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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:
2003-07-31 to 2003-09-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guideline
Qualifier:
according 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
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
Source of the test substance was not reported. Batch No: 0003A/RCC 0122A.
- Expiration date of the lot/batch:
October 2004
- Purity test date:
10 October 2002

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
At room temperature in the dark (ambient humidity).
- Stability under test conditions:
Not reported
- Solubility and stability of the test substance in the solvent/vehicle:
Solutions were freshly prepared for each experiment. The stability of the test substance can be assumed.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:Not applicable.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
Solutions were freshly prepared for each experiment.
- Preliminary purification step (if any): No details reported.
- Final dilution of a dissolved solid, stock liquid or gel:
No details reported.
- Final preparation of a solid:
No details reported.

Method

Target gene:
Strain TA1537 (his C3076)(no R factor) detects frameshift mutations
Strain TA98 (his D3052) R factor pKM101 detects frameshift mutations
Strain TA100 (hisG46) R factor pK,101 detects basepair substitutions
Strain TA 1535 (his G46) (no R factor) detects basepair subsitutions
Strain TA 102 (hist G428) R factor pKM101, pAQ1 detects basepair substitutions.
Species / strain
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
100, 300,1000, 3000, 5000 µg/plate with and without metabolic activation (experiment 1)
500, 1000, 1500 µg/plate with and without metabolic activation (experiment 2, only with strain TA 1537)
All concentrations are given as free base equivalent.
Vehicle:
- Vehicle(s) used: DMSO and dist. water
Controls
Negative controls:
no
Solvent controls:
yes
Remarks:
in experiment 2, two independent experiments were conducted, one with DMSO as solvent and as solvent control and one with distilled water as solvent and as solvent control.
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
other: 2-aminoanthracene
Remarks:
without S9: 2-nitrofluorene, sodium azide, mitomycin C, 9-aminoacridine; with S9: 2-aminoanthracene
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

EXPOSURE DURATION: 48 hrs

NUMBER OF REPLICATIONS: 3 each per concentration level and positive control. 6 per vehicle control


DETERMINATION OF CYTOTOXICITY
- Method: bacterial background lawn, microcolonies
Evaluation criteria:
A reproducible, concentration-dependent increase in the number of revertants of at least one tester strain over the vehicle control value and/or outside the historical control range is indicative of genotoxic activity. Since the results were unequivocal, no detailed statistical analysis was performed.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 98 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
TA 98 without metabolic activation, TA 102 with metabolic activation
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes

Any other information on results incl. tables

No precipitation was observed up to the top concentration tested. There was, however, a concentration-dependent and strain-specific bacteriotoxicity, as seen by a reduced background lawn and/or a decrease of absolute revertants.

 

Mutagenicity

The test item did not increase consistently the number of revertant colonies in the tester strains S. typhimurium TA 1535, TA 98 and TA 102 in the concentration range from 100 to 5000 µg/plate (free base) compared to the negative control in the standard plate test. There was no substantial difference in the mutation frequency in the presence and absence of metabolic activation.

There was a slight mutagenic response (up to 1.6-fold) observed in S. typhimurium TA 100 (nonactivation) at 1000 µg/plate. A marked increase of revertants (up to 22.7-fold) was seen in TA 1537 (with metabolic activation) at 500 µg/plate and above, using the standard plate test, although no dose response was observed. The effect was more pronounced in the absence of metabolic activation. This positive result in TA 1537 was confirmed in a repeat assay.

All revertant counts were within the historical background data. The positive controls caused the expected mutagenic response showing the sensitivity and validity of the assay.

Table 1: Mutagenic activity of the test item. Mean values Experiment 1 (Plate incorporation). Without metabolic activation

Compound

(µg/plate)

Mean Revertants/Plate

S. typhimurium

TA 1535

TA 1537

TA 98

TA 100

TA 102

DMSO

10

11

32

77

301

100

10

90

26

84

289

300

11

153

38

91

315

1000

16

250

17 T

127

308

3000

18

1 T

0 T

51 T

343

5000 T

0

0

0

0

231

NaN3 NaN3

5

538

-

-

703

-

9-AA

50

-

238

-

-

-

2-Nf

10

-

-

488

-

-

MMC

0.5

-

-

-

-

737

Table 2: Mutagenic activity of the test item. Mean values Experiment 1 (Plate incorporation). With metabolic activation

Compound

(µg/plate)

Mean Revertants/Plate

S. typhimurium

TA 1535

TA 1537

TA 98

TA 100

TA 102

DMSO

15

13

34

82

285

100

15

15

35

89

289

300

12

23

40

90

334

1000

15

49

39

76

232

3000

16

31T

50

89

219 T

5000

14

18 T

31

68 T

50 T

9-AA

4

257

215

797

985

-

9-AA

10

 

 

 

 

1131

P: Precipitation; T: Toxic; -: Not used; Underlined values are regarded as increased

Historical Range

8 - 15

5 -21

32 - 54

49 - 119

285 - 418

Table 3: Mutagenic activity of the test item. Mean values Experiment 2 (Plate incorporation). Without metabolic activation

Compound

(µg/plate)

Mean Revertants/Plate

S. typhimurium TA 1537

DMSO

Dist. Water

Control

9

8

500

144

126

1000

164

151

1500

147

137

9-AA

50

187

186

Table 4: Mutagenic activity of the test item. Mean values Experiment 2 (Plate incorporation). With metabolic activation

Compound

(µg/plate)

Mean Revertants/Plate

S. typhimurium TA 1537

DMSO

Dist. Water

Control

11

9

500

19

30

1000

27

34

1500

27

44

9-AA

4

172

169

Historical Range

5 - 21

Applicant's summary and conclusion

Conclusions:
Based on the results of this study it is concluded that the test substance, when tested up to bacteriotoxic concentrations, caused no mutations in the tester strains S. typhimurium TA 1535, TA 98 and TA 102 in the presence and absence of metabolic activation. However, there was a weak increase of revertants in TA 100 and a reproducible mutagenic response in S. typhimurium TA 1537.
Based on the results of this study, it was concluded that the test susbtance was mutagenic.