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Toxicological information

Genetic toxicity: in vitro

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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:
05 Jun - 28 Jun 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)
Version / remarks:
adopted May 26, 1983 (2)
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)
Version / remarks:
adopted March 2, 1983
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
- Chemical denomination: fatty acids, C14-18 and C16-18 unsaturated, ester with propylenglycol
- Physical state: yellowish liquid
- Analytical purity: 100% content of active substance
- Lot/batch No.: 0130
- Stability under test conditions: pure: 18 months; in solvent: not given
- Expiration date of the lot/batch: 1992-07-16
- Storage condition of test material: at room temperature

Method

Target gene:
his operon
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
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 nor precipitates, but tested up to recommended limit concentrations
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 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
- Other confounding effects: Contamination of 1 plate of strain TA98 in the second experiment (1000 µg/plate), therefore only 2 individual values were used for calculation of the mean number of revertant colonies at this concentration.

COMPARISON WITH HISTORICAL CONTROL DATA:
- see Table 1 and Table 2 under "Any other information on results incl. tables".
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

9.7 ± 3.1

97.7 ± 2.1

6.3 ± 3.2

6.7 ± 1.2

46.7 ± 3.2

Solvent control

7.7 ± 3.5

94.3 ± 17.4

8.0 ± 1.7

7.3 ± 2.3

46.3 ± 6.0

8

8.3 ± 2.1

70.0 ± 7.8

9.0 ± 1.0

6.7 ± 1.2

33.7 ± 0.6

40

11.0 ± 2.7

74.3 ± 3.2

7.3 ± 2.1

5.3 ± 0.6

40.3 ± 4.9

200

12.0 ± 4.6

67.3 ± 11.2

6.3 ± 1.2

5.7 ± 1.2

37.3 ± 3.2

1000

10.0 ± 4.0

62.7 ± 10.8

10.3 ± 1.2

4.3 ± 1.5

42.7 ± 3.1

5000

9.0 ± 1.7

83.0 ± 6.9

5.7 ± 1.5

4.3 ± 0.6

48.3 ± 3.5

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)

744.7 ± 49.3

894.0 ± 62.0

939.7 ± 181.4

1541.0 ± 96.0

1410.7 ± 154.4

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)

9.0 ± 1.7

88.7 ± 17.7

7.0 ± 2.7

10.3 ± 1.5

46.0 ± 8.7

+

Negative control

5.7 ± 0.6

80.3 ± 15.3

8.7 ± 2.9

11.3 ± 2.3

53.3 ± 3.2

+

Solvent control

9.7 ± 4.6

80.3 ± 14.4

9.3 ± 1.2

13.7 ± 2.3

44.3 ± 6.7

+

8

12.3 ± 3.1

82.3 ± 12.1

7.7 ± 3.5

14.7 ± 2.9

49.7 ± 4.2

+

40

11.3 ± 4.6

92.0 ± 11.8

6.3 ± 0.6

13.0 ± 4.2

47.7 ± 4.0

+

200

8.0 ± 2.0

84.3 ± 4.9

8.0 ± 1.0

16.3 ± 1.5

53.3 ± 4.0

+

1000

10.0 ± 2.0

70.7 ± 9.7

9.3 ± 3.8

13.0 ± 1.0

50.5 ± 3.5C

+

5000

10.0 ± 3.6

74.7 ± 11.0

7.0 ± 1.7

13.0 ± 3.0

54.0 ± 7.0

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)

213.3 ± 19.8

230.0 ± 4.6

283.3 ± 27.8

1434.0 ± 68.9

1307.6 ± 68.3

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)

186.3 ± 19.4

1328.3 ± 166.5

52.3 ± 1.5

1711.3 ± 25.0

1587.7 ± 237.7

SA = sodium azide

4NPD = 4-nitro-o-phenylendiamine

9AA = 9-aminoacridine

2AA = 2-Amino-anthracene

C = Contamination, only 2 individual values

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

13.3 ± 1.5

117.7 ± 10.0

7.7 ± 2.9

9.0 ± 4.4

33.0 ± 1.0

Solvent control

10.3 ± 5.8

111.7 ± 5.0

7.3 ± 0.6

9.3 ± 1.5

30.0 ± 6.3

8

11.3 ± 2.3

118.3 ± 12.5

7.3 ± 1.5

13.0 ± 2.0

32.0 ± 12.5

40

14.0 ± 2.7

105.0 ± 17.4

9.3 ± 0.6

9.3 ± 0.6

25.0 ± 6.2

200

13.0 ± 1.0

97.7 ± 2.9

8.0 ± 1.7

7.7 ± 2.1

32.0 ± 6.6

1000

13.3 ± 2.1

111.7 ± 9.0

9.7 ± 4.0M

8.7 ± 4.0

41.3 ± 4.2

5000

12.0 ± 1.0

125.3 ± 4.9

7.3 ± 2.1

11.0 ± 4.6

30.7 ± 2.5

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)

807.3 ±10.6

945.3 ± 37.5

1156.0 ± 216.5

2159.3 ± 106.6

226.7 ± 24.7

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)

12.3 ± 5.5

110.3 ± 13.1

9.3 ± 3.8

22.0 ± 6.1

29.3 ± 3.8

+

Negative control

12.3 ± 5.8

121.7 ± 10.3

9.3 ± 2.3

14.0 ± 7.2

39.7 ± 3.1

+

Solvent control

12.3 ± 3.1

120.3 ± 17.0

8.7 ± 1.2

11.3 ± 1.5

45.0 ± 1.0

+

8

15.0 ± 3.5

122.3 ± 12.7

6.7 ± 2.5

9.7 ± 1.2

40.1 ± 2.3

+

40

13.3 ± 2.3

123.0 ± 7.6

8.7 ± 5.0

13.7 ± 6.7

41.3 ± 8.3

+

200

16.0 ± 3.0

122.3 ± 13.3

9.0 ± 3.6

9.7 ± 6.4

45.0 ± 10.2

+

1000

16.0 ± 4.6

104.0 ± 19.3

8.3 ± 1.2

12.3 ± 3.2

41.7 ± 6.4

+

5000

16.0 ± 1.7

95.0 ± 8.5

11.7 ± 3.5

12.0 ± 5.6

39.0 ± 13.1

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)

232.0 ± 15.1

286.0 ± 29.2

748.0 ± 139.2

455.3 ± 14.2

531.3 ± 22.7

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)

131.0 ± 5.2

1735.0 ± 84.5

81.3 ± 8.4

585.3 ± 32.1

594.3 ± 56.6

SA = sodium azide

4NPD = 4-nitro-o-phenylendiamine

9AA = 9-aminoacridine

2AA = 2-Amino-anthracene

M = Manual evaluation

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative