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:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
No details on analytical purity given

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
his operon (S. typhimurium) and trp operon (E. coli)
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male Sprague Dawley rats treated i.p. with a single dose of 500 mg/kg bw Arochlor 1254
Test concentrations with justification for top dose:
Range-finding toxicity study (in TA 100 and WP2 uvrA): 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3330 and 5000 µg/plate, with and without metabolic activation
Main study (all strains): 33.3, 100, 333, 1000, 3330 and 5000 µg/plate, with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
-S9: 2-NF (1 µg/plate, TA 98); SA (2 µg/plate, TA 100 and TA 1535); ICR-191 (2 µg/plate, TA 1537); 4-NQO (1 µg/plate, WP2 uvrA); +S9: BP (2.5 µg/plate, TA 98); 2-AA (2.5-25 µg/plate, TA 100, TA 1535, TA 1537 and WP2 uvrA)
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: 2-aminoanthracene; ICR-191
Remarks:
2-NF: 2-nitrofluorene; SA: sodium azide; 4-NQO: 4-nitroquinoline-N-oxide; BP: benzo(a)pyrene; 2-AA: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 52 ± 4 h

NUMBER OF REPLICATIONS: triplicates each in one experiment

DETERMINATION OF CYTOTOXICITY
- Method: inspection of bacterial background lawn
Evaluation criteria:
The results of the test were considered positive, if the following criteria were met:
- tester strains TA 98, TA 100 and WP2 uvrA: for a test article to be considered positive, it must produce at least a 2-fold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate must be accompanied by a dose response to increasing concentrations of the test article.
- tester strains TA 1535 and TA 1537: for a test article to be considered positive, it must produce at least a 3-fold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate must be accompanied by a dose response to increasing concentrations of the test article.
Statistics:
Mean values and standard deviations of revertants per plate were calculated.

Results and discussion

Test resultsopen allclose all
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: in the absence of S9 mix, slight precipitation of the test substance was observed in all experiments at concentrations ≥ 100 µg/plate. In the absence of S9 mix, slight precipitates were noted at ≥ 333 µg/plate in the preliminary cytotoxicity study and at ≥ 1000 µg/plate in the main study.

RANGE-FINDING/SCREENING STUDIES: in a preliminary cytotoxicity study, the tester strains TA 100 and WP2 uvrA were treated with the test substance at concentrations ranging from 6.67 to 5000 µg/plate in the presence and absence of metabolic activation (S9 mix). No cytotoxicity was observed in these strains up to the limit dose of 5000 µg/plate, neither with nor without addition of S9 mix.

Any other information on results incl. tables

Table 1. Test results of experiment (plate incorporation)

Bacterial Reverse Mutation Assay, mean revertant colonies/plate (mutation factor) (n=3 ± SD)

EXPERIMENT

S9-Mix

Without

 

Concentration (per plate)

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

SC

25 ± 2

80 ± 9

14 ± 8

3 ± 1

26 ± 3

Test material

 

33.3 µg

19 ± 4

88 ± 5

12 ± 5

4 ± 4

19 ± 2

100 µg

21 ± 7

96 ± 12

11 ± 2

5 ± 4

19 ± 4

333 µg

22 ± 2

92 ± 9

13 ± 2

5 ± 5

23 ± 10

1000 µg

25 ± 6

97 ± 11

25 ± 2

5 ± 3

30 ± 3

3330 µg

22 ± 1

101 ± 3

10 ± 1

3 ± 2

32 ± 2

5000 µg

20 ± 6

85 ± 6

15 ± 3

4 ± 2

27 ± 3

PC

 

2-NF

206 ± 42

-

-

-

-

SA

-

549 ± 71

480 ± 3

-

-

ICR-191

-

-

-

277 ± 33

-

4-NQO

 -

 -

 -

260 ± 43

S9-Mix

 

With

Concentration (per plate)

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

SC

36 ± 2

102 ± 2

19 ± 1

7 ± 1

25 ± 5

Test material

 

33.3 µg

36 ± 7

93 ± 9

15 ± 2

8 ± 4

22 ± 5

100 µg

34 ± 7

97 ± 17

16 ± 4

9 ± 2

22 ± 6

333 µg

34 ± 6

87 ± 6

17 ± 2

9 ± 4

27 ± 3

1000 µg

39 ± 9

94 ± 17

17 ± 3

8 ± 2

26 ± 6

3330 µg

38 ± 8

92 ± 6

16 ± 4

6 ± 3

29 ± 11

5000 µg

34 ± 5

139 ± 5

20 ± 3

3 ± 2

24 ± 4

PC

 

 

 

 

 

BP

471 ± 14

-

-

-

-

2-AA

-

918 ± 296

124 ± 6

171 ± 32

278 ± 24

SC = Solvent control; PC = Positive control substances; SD = standard deviation;

2-NF: 2-nitrofluorene; SA: sodium azide; 4-NQO: 4-nitroquinoline-N-oxide; BP: benzo(a)pyrene; 2-AA: 2-aminoanthracene

Applicant's summary and conclusion

Conclusions:
Based on the results of the conducted study the test substance did not exhibit mutagenic properties in bacterial cells.