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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:
21 December 1998 to 21 January 1999
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was conducted in 1998 to 1999 in accordance with OECD Guideline 471/472 on the appropriate strains of bacteria (S. typhimuriumTA1535, TA100, TA1537 and TA98 as well as E. coli WP2uvrA in both the presence and absence of metabolic activation. Whilst not conducted to GLP the report does include a signature page confirming that the report accurately reflects the proceedings of the experiment signed by the laboratory manager. Neither the batch details nor the purity of the substance are reported. Concurrent controls were run as a part of the experiment. Six dose levels were tested during the study, however the experiments were run on duplicate rather than triplicate plates and whilst the study result in an unequivocally negative result, the experiment was not duplicated and no justification for this was given in the report. Despite its basic nature and some deviations from the approved guidelines the study can be considered to be reliable with restrictions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Plates were tested in duplicate not triplicate and the negative result was not confirmed
Qualifier:
according to
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Deviations:
yes
Remarks:
Plates were tested in duplicate not triplicate and the negative result was not confirmed
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Method

Target gene:
Histidine synthesis in S. typhimurium strains, and tryptophan synthesis in the E. coli strain tested.
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
- Type and identity of media: Nutrient Broth No. 2 (Oxoid)
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
- Type and identity of media: Nutrient Broth No. 2 (Oxoid)
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Range finding test: 0, 0.305, 1.22, 4.88, 19.5, 78.1, 313, 1250 and 5000 µg/plate
Main test: 0, 39.1, 78.1, 156, 313, 625 and 1250 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 2-(2-furyl)-3- (5-nitro-2-furyl) acrylamide; 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

NUMBER OF REPLICATIONS: Performed in duplicate

DETERMINATION OF CYTOTOXICITY
- Method: assessment of the bacterial lawn growth
Evaluation criteria:
Plates were assessed for a significant increase in the number of revertant colonies.

Results and discussion

Test resultsopen allclose all
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:
Observed at the two highest doses tested
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Observed at the two highest doses tested
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
Based on the results of the range finding test, the maximum concentration selected for the main test was 1250 µg/plate as the test material caused a reduction in the growth of the bacterial lawn at concentrations above this dose.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Dose response curves for the number of revertants both with and without metabolic activation are presented in the attached figures.

Table 1: Results of the main test

Metabolic activation

Test material concentration

Replicate

Number of revertant colonies per plate

Base-pair substitution

Frameshift

TA 100

TA 1535

WP-2uvrA

TA 98

TA 1537

- S9 Mix

Solvent control

R1

148

9

17

16

9

R2

156

7

17

12

14

Mean

(152)

(8)

(17)

(14)

(12)

39.1

R1

138

11

14

14

13

R2

160

13

15

17

6

Mean

(149)

(12)

(15)

(16)

(10)

78.1

R1

159

6

14

17

13

R2

155

11

18

15

11

Mean

(157)

(9)

(16)

(16)

(12)

156

R1

152

6

16

22

6

R2

152

8

22

14

9

Mean

(152)

(7)

(19)

(18)

(8)

313

R1

151

9

17

7

7

R2

166

8

19

7

8

Mean

(159)

(9)

(18)

(7)

(8)

625

R1

62*

6*

13*

0*

2*

R2

82*

4*

10*

5*

2*

Mean

(72)

(5)

(12)

(3)

(2)

1250

R1

0*

0*

0*

0*

0*

R2

0*

0*

0*

0*

0*

Mean

(0)

(0)

(0)

(0)

(0)

+ S9 Mix

Solvent control

R1

173

9

19

24

12

R2

165

9

19

23

11

Mean

(169)

(9)

(19)

(24)

(12)

39.1

R1

152

8

16

31

11

R2

167

8

1

28

11

Mean

(160)

(8)

(16)

(30)

(11)

78.1

R1

158

13

30

33

9

R2

198

16

11

26

13

Mean

(178)

(15)

(21)

(30)

(11)

156

R1

152

12

23

31

19

R2

164

16

26

30

9

Mean

(158)

(14)

(25)

(31)

(14)

313

R1

170

3

14

19

16

R2

163

8

11

28

15

Mean

(167)

(6)

(13)

(24)

(16)

625

R1

150*

7*

18*

19*

9*

R2

128

5*

23*

18*

4*

Mean

(117)

(6)

(21)

(19)

(7)

1250

R1

122*

7*

12*

15*

3*

R2

125

4*

8*

20*

1*

Mean

(124)

(6)

(10)

(18)

(2)

Positive controls

- S9 Mix

 

 

 

Positive control substance

AF-2

NaN3

AF-2

AF-2

9-AA

Concentration (µg/plate)

0.01

0.5

0.01

0.1

80

 

R1

486

488

93

264

259

R2

516

491

85

263

285

Mean

(501)

(490)

(89)

(264)

(272)

Positive controls

+ S9 Mix

 

 

 

Positive control substance

2-AA

2-AA

2-AA

2-AA

2-AA

Concentration (µg/plate)

1.0

2.0

10

0.5

2.0

 

R1

826

286

1292

239

116

R2

867

311

1369

255

112

Mean

(847)

(299)

(1331)

(247)

(114)

AF-2 = 2-(2-furyl)-3- (5-nitro-2-furyl) acrylamide

NaN3= sodium azide

9-AA = 9-aminoacridine

2-AA = 2 -aminoanthracene

*Inhibition of bacterial growth

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
negative Both with and without metabolic activation

Under the conditions of the test, the test material was found to be negative for mutagenicity in both the S. typhimurium and E. coli strains tested in the presence and absence of metabolic activation.
Executive summary:

The mutagenicity of the test material was assessed using a bacterial reverse mutagenicity assay (Ames test). The study was performed in compliance with the current OECD guidelines 471 and 472 with a few minor deviations from the standard protocol. Under the conditions of the test, the test material was found to be negative in all strains tested both with and without metabolic activation.