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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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
26 Mar - 05 Apr 1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline study with acceptable restrictions. No S. typhimurium TA102 or E. coli strain was included.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report Date:
1991

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
no S. typhimurium TA102 or E. coli strain was included
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
no S. typhimurium TA102 or E. coli strain was included
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): [trade name], Decanoic acid, mixed diesters with octanoic acid and propylene glycol
- Analytical purity: 100%
- Physical state: colourless liquid
- Lot/batch No.: 3409
- Production date: November 04, 1990
- Expiration date of the lot/batch: May 04, 1992
- Storage condition of test material: at room temperature
- Stability:
Pure: 18 months
In Solvent: not given
- Receipt of the test substance: February 08, 1991

Method

Target gene:
his operon
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
- Type and identity of media: Standard I nutrient broth for microbiology
Additional strain / cell type characteristics:
other: rfa-; uvrB- (R+ for TA98 and TA100)
Species / strain / cell type:
S. typhimurium TA 1538
Details on mammalian cell type (if applicable):
- Type and identity of media: Standard I nutrient broth for microbiology
Additional strain / cell type characteristics:
other: rfa-; uvrB-
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254.
Test concentrations with justification for top dose:
First and second experiment: 8, 40, 200, 1000 and 5000 µg/plate with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Suspension medium Tween 80/bidest. water
- Justification for choice of solvent/vehicle: According to the author, the suspension medium was chosen based on the solubility properties preliminary tested before start of the study.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Tween 80/bidest. water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide (2 µg/plate, ±S9, TA1535 and TA100); 9-Aminoacridine (80 µg/plate, ±S9, TA1537); 4-Nitro-o-phenylendiamine (40 µg/plate, ±S9,TA98 and TA1538); 2 Amino-anthracene (2.5 or 5 µg/plate, ±S9, TA1535 and TA1357; TA100, TA1538 and TA98)
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
Evaluation criteria:
The test material may be considered positive in this test system if a combination of the following criteria are met:
- the plate background of non-reverted bacteria does not show any growth reduction versus the respective negative controls.
- the spontaneous mutation rates of each tester strain per plate are within the characteristic spontaneous mutation range.
- the positive controls show mutation rates exceeding the control values of TA100 at least two fold and those of the other strains at least by the factor 3.
- at more than one dose tested, at least a two-fold (or more) increase in comparison with the negative controls in the tester strain TA100. For the other strains, an increase in the mutation rate of more than 3 above the corresponding negative controls.
Statistics:
Mean values and standard deviation were 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 / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
COMPARISON WITH HISTORICAL CONTROL DATA:
- see Table 1 and Table 2
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

COMPARISON WITH HISTORICAL DATA

Table 1. Characteristic spontaneous mutation range of the test batches without S9-mix.

Tester strains

TA1535

TA100

TA1537

TA1538

TA98

Historical laboratory values (Mean values)

Extreme values

 

6

1-25

 

87

35-201

 

7

1-24

 

15

6-27

 

20

5-39

Ames et al.*

(Mean values)

Extreme values

 

20

5-50

 

160

60-220

 

7

3-25

 

25

5-40

 

40

15-75

 *Ames, BN, McCann, J, Yamasaki, E 1977, ´Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsome mutagenicity test.`in Kilbey et al. Handbook of Mutagenicity Test Procedures, Elsevier, Amsterdam, pp. 1 -17.

Table 2. Characteristic spontaneous mutation range of the test batches containing S9-mix.

Tester strains

TA1535

TA100

TA1537

TA1538

TA98

Historical laboratory values (Mean values)

Extreme values

 

8

1-25

 

105

54-252

 

6

1-23

 

18

3-48

 

27

7-76

STUDY RESULTS

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

TA1535

TA100

TA1537

TA1538

TA98

Negative control

11.7 ± 2.3

99.7 ± 11.7

6.7 ± 3.8

8.7 ± 0.6

22.7 ± 1.5

Solvent control

12.7 ± 5.5

113.3 ± 6.4

6.3 ± 1.5

11.30 ± 2.1

27.0 ± 3.5

8

13.7 ± 1.5

103.3 ± 3.1

8.0 ± 3.6PM

7.7 ± 1.5

30.0 ± 5.3

40

9.3 ± 2.1

104.7 ± 12.0

6.3 ± 1.5

10.7 ± 3.8

26.3 ± 6.7

200

8.7 ± 3.1

113.0 ± 7.0

8.0 ± 2.0

11.0 ± 2.0

26.0 ± 2.7

1000

13.7 ± 4.2

104.7 ± 7.1

8.0 ± 3.0

13.3 ± 4.0

27.3 ± 3.5

5000

9.7 ± 0.6

110.0 ± 8.9

7.0 ± 3.6

7.3 ± 2.3

24.3 ± 2.1

Positive

controls,

–S9

Name

SA

SA

9AA

4NPD

4NPD

Concentrations

(μg/plate)

2

2

80

40

40

Mean No. of colonies/plate

(average of 3 ± SD)

534.7 ± 38.4

324.0 ± 19.2

352.0 ± 57.1

1428.0 ± 19.0

699.0 ± 99.0

Name

2AA

2AA

2AA

2AA

2AA

Concentrations

(μg/plate)

2.5

5

2.5

5

5

Mean No. of colonies/plate

(average of 3 ± SD)

