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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:
14 February 1989 - 3 March 1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Read across to similar substance. Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Justification for type of information:
See read-across justification in Section 13.
Cross-reference
Reason / purpose:
other: Target substance
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted on read across material
Justification for type of information:
See read-across justification in Section 13.
Reason / purpose:
read-across source
Species / strain:
S. typhimurium, other: TA 1535, TA100, TA 1537, TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Report Date:
1989

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Description: White powder

Method

Target gene:
S. typhimurium: Histidine locus
Species / strain
Species / strain / cell type:
S. typhimurium, other: TA1535, TA100, TA1537, TA1538 and TA98
Additional strain / cell type characteristics:
other: TA1535 and TA100 are sensitive to agents inducing base-pair substitutions. TA1537, TA1538 and TA98 are sensitive to agents inducing frame-shift mutations.
Metabolic activation:
with and without
Metabolic activation system:
S9. Prepared from the livers of male Sprague-Dawley rats that had each recieved a single i.p. injection of Aroclor 1254 at 500 mg/kg 5 days before S9 preparation.
Test concentrations with justification for top dose:
Experiment 1 ± S9: 0, 8, 40, 200, 1000, 5000 µg/plate
Experiment 2 ± S9: 0, 312.5, 625, 1250, 2500, 5000 µg/plate
Vehicle / solvent:
The test material was accurately weighed and dissolved in sterile distilled water and appropriate dilutions made.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Remarks:
A solvent treatment group was used as the negative control
Positive controls:
yes
Positive control substance:
other: N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG); 9-Aminoacridine (9AA); 4-Nitro-O-phenylenediamine (4NOPD) and 2-Aminoanthracene (2AA)
Details on test system and experimental conditions:
Test Procedure
a) Preliminary Toxicity Study
In order to select appropriate dose levels for use in the main study, a preliminary test was carried out to determine the toxicity of the test material to the tester organisms. 0.1 mL of bacterial suspension (TA100) and 0.L ml of test solution were added to 2 mL of molten, trace histidine supplemented media (histidine/biotin & top agar) and overlayed onto sterile plates of Vogel-Bonner agar (minimal agar - 25 mL/ plate). Five doses of the test compound and a solvent control (distilled water) were tested in duplicate. After 48 - 72 hours incubation the plates were scored for revertant colonies and examined for a thinning of the background lawn.
b) Mutation Study
EXPERIMENT 1
Five concentrations of the test material were assayed in triplicate against each tester strain, using the direct plate incorporation method in accordance with the standards for mutagenicity tests using microorganisms.
Test Material and Negative Controls
0.1 mL of the appropriately diluted test material or negative control solution was placed in sets of sterile test tubes containing 2.0 mL of molten, trace histidine supplemented, top agar at 45°C. These sets comprised of two test tubes for each bacterial tester strain. A 0.1 mL aliquot of one of the bacterial suspensions was also added to each of the two test tubes. Into one of the test tubes was placed 0.5 mL of the S9 liver microsome mix; in the other tube 0.5 mL of pH 7.4 buffer was added. This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material.
Positive Controls
Without Activation
0.1 mL of one of the positive control solutions (MNNG, 9AA, or 4NOPD) was added to a test tube containing 2.0 mL of molten, trace histidine supplemented, top agar. 0.1 mL of the appropriate bacterial suspension and 0.5 mL of pH 7.4 buffer was also added to the test tube. This procedure was then repeated, in triplicate, for each of the positive controls.
With Activation
0.1 mL of 2AA solution was added to a test tube containing 2.0 mL of molten, trace histidine supplemented, top agar. 0.1 mL of one of the test bacterial suspensions and 0.5 mL of S9 mix were also added to the test tube. The procedure was then repeated, in triplicate, for each tester strain.

The contents of each test tube were equally distributed onto the surface of Vogel-Bonner agar plates (one tube per plate). These plates were incubated at 37°C for at least 48 hours and the number of revertant colonies counted.
EXPERIMENT 2
The complete experiment was repeated using fresh bacterial cultures, test material and control solutions.
Statistics:
For a substance to be considered positive in this test system, it should have induced a dose-related and statistically significant increase in mutation rate (of at least twice the spontaneous reversion rate) in one or more strains of bacteria in the presence and/or absence of the S9 microsomal enzymes in both experiments. To be considered negative the number of induced revertants compared to spontaneous revertants should be less than twofold at each dose level employed, the intervals of which should be between 2 and 5 fold and extend to the limits imposed by toxicity, solubility or up to the maximum recommended dose of 5000 µg/plate. In this case the limiting factor was the maximum recommended dose.

Results and discussion

Test results
Species / strain:
S. typhimurium, other: TA1535, TA100, TA1537, TA1538 and TA98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
a) Preliminary Toxicity Study
The dose range of Substance 1658/5 used in the preliminary toxicity study was 0, 312.5, 625, 1250, 2500 and 5000 µg/plate. The test material was nontoxic in the strain of Salmonella used (TA100).
b) Mutation Study
Checks were done on viability and spontaneous reversion rate for each tester strain. The overnight culture of each strain was found to be in the required range of 10^7 – 10^9 bacteria per mL and the spontaneous reversion rate for each was found to be within the expected range.
No toxicity was exhibited to any of the strains of Salmonella used.
No significant increases in the numbers of revertant colonies of bacteria were recorded for any of the strains of Salmonella, at any dose level used, either with or without metabolic activation.
The positive control substances all produced marked increases in the number of revertant colonies and the activity of the S9 fraction was found to be satisfactory.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Experiment 1

 

Mutation Study Mean and Individual Plate Counts for Test Material and Negative Controls

Strain of Salmonella typhimurium

Concentration (µg/plate)

Metabolic Activation

Mean Number of Revertants

 

 

 

 

 

 

TA1535

5000

1000

200

40

8

0

 

5000

1000

200

40

8

0

-

-

-

-

-

-

 

+

+

+

+

+

+

14

18

13

15

20

15

 

11

12

13

15

10

13

 

 

 

 

 

 

TA1537

5000

1000

200

40

8

0

 

5000

1000

200

40

8

0

-

-

-

-

-

-

 

+

+

+

+

+

+

6

4

3

3

6

5

 

9

8

9

10

7

9

 

 

 

 

 

 

TA1538

5000

1000

200

40

8

0

 

5000

1000

200

40

8

0

-

-

-

-

-

-

 

+

+

+

+

+

+

8

13

11

9

8

11

 

16

18

14

19

20

18

 

 

 

 

 

 

TA98

5000

1000

200

40

8

0

 

5000

1000

200

40

8

0

-

-

-

-

-

-

 

+

+

+

+

+

+

17

23

15

17

19

20

 

25

29

20

26

21

28

 

 

 

 

 

 

TA100

5000

1000

200

40

8

0

 

5000

1000

200

40

8

0

-

-

-

-

-

-

 

+

+

+

+

+

+

85

84

95

89

93

98

 

94

93

102

104

102

85

 

Experiment 2

Mutation Study Mean and Individual Plate Counts for Test Material and Negative Controls

Strain of Salmonella typhimurium

Concentration (µg/plate)

Metabolic Activation

Mean Number of Revertants

 

 

 

 

 

 

TA1535

5000

2500

1250

625

312.5

0

 

5000

2500

1250

625

312.5

0

-

-

-

-

-

-

 

+

+

+

+

+

+

11

11

13

10

10

10

 

15

10

12

13

17

13

 

 

 

 

 

 

TA1537

5000

2500

1250

625

312.5

0

 

5000

2500

1250

625

312.5

0

-

-

-

-

-

-

 

+

+

+

+

+

+

7

8

8

7

5

8

 

9

11

8

8

9

11

 

 

 

 

 

 

TA1538

5000

2500

1250

625

312.5

0

 

5000

2500

1250

625

312.5

0

-

-

-

-

-

-

 

+

+

+

+

+

+

10

8

9

13

14

13

 

14

10

13

11

11

15

 

 

 

 

 

 

TA98

5000

2500

1250

625

312.5

0

 

5000

2500

1250

625

312.5

0

-

-

-

-

-

-

 

+

+

+

+

+

+

17

15

16

16

15

20

 

23

25

24

24

21

26

 

 

 

 

 

 

TA100

5000

2500

1250

625

312.5

0

 

5000

2500

1250

625

312.5

0

-

-

-

-

-

-

 

+

+

+

+

+

+

87

74

72

82

88

81

 

93

85

93

90

86

94

 

Positive Controls

Experiment 1

Mean and Individual Plate Counts for Concurrent Positive Controls

Strain of Salmonella typhimurium

Test Material

Concentration (µg/plate)

Metabolic Activation

Mean Number of Revertants

TA1535

TA1537

TA1538

TA98

TA100

TA1535

TA1537

TA1538

TA98

TA100

MNNG

9AA

4NOPD

4NOPD

MNNG

2AA

2AA

2AA

2AA

2AA

2

50

10

10

2

3.3

3.3

3.3

3.3

3.3

                -

-

-

-

-

+

+

+

+

+

45

316

655

751

519

65

41

241

551

970

 

Experiment 2

Mean and Individual Plate Counts for Concurrent Positive Controls

Strain of Salmonella typhimurium

Test Material

Concentration (µg/plate)

Metabolic Activation

Mean Number of Revertants

TA1535

TA1537

TA1538

TA98

TA100

TA1535

TA1537

TA1538

TA98

TA100

MNNG

9AA

4NOPD

4NOPD

MNNG

2AA

2AA

2AA

2AA

2AA

2

50

10

10

2

3.3

3.3

3.3

3.3

3.3

                -

-

-

-

-

+

+

+

+

+

103

401

509

678

394

124

219

335

375

558

 

REFERENCES

Ames, B.N., Durston, W.E., Yamasaki, E., and Lee, F.D. Proc. Nat. Acad. Sci. USA (1970), 70, 2285

Ames, B.N., McCann, J. and Yamasaki, E., Mutation Research (1975), 31, 347.

McCann, J., Coi, E., Yamasaki, E., and Ames, B.N. Proc. Nat. Acad. Sci. USA (1975) 75, 5135.

Garner, R.C., Miller, E.C., and Miller, J.A. Cancer Res. (1972), 33, 2058.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test material was found to be non-mutagenic under the conditions of this test.
Executive summary:

The test material, SUBSTANCE 1658/5, was found to be non-mutagenic under the conditions of this test, in both the presence and absence of metabolic activation. The method used conforms with the OECD Guidelines for the Testing of Chemicals, Protocol No. 471 and also with Method B14 in Annex V of EEC Commission Directive 84/449/EEC.