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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:
1980
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1980
Report Date:
1980

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(adopted 1981)
Deviations:
yes
Remarks:
, strain TA102 or E. coli is lacking (OECD TG 471, adopted 1997)
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
HIS
Species / strain
Species / strain / cell type:
other: S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 1538
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix from Aroclor1254-induced rats
Test concentrations with justification for top dose:
25, 75, 225, 675 and 2025 µg per plate
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see details on test system and conditions
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
Each Petri dish contained: 1) approx. 20 mL of minimum agar (Agar, Difco Laboratories, Detroit, Michigan, U.S.A., plus salts (Vogel-Bonner Medium E) and glucose), 2) 0.1 mL of the solution of the test substance or the vehicle and 0.1 mL of a bacterial culture (in nutrient broth, Difco Laboratories, Detroit, Michigan, U.S.A., 0.8% plus 0.5% NaCl) in 2.0 mL of soft agar. The soft agar was composed of: 100 mL of 0.6% agar solution with 0.6% NaCl and 10 mL of a solution of l-histidine, 0.5 mM (Fluka, Buchs, Switzerland) and +biotin 0.5 mM (Fluka, Buchs, Switzerland). In the experiments in which the substance was metabolically activated, 0.5 mL of an activation mixture was added also. One mL activation mixture contained: 0.3 mL S9 fraction of liver from rats induced with Aroclor 1254 (Analabs., Inc., North Haven, Connecticut, U.S.A.) and 0.7 mL of a solution of co-factors.

DURATION
The plates were incubated for about 48 hours at 37°C in darkness.

NUMBER OF REPLICATIONS
3 plates per dose

DETERMINATION OF CYTOTOXICITY
No data

POSITIVE CONTROLS
TA 98: daunorubicin-HCl (5 and 10 µg/plate)
TA 100: 4-nitroquinoline-N-oxide (0.125 and 0.25 µg/plate)
TA 1535: N-Methyl-N'-nitro-N-nitrosoguanidine (3 and 5 µg/plate)
TA 1537: 9(5)aminoacridine hydrochloride monohydrate (50 and 100 µg/plate)
TA 1538: 2-nitrofluorene (5 and 10 µg/plate)
The activation mixture was tested with Strain TA 1535 and cyclophosphamide (250 µg/plate)
Evaluation criteria:
When the colonies had been counted, the arithmetic mean was calculated. The test substance is generally considered to be non-mutagenic if the colony count in relation to the negative control is not doubled at any concentration.

Results and discussion

Test results
Species / strain:
other: S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At the concentrations of 675 and 2025 µg/plate the substance precipitated in soft agar
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Number (arithmetic mean) of colonies of histidine-prototrophic back-mutants in experiments without microsomal activation.

Concentration

(µg/plate)

TA98

TA100

TA1535

TA1537

TA1538

0

11

136

8

4

12

25

20

129

10

3

8

75

18

158

10

7

11

225

13

145

10

6

6

675

16

156

7

2

12

2025

17

159

7

6

9

Daunorubicin-HCl

19

(control)

 

 

 

 

5

134

 

 

 

 

10

293

 

 

 

 

4-NQO

 

156

(control)

 

 

 

0.125

 

650

 

 

 

0.250

 

>1000

 

 

 

N-methyl-N´-nitro-N-nitrosoguanidine

 

 

8

(control)

 

 

3

 

 

349

 

 

5

 

 

1470

 

 

9(5) Aminoacridine-HCl

 

 

 

6

(control)

 

50

 

 

 

85

 

100

 

 

 

520

 

2-Nitrofluorene

 

 

 

 

11

(control)

5

 

 

 

 

660

10

 

 

 

 

823

Table 2: Number (arithmetic mean) of colonies of histidine-prototrophic back-mutants in experiments with microsomal activation.

Concentration

(µg/plate)

TA98

TA100

TA1535

TA1537

TA1538

0

26

141

4

3

15

25

24

136

7

4

15

75

21

141

12

5

19

225

26

128

7

5

16

675

24

112

7

3

16

2025

21

130

8

5

17

Cyclophosphamide

 

 

 

 

 

0

 

 

13

 

 

250

 

 

214

 

 

Applicant's summary and conclusion

Conclusions:
The test substance showed no mutagenic potential in bacterial cells.
Executive summary:

The mutagenic potential of the test material was assessed in an Ames assay with TA98, TA100, TA1535, TA1537 and TA1538 as tester strains. The bacteria were treated with 25, 75, 225, 675, and 2025 µg test substance per plate, with and without microsomal activation. Adequate positive controls and a solvent control were also applied. In the experiments performed with and without microsomal activation, comparison of the number of histidine-prototrophic mutants in the controls and after treatment with the test substance revealed no marked differences. The slight increase in the number of back-mutant colonies in the experiment on Strain TA 1535 with microsomal activation is attributed to variations in the rate of spontaneously occurring back-mutants.