9.7 ± 0.6

110.7 ± 7.6

10.7 ± 2.1

18.7 ± 6.1

27.3 ± 1.2

+

Negative control

12.7 ± 3.1

101.7 ± 7.8

9.3 ± 2.1

18.7 ±3.2

42.3 ± 5.0

+

Solvent control

14.7 ± 3.5

107.0 ± 15.4

9.3 ± 3.2

15.3 ± 2.1

45.0 ± 5.0

+

8

14.0 ± 4.4

103.7 ± 23.0

8.0 ± 1.7

12.7 ± 2.1

42.7 ± 4.7

+

40

14.3 ± 2.1

104.3 ± 12.7

6.3 ± 1.2

15.7 ± 2.9

49.3 ± 2.1

+

200

11.7 ± 1.5

115.0 ± 10.4

7.0 ± 1.0

14.7 ± 4.7

38.7 ± 2.3

+

1000

16.0 ± 2.0

116.7 ± 11.9

5.3 ± 1.5

17.0 ± 2.0

45.3 ± 8.1

+

5000

6.3 ± 0.6

81.0 ± 4.6

6.3 ± 3.2

16.0 ± 2.7

31.3 ± 0.6

Positive controls, +S9

Name

SA

SA

9AA

4NPD

4NPD

Concentrations

(μg/plate)

2

2

80

40

40

Mean No. of colonies/plate

(average of 3 ± SD)

143.0 ± 17.8

180.3 ± 8.1

268.3 ± 18.5

1170.3 ± 84.0

617.3 ± 169.5

Name

2AA

2AA

2AA

2AA

2AA

Concentrations

(μg/plate)

2.5

5

2.5

5

5

Mean No. of colonies/plate

(average of 3 ± SD)

181.0 ± 19.3

1059.3 ± 129.7

95.7 ± 16.3

1219.7 ± 152.3

875.0 ± 40.5

SA = sodium azide

4NPD = 4-nitro-o-phenylendiamine

9AA = 9-aminoacridine

2AA = 2-Amino-anthracene

PM = in part manual evaluation

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

TA1535

TA100

TA1537

TA1538

TA98

Negative control

9.7 ± 1.5

88.3 ± 10.8

10.3 ± 4.0

9.7 ± 2.5

28.7 ± 2.5

Solvent control

8.3 ± 1.5

86.7 ± 19.7

8.7 ± 1.2

10.7 ± 1.2

26.3 ± 4.0

8

9.3 ± 1.5

86.0 ± 3.5

7.3 ± 0.6

9.0 ± 1.0

24.7 ± 4.0

40

10.3 ± 0.6

91.7 ± 18.3

8.7 ± 2.1

9.3 ± 1.5

27.0 ± 6.6

200

6.7 ± 2.5

92.7 ± 4.5

9.3 ± 3.1

8.0 ± 2.7

20.7 ± 1.5

1000

10.3 ± 3.1

92.7 ± 5.9

8.0 ± 4.6

8.7 ± 5.5

31.3 ± 1.2

5000

7.7 ± 3.1PM

92.3 ± 20.0

9.0 ± 4.0

8.7 ± 1.2

28.3 ± 5.1

Positive

controls,

–S9

Name

SA

SA

9AA

4NPD

4NPD

Concentrations

(μg/plate)

2

2

80

40

40

Mean No. of colonies/plate

(average of 3 ± SD)

357.3 ± 26.8

216.7 ± 15.3

692.0 ± 186.2

1242.3 ± 56.2

535.0 ± 49.7

Name

2AA

2AA

2AA

2AA

2AA

Concentrations

(μg/plate)

2.5

5

2.5

5

5

Mean No. of colonies/plate

(average of 3 ± SD)

9.3 ± 2.1

80.3 ± 8.2

6.7 ± 2.9

14.3 ± 2.5

32.3 ± 4.6

+

Negative control

7.0 ± 2.7

72.3 ± 8.0

7.0 ± 1.0

12.7 ± 0.6M

30.0 ± 7.6M

+

Solvent control

9.7 ± 2.3

80.0 ± 12.1

7.0 ± 1.0

10.7 ± 0.6M

37.3 ± 4.7M

+

8

6.7 ± 1.2

64.7 ± 5.5

8.7 ± 1.5M

14.3 ± 1.5M

36.3 ± 6.7M

+

40

5.3 ± 0.6

72.3 ± 9.7

7.3 ± 1.2M

11.7 ± 1.5M

31.7 ± 3.2M

+

200

8.7 ± 1.5

77.0 ± 14.8

7.7 ± 2.1M

11.3 ± 2.5M

30.7 ± 9.5M

+

1000

7.7 ± 1.5

69.7 ± 4.0

6.0 ± 1.0M

11.0 ± 1.0M

30.7 ± 8.5M

+

5000

5.0 ± 1.0

58.0 ± 2.0

3.3 ± 1.5M

6.3 ± 0.6M

22.7 ± 4.0M

Positive controls, +S9

Name

SA

SA

9AA

4NPD

4NPD

Concentrations

(μg/plate)

2

2

80

40

40

Mean No. of colonies/plate

(average of 3 ± SD)

57.0 ± 3.6

98.0 ± 11.4

428.3 ± 29.3

986.0 ± 89.9M

720.0 ± 16.0M

Name

2AA

2AA

2AA

2AA

2AA

Concentrations

(μg/plate)

2.5

5

2.5

5

5

Mean No. of colonies/plate

(average of 3 ± SD)

90.3 ± 20.8

467.0 ± 22.5

105.3 ± 12.1

1072.0 ±57.7M

909.3 ± 159.8M

SA = sodium azide

4NPD = 4-nitro-o-phenylendiamine

9AA = 9-aminoacridine

2AA = 2-Amino-anthracene

M = manual evaluation

PM = in part manual evaluation

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative