding Test)
Salmonella strains: 5, 15, 50, 150, 500, 1500 and 5000 μg/plate.
E.coli strain WP2uvrA-: 50, 150, 500, 1500 and 5000 μg/plate.

Experiment 2 (Main Test)
Salmonella strains (with and without S9) & E.coli strain WP2uvrA- (without S9 only): 15, 50, 150, 500, 1500 and 5000 μg/plate.
E.coli strain WP2uvrA- (with S9): 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:
The test material may be considered 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: Immiscible in water
- Precipitation: None
- Other confounding effects: None

PRELIMINARY TOXICITY STUDY: The test material exhibited toxicity (as a significant weakening of the bacterial background lawn) from 1500 μg/plate to TA 100 and was non-toxic 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 to all of the Salmonella strains both with and without S9 from 1500 μg/plate, although less toxicity was observed to TA98. However, no toxicity was noted to E.coli strain WP2uvrA- at any test material dose level.
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

118

120

140

125

111

115

103

149

118

91*

13*

+ S9

TA 100

137

101

105

108

107

110

116

107

95

54

10*

- S9

WP2 uvr A

17

20

18

16

16

20

11

10

8

18

22

+ S9

WP2 uvr A

25

19

19

22

18

22

24

18

21

20

23

 

*: Partial absence of bacterial background lawn

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 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 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. The experiment was repeated on a separate day using an amended dose range (15 to 5000 µg/plate), fresh cultures of the bacterial strains and fresh test material formulations. 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 to all of the Salmonella strains both with and without S9 from 1500 μg/plate, although less toxicity was observed to TA 98. However, no toxicity was noted to E.coli strain WP2uvrA- at any test material dose level. The test material was 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, 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.