Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 1999-01-19 to 1999-02-16
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

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999
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
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Program (inspection date: 1998-03-23
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
impurity
Test material form:
liquid
Details on test material:
- Physical state: colourless liquid
- Storage condition of test material: room temperature in the dark under nitrogen

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
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (10% S9-fraction from liver of male Sprague-Dawley rat injected with Aroclor 1254)
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.
- Mutation study, Experiment 1 & 2: 50, 150, 500, 1500 and 5000 µg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: suspension observed with water at 50 mg/mL. Good solution with DMSO at 50 mg/mL.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
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:
DMSO
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
- Exposition duration: 48 hours

NUMBER OF REPLICATIONS: Triplicate plate per dose level

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

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 for 1997).
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.
Rationale for test conditions:
Tested up to the recommended maximum test concentration for soluble non-cytotoxic substances, i.e. 5000 µg/plate.
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 significant increase in the revertant count in at least one strain of bacteria.
Statistics:
Dunnet's method of linear regression

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
At 5000 µg/plate in the presence of S9-mix only
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
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
- Precipitation: observed at 5000 µg/plate

RANGE-FINDING/SCREENING STUDIES: The test material was toxic to TA100 at 5000 µg/plate, with S9-mix only, but was non toxic to TA100, without S9-mix, and E. coli strain WP2uvrA-, both with and without S9-mix. 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 untreated controls. The comparison was made with the historical control ranges for 1997 of the corresponding Testing Laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY: The test material caused a visible reduction in the growth of the bacterial lawn in all of the Salmonella tester strains at 5000 µg/plate in the presence of Aroclor-induced rat liver S9 only. No toxicity was observed in E. coli strain WP2uvrA-, either with or without S9. An oily precipitate was observed at 5000 µg/plate, this did not prevent the scoring of revertant colonies.

Any other information on results incl. tables

Table 7.6.1/2 : Preliminary toxicity results

S9-mix

Strain

Dose (µg/plate

0

0.15

0.5

1.5

5

15

50

150

500

1500

5000

+

TA100

97

100

93

102

110

79

96

106

94

92

89PS

-

113

95

91

C

110

85

105

133

100

85

91P

+

WP2uvrA-

38

23

29

26

40

25

22

32

27

38

25P

-

29

37

31

38

33

18

32

28

27

28

34P

P = precipitate

S = sparse background lawn

C = contaminated

Mutation study: result tables are included in "Attached background material"

Applicant's summary and conclusion

Conclusions:
The test item was not mutagenic both in the presence and absence of 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, TA 100 and E.coli strain WP2 uvrA- were exposed the test material diluted in DMSO 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 main tests was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test material formulations.

The vehicle (dimethyl sulphoxide) 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 lawn in all of the Salmonella tester strains at 5000 µg/plate in the presence of Aroclor-induced rat liver S9 only. No toxicity was observed in E. coli strain WP2uvrA- , either with or without S9. An oily precipitate 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, ST 08 C 98 was not mutagenic with and without metabolic activation to 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.