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

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1983
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Version / remarks:
1996
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
testing lab.
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S-9 mix
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S-9 mix
Test concentrations with justification for top dose:
20, 100, 500, 2500, 5000 µg/plate (standard plate test)
125, 250, 500, 1000, 1500 µg/plate (preincubation test)
Vehicle:
water
Controls
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: with S9-mix: 2-aminoanthracene (all strains); without S9-mix: 2-aminoanthracene (all strains), N-methyl-N'-nitro-N-nitrosoguanidine (TA1535, 100), 4-nitro-o-phenylenediamine (TA98), 9-aminoacridine (TA1537) and N-ethyl-N'-nitro-N-nitrosoguanidine (E.coli)
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 min
- Expression time (cells in growth medium): 48-72 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:
The test chemical is considered positive in this assay if the following criteria are met:
A dose-related and reproducable increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S-9 mix or after adding a metabolizing system.
A test substance is generally considered nonmutagenic in this test if:
The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.
Statistics:
The arithmetic mean of the counted colonies per concentration was calculated.

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:
yes
Remarks:
A bacteriotoxic effect was observed in the preincubation test depending an the strain and test conditions at about > 1000 µg/plate.
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
other: A bacteriotoxic effect was observed in the preincubation test depending an the strain and test conditions at about > 1000 ug/plate.
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
An increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S-9 mix or after the addition of a metabolizing system.

Any other information on results incl. tables

Table 1: Number of revertants per plate (mean of three plates), test 1

strain TA98

strain TA100

strain TA1535

strain TA1537

conc. [µg/mL]

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

0 (water)

27

46

159

158

20

20

12

 

20

27

46

157

158

20

22

10

 

100

26

37

157

159

19

20

11

 

500

22

29

127

157

20

16

10

 

2500

 

8

 

83

 

8

 

 

5000

 

 

 

 

 

 

 

 

4-nitro-o-phenyleridiamine 10 µg

1266

 

 

 

 

 

 

 

2-Aminoanthracene 2.5 µg

 

1215

 

1536

 

135

 

147

N-methyl-N' -nitro-N-nitrosoguanidine

5 µg

 

 

2239

 

1424

 

 

 

9-aminoacridine

100 µg

 

 

 

 

 

 

472

 

 

 

 

Table 2: Number of revertants per plate (mean of three plates), test 2

strain TA98

strain TA100

strain TA1535

strain TA1537

E. coli WP2 uvrA

conc. [µg/mL]

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

0 (water)

30

48

137

151

13

12

10

11

33

38

125

28

39

141

154

13

15

13

11

32

39

250

35

46

136

152

16

12

9

10

33

35

500

35

46

158

139

12

15

9

12

35

41

1000

28

46

104

119

10

15

6

9

22

37

1500

34

38

108

135

-

12

1

12

17

27

4-nitro-o-phenyleridiamine 10 µg

1113

 

 

 

 

 

 

 

 

 

2-Aminoanthracene 2.5 µg*

 

786

 

662

 

88

 

129

 

112

N-methyl-N' -nitro-N-nitrosoguanidine

5 µg

 

 

1297

 

1083

 

 

 

 

 

9-aminoacridine

100 µg

 

 

 

 

 

 

346

 

 

 

N-ethylN-nitro-N-nitrosoguanidine

10 µg

 

 

 

 

 

 

 

 

772

 

  *except for E.coli: 60 µg

 

 

 

 

 

 

 

 

 

Applicant's summary and conclusion