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Toxicological information

Genetic toxicity: in vitro

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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:
8. Dec. 1998 - 19. Jan. 1999
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see "remarks"
Remarks:
GLP-Guideline study. According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (Dec 2012)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.
Justification for type of information:
refer to analogue justification provided in IUCLID section 13

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Safepharm Laboratories Limited, Derby, UK
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
131459-39-7
Cas Number:
131459-39-7
IUPAC Name:
131459-39-7

Method

Target gene:
his operon and trp operon
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: incapable of DNA excision repair (uvrB-); increased permeability of the cell wall (rfa)
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 Sprague-Dawley rats treated with Aroclor 1254
Test concentrations with justification for top dose:
50, 150, 500, 1500, 5000 µg/plate (first and second experiment)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: without S9: N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG; 3 µg/plate TA100, 5 µg/plate TA1535, 2 µg/plate WP2uvrA-), 9-Aminoacridine (9-AA; 80 µg/plate TA 1535), 4-Nitroquinoline-1 (4NQO; 0.2 µg/plate TA98)
Positive controls:
yes
Positive control substance:
other: with S9 mix: 2-Aminoanthracene (2AA; 1 µg/plate TA 100, 2 µg/plate TA 1535 and TA 1537, 10 µg/plate WP2uvrA-); Benzo(a)pyrene (BP; 5 µg/plate TA 98)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: inspection of bacterial background lawn, number of revertant colonies
Evaluation criteria:
The test material may be considered to be positive in this test system if the following criteria are met: All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (see table 1); Each positive control value was at least two times the respective vehicle control value for each strain (for historical values see table 2); A minimum of 4 non-toxic dose levels were achieved; 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:
Dunett's method of linear regression, Kirkland DJ (1989), Statistical Evaluation of Mutagenicity Test Data. UKEMS Sub-comittee on Guidelines for Mutagenicity Testing. Report - Part III - Cambridge University Press

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
Remarks:
oily precipitate at 5000 µg/plate which did not prevent scoring of revertant colonies
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
Remarks:
oily precipitate at 5000 µg/plate which did not prevent scoring of revertant colonies
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: oily precipitate at 5000 µg/plate

RANGE-FINDING/SCREENING STUDIES: the dose range of the test material used in the preliminary toxicity study was 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/plate. The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-).

COMPARISON WITH HISTORICAL CONTROL DATA: Values of positive and negative controls were in range of historical control data.

Any other information on results incl. tables

Table 3. Test results of experiment 1 (plate incorporation).

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates ± Standard deviation)

Base-pair substitution type

Frameshift type

TA 100

TA1535

WP2uvrA-

TA98

TA1537

0

74 ± 9.6

21 ± 2.3

22 ± 2.0

23 ± 4.0

8 ± 2.0

50

75 ± 7.1

20 ± 2.1

24 ± 2.1

24 ± 4.0

6 ± 2.0

150

79 ± 13.1

20 ± 6.8

22 ± 1.5

19 ± 2.6

10 ± 1.0

500

76 ± 4.2

15 ± 4.0

23 ± 7.0

21 ± 4.9

9 ± 3.8

1500

71 ± 6.1

20 ± 3.8

18 ± 5.5

21 ± 5.7

10 ± 3.5

5000

73 ± 1.5 P

21 ± 5.5 P

24 ± 6.0 P

21 ± 5.9 P

9 ± 2.5 P

Positive controls, –S9

Name

ENNG

ENNG

ENNG

4NQO

9AA

Concentrations

(μg/plate)

3

5

2

0.2

80

Mean No. of colonies/plate

(average of 3 ± SD)

323 ± 11.7

283 ± 10.0

503 ± 24.2

166 ± 5.3

1027 ± 358.2

+

0

87 ± 1.5

16 ± 1.5

28 ± 2.3

34 ± 6.7

18 ± 0.6

+

50

94 ± 6.1

13 ± 1.2

29 ± 5.8

31 ± 0.6

23 ± 7.4

+

150

88 ± 4.0

13 ± 2.6

25 ± 5.6

35 ± 6.4

19 ± 7.0

+

500

87 ± 3.5

12 ± 1.2

29 ± 3.8

32 ± 9.5

20 ± 5.5

+

1500

81 ± 6.5

10 ± 0.6

30 ± 3.6

33 ± 3.8

15 ± 4.6

+

5000

77 ± 5.1 P

12 ± 0.6 P

30 ± 2.6 P

31 ± 4.7 P

18 ± 3.8 P

Positive controls, +S9

Name

2AA

2AA

2AA

BP

2AA

Concentrations

(μg/plate)

1

2

10

5

2

Mean No. of colonies/plate

(average of 3 ± SD)

920 ± 153.0

198 ± 9.3

555 ± 27.8

511 ± 132.0

196 ± 6.5

ENNG = N-ethyl-N-nitro-N-nitrosoguanidine

4NQO = 4-nitroquinoline-N-oxide

9AA = 9-aminoacridine

2AA = 2 -aminoanthracene

BP = Benzo(a)pyrene

P = Precipitate

Table 4. Test results of experiment 2 (plate incorporation).

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates ± Standard deviation)

Base-pair substitution type

Frameshift type

TA 100

TA1535

WP2uvrA-

TA98

TA1537

0

103 ± 5.3

26 ± 3.8

31 ± 8.5

29 ± 7.0

12 ± 2.9

50

105 ± 10.8

21 ± 4.9

30 ± 7.4

28 ± 7.1

11 ± 1.0

150

97 ± 7.0

26 ± 1.0

35 ± 2.9

22 ± 1.2

13 ± 3.5

500

104 ± 9.1

25 ± 4.6

34 ± 5.1

26 ± 5.6

15 ± 1.7

1500

110 ± 9.5

23 ± 6.0

25 ± 0.6

27 ± 3.8

13 ± 1.7

5000

112 ± 5.7 P

28 ± 0.6 P

35 ± 2.6 P

20 ± 1.0 P

11 ± 1.5 P

Positive controls, –S9

Name

ENNG

ENNG

ENNG

4NQO

9AA

Concentrations

(μg/plate)

3

5

2

0.2

80

Mean No. of colonies/plate

(average of 3 ± SD)

880 ± 31.5

257 ± 34.4

1191 ± 59.3

166 ± 4.6

1384 ± 263.4

+

0

103 ± 8.6

13 ± 3.6

37 ± 2.1

27 ± 6.2

20 ± 3.8

+

50

92 ± 21.0

12 ± 1.5

33 ± 4.0

27 ± 7.2

20 ± 2.6

+

150

91 ± 7.5

14 ± 1.5

34 ± 2.6

29 ± 4.0

22 ± 2.6

+

500

113 ± 15.0

14 ± 2.3

39 ± 2.1

27 ± 6.7

15 ± 3.2

+

1500

110 ± 7.6

13 ± 2.6

36 ± 3.2

31 ± 4.9

21 ± 3.1

+

5000

97 ± 9.0 P

17 ± 5.8 P

30 ± 2.5 P

28 ± 4.7 P

22 ± 3.0 P

Positive controls, +S9

Name

2AA

2AA

2AA

BP

2AA

Concentrations

(μg/plate)

1

2

10

5

2

Mean No. of colonies/plate

(average of 3 ± SD)

865 ± 43.2

246 ± 7.5

1051 ± 90.0

611 ± 51.5

182 ± 7.0

ENNG = N-ethyl-N-nitro-N-nitrosoguanidine

4NQO = 4-nitroquinoline-N-oxide

9AA = 9-aminoacridine

2AA = 2 -aminoanthracene

BP = Benzo(a)pyrene

P = Precipitate

Applicant's summary and conclusion

Conclusions:
negative