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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report date:
1998

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
(only 2-aminoanthracene as positve control with metabolic activation)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Imidazole-1-propionitrile(8CI);1-(2-Cyanoethyl)imidazole
EC Number:
607-301-1
Cas Number:
23996-53-4
Molecular formula:
C6H7N3
IUPAC Name:
Imidazole-1-propionitrile(8CI);1-(2-Cyanoethyl)imidazole
Details on test material:
- Name of test substance (as cited in study report): Cyanethylimidazol
- Analytical purity: > 95%
- Batch No.: K 94/1
- Substance No.: 96/124
- Physical state: liquid (brownish, clear)
- Storage: refrigerator

Method

Target gene:
His- and Trp-operon
Species / strain
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E.coli WP2 uvrA
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplement post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254.
Test concentrations with justification for top dose:
20, 100, 500, 2500 and 5000 µg/plate (experiment 1 and 2)
Vehicle / solvent:
water
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: (+ S9): 2-aminoanthracene (2AA; all strains); (- S9): N-methyl-N'-nitro-N-nitroso-guanidine (MNNG; TA 100 and TA 1535), 4-nitro-o-phenylendiamine (NOPD; TA 98), 9-aminoacridine (AAC; TA 1537), N-ethyl-N'-nitro-N-nitroso-guanidine (ENNG; E. coli WP2 uvrA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) (experiment 1)

DURATION
- Exposure duration: 48 - 72 h at 37 °C

METHOD OF APPLICATION: preincubation (experiment 2)

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 - 72 h at 37 °C

NUMBER OF REPLICATIONS: 3 plates per concentration per experiment

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of revertant colonies; clearing or diminution of the background lawn; degree of survival of treated cultures
Evaluation criteria:
In general, a substance has to fulfill the following criteria to be characterized as positive in the bacterial tests:
- doubling of the spontaneous mutation rate (compared to control)
- dose-response realtionship
- reproducibility of the results.

Results and discussion

Test results
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E.coli WP2 uvrA
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: no precipitation occurred up to a concentration of 5000 µg/plate.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1. 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

E. coli WP2 uvrA

TA98

TA1537

0

 119 ± 10

18 ± 1

33 ± 8

25 ± 4

8 ± 1

20

122 ± 3

15 ± 1

36 ± 3

30 ± 1

9 ± 1

100

121 ± 11

20 ± 4

39 ± 1

20 ± 2

8 ± 3

500

134 ± 6

18 ± 2

37 ± 8

24 ± 4

9 ± 1

2500

129 ± 8

18 ± 3

36 ± 6

27 ± 5

10 ± 2

5000

128 ± 22

15 ± 3

47 ± 3

26 ± 5

6 ± 4

Positive controls, –S9

Name

MNNG

MNNG

ENNG

NOPD

AAC

Concentrations

(μg/plate)

5

5

10

10

100

Mean No. of colonies/plate

(average of 3 ± SD)

1214 ± 154

1096 ± 139

560 ± 55

692 ± 113

777 ± 131

+

0

126 ± 9

19 ± 3

34 ± 6

39 ± 3

9 ± 1

+

20

142 ± 7

16 ± 2

40 ± 6

36 ± 4

12 ± 1

+

100

135 ± 15

15 ± 5

38 ± 1

41 ± 1

12 ± 2

+

500

112 ± 9

16 ± 4

41 ± 2

45 ± 4

10 ± 1

+

2500

115 ± 1

17 ± 2

40 ± 10

40 ± 2

9 ± 2

+

5000

123 ± 7

14 ± 7

45 ± 3

27 ± 7

7 ± 1

Positive controls, +S9

Name

2AA

2AA

2AA

2AA

2AA

Concentrations

(μg/plate)

2.5

2.5

60

2.5

2.5

Mean No. of colonies/plate

(average of 3 ± SD)

1531 ± 80

1033 ± 45

262 ± 49

864 ± 102

224 ± 12

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

MNNG = N-methyl-N-nitro-N-nitrosoguanidine

NOPD = 4-nitroquinoline-N-oxide

AAC = 9-aminoacridine

2AA = 2-Aminoanthracene

 

Table 2. Test results of experiment 2 (preincubation test).

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

E. coli WP2 uvrA

TA98

TA1537

0

128 ± 5

21 ± 2

35 ± 5

29 ± 6

13 ± 2

20

105 ± 6

16 ± 1

33 ± 5

28 ± 5

8 ± 3

100

100 ± 5

19 ± 2

29 ± 2

28 ±2

11 ± 4

500

113 v 11

18 ± 2

34 ± 5

26 ± 4

10 ± 2

2500

109 ± 12

16 ± 2

28 ± 3

29 ± 8

11 ± 2

5000

113 ± 9

18 ± 2

30 ± 1

28 ± 7

10 ± 3

Positive controls, –S9

Name

MNNG

MNNG

ENNG

NOPD

AAC

Concentrations

(μg/plate)

5

5

10

10

100

Mean No. of colonies/plate

(average of 3 ± SD)

1132 ± 25

1200 ± 54

530 ± 19

1466 ± 25 

457 ± 49

+

0

138 ± 4

21 ± 2

45 ± 5

48 ± 4

13 ± 1

+

20

107 ± 7

16 ± 3

37 ± 4

50 ± 3

15 ± 1

+

100

110 ± 9

15 ± 2

41 ± 5

45 ± 6

15 ± 3

+

500

114 ± 9

12 ± 3

38 ± 1

53 ± 4

11 ± 3

+

2500

111 ± 11

16 ± 1

48 ± 1

45 ± 7

10 ± 3

+

5000

106 ± 7

17 ± 3

42 ± 5

49 ± 6

11 ± 2

Positive controls, +S9

Name

2AA

2AA

2AA

2AA

2AA

Concentrations

(μg/plate)

2.5

2.5

60

2.5

2.5

Mean No. of colonies/plate

(average of 3 ± SD)

1097 ± 36

124 ± 7

204 ± 7

925 ± 40

112 ± 8

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

MNNG = N-methyl-N-nitro-N-nitrosoguanidine

NOPD = 4-nitroquinoline-N-oxide

AAC = 9-aminoacridine

2AA = 2-Aminoanthracene

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative