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Diss Factsheets

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:
weight of evidence
Study period:
06 - 15 Sep 1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP-guideline study with acceptable restrictions. No TA 102 or E.coli strains were tested.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report date:
1989

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
no TA 102 or E.coli strains were tested
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
no TA 102 or E.coli strains were tested
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Fatty acids, C8-10, C12-18-alkyl esters
EC Number:
306-082-7
EC Name:
Fatty acids, C8-10, C12-18-alkyl esters
Cas Number:
95912-86-0
IUPAC Name:
95912-86-0
Details on test material:
- Name of test material (as cited in study report): fatty acid alkylester C12-18
- Analytical purity: no data
- Physical state: clear liquid
- Lot/batch No.: 190

Method

Target gene:
his operon
Species / strain
Species / strain / cell type:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and 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 500 mg/kg bw Aroclor 1254
Test concentrations with justification for top dose:
Experiment 1: 8, 40, 200, 1000 and 5000 µg/plate without metabolic activation; 16, 80, 400, 1000 and 5000 µg/plate with metabolic activation
Experiment 2: 8, 40, 200, 1000 and 5000 µg/plate with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Tween 80/aqua bidest.
- Justification for choice of solvent/vehicle: the vehicle was chosen according to the solubility properties tested before the start of the study
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide (2.5 µg/plate, ± S9, TA 100, TA 1535); 9 -aminoacridine (80 µg/plate, ± S9, TA 1537); 4 -nitro-o-phenylenediamine (40 µg/plate, ± S9, TA98, TA 1538); 2 -aminoanthracene (5.0 µg/plate ± S9, TA 1535, TA 1537; 2.5 µg/plate, ± S9, TA 98, TA 100)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 12 h
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER: The test batches containing S9-mix were controlled for sterility by adding 0.5 ml S9-mix to untreated agar plates. The microsomal enzyme activity was examined with 2-amino-anthracene and benzo(a)pyrene on TA 98 and in a cytogenetic test with cyclophosphamide.
Evaluation criteria:
A combination of the following criteria was considered as a positive result:
- The plate background of non-reverted bacteria did not show any growth reduction versus the respective negative controls
- The spontaneous mutation rates of each tester strain per plate were within the characteristic spontaneous mutation range (see Table 1 under 'any other information on materials and method including tables')
- As a rule, the positive control showed mutation rates exceding the control values of TA 100 at least by the factor 2.0 and those of the other tester strains at least by the factor 3.0
- At more than one dose tested, the test substance caused at least a 2.0 fold increase in comparison with the negative controls in the tester strain S. typhimurium TA 100. For the other tester strains used, an increase in the mutation rate of more than 3.0 above the corresponding negative controls was considered positive.

Results and discussion

Test results
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
COMPARISON WITH HISTORICAL CONTROL DATA: Yes, the spontaneous mutation rate of each tester strain per plate were within the characteristic spontaneous mutation range (see Table 1).

ADDITIONAL INFORMATION ON CYTOTOXICITY: cytotoxicity was observed at 5000 µg/plate

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 2: Test results of experiment 1

 

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates ± SD)

Base-pair substitution type

Frameshift type

TA 100

TA 1535

TA 1538

TA 98

TA 1537

Negative controls, –S9

Culture medium and Tween 80/aqua bidest.

64.4 ± 11.4

10.0 ± 3.5

9.6 ± 2.9

23.8 ± 3.1

5.5 ± 1.3

8

67.6 ± 4.5

13.3 ± 2.5

10.0 ± 1.0

29.0 ± 4.3

9.6 ± 2.8

-

40

78.3 ± 12.0

9.0 ± 4.0

10.0 ± 1.0

29.3 ± 1.5

5.0 ± 2.6

-

200

79.5 ± 23.3*

7.0 ± 3.0

9.6 ± 2.5

22.6 ± 4.7

8.0 ± 6.0

1000

80.6 ± 17.0

8.6 ± 3.5

11.6 ± 3.2

31.0 ± 2.6

6.0 ± 1.0

5000

55.6 ± 9.4

8.6 ± 2.3

8.3 ± 0.5

29.6 ± 5.5

8.6 ± 2.8

Positive controls, –S9

Name

SA

SA

4-NP

4-NP

9-AA

Concentrations

(μg/plate)

2

2

40

40

80

Mean No. of colonies/plate

(average of 3 ± SD)

266.3 ± 32.3

573.0 ± 32.6

1871.3 ± 40.8

968.6 ± 144.3

218 ± 100.9

Positive controls, –S9

Name

2AA

2AA

2AA

2AA

2AA

Concentrations

(μg/plate)

5

2.5

5

5

2.5

Mean No. of colonies/plate

(average of 3 ± SD)

56.5 ± 2.1*

10.6 ± 1.1

16.6 ± 4.0

26.3 ± 7.2

7.0 ± 3.4

Negative controls, +S9

Culture medium and Tween 80/aqua bidest.

84.6 ± 17.9

12.0 ± 2.5

11.3 ± 1.8

18.0 ± 3.5

7.5 ± 3.3

+

16

71.3 ± 2.0

14.0 ± 2.6

14.3 ± 0.5

22.3 ± 3.5

4.6 ± 2.0

+

80

81.3 ± 11.5

10.3 ± 1.5

9.3 ± 0.5

23.6 ± 2.0

7.0 ± 3.0

+

400

89.3 ± 2.0

13.3 ± 5.5

13.6 ± 4.9

24.0 ± 3.6

7.0 ± 1.7

+

1000

94.6 ± 12.5

13.0 ± 2.6

9.0 ± 2.0

22.6 ± 2.0

4.0 ± 2.6

+

5000

100.0 ± 3.6

15.6 ± 3.0

9.0 ± 1.0

15.0 ± 3.0

5.0 ± 4.0

Positive controls, +S9

Name

SA

SA

4-NP

4-NP

9AA

Concentrations

(μg/plate)

2

2

40

40

80

Mean No. of colonies/plate

(average of 3 ± SD)

140.0 ± 18.7

20.6 ± 2.5

19.6 ± 2.8

721.3 ± 38.8

196.0 ± 67.6

Positive controls, +S9

Name

2AA

2AA

2AA

2AA

2AA

Concentrations

(μg/plate)

5

2.5

5

5

2.5

Mean No. of colonies/plate

(average of 3 ± SD)

1100.3 ± 55.1

438.0 ± 33.4

162.0 ± 22.5

884.0 ± 114.0

46.3 ± 9.0

9-AA = 9-aminoacridine

4-NP = 4-nitro-o-phenylenediamine

SA = sodium azide

2AA = 2-Aminoanthracene

* contamination of 1 plate

Table 3: Test results of experiment 2

 

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates ± SD)

Base-pair substitution type

Frameshift type

TA 100

TA 1535

TA 1538

TA 98

TA 1537

Negative controls, –S9

Culture medium and Tween 80/aqua bidest.

113.1± 12.7

9.8± 3.7

11.8± 3.3

47.6± 4.4

8.0± 1.0

 

8

108.6± 7.0

10.0± 2.8

9.6± 3.7

48.6± 12.5

7.6± 3.0

-

40

109.6± 6.4

9.3± 4.3

11.3± 0.5

43.3± 8.6

6.6± 1.1

-

200

99.5± 6.3

14.6± 5.6

13.0± 6.0

47.0± 11.5

7.0± 2.6

1000

115.0± 14.1

11.0± 1.1

12.0± 3.4

49.0± 7.2

9.0± 2.0

5000

114.3± 9.2

6.3± 2.8

9.6± 2.0

44.0± 7.2

11.0± 3.6

Positive controls, –S9

Name

SA

SA

4-NP

4-NP

9-AA

Concentrations

(μg/plate)

2

2

40

40

80

Mean No. of colonies/plate

(average of 3 ± SD)

645.3± 56.1

388.6± 15.9

2167.3± 101.2

1103.3± 162.4

91.0± 19.5

Positive controls, –S9

Name

2AA

2AA

2AA

2AA

2AA

Concentrations

(μg/plate)

5

2.5

5

5

2.5

Mean No. of colonies/plate

(average of 3 ± SD)

107.3± 6.6

7.6± 2.3

18.6± 8.5

54.6± 3.2

9.6± 2.5

Negative controls, +S9

Culture medium and Tween 80/aqua bidest.

109.0± 10.0

15.1± 4.0

24.1± 1.9

61.3± 7.9

6.8± 2.9

+

8

92.3± 17.0

11.0± 2.6

27.3± 2.5

69.6± 14.5

9.6± 0.5

+

40

116.0± 7.0

12.3± 3.2

29.6± 1.5

59.3± 3.2

7.0± 1.7

+

200

104.0± 16.3

12.6± 4.9

27.0± 6.5

68.6± 8.0

7.3± 1.1

+

1000

118.6± 5.1

14.6± 5.5

25.3± 3.5

70.6± 8.0

7.6± 3.0

+

5000

113.6± 25.7

9.3± 3.0

28.6± 1.5

75.3± 3.2

12.6± 1.5

Positive controls, +S9

Name

SA

SA

4-NP

4-NP

9AA

Concentrations

(μg/plate)

2

2

40

40

80

Mean No. of colonies/plate

(average of 3 ± SD)

202.0± 16.0

178.3± 7.6

1487.0± 128.0

736.3± 7.9

65.0± 15.0

Positive controls, +S9

Name

2AA

2AA

2AA

2AA

2AA

Concentrations

(μg/plate)

5

2.5

5

5

2.5

Mean No. of colonies/plate

(average of 3 ± SD)

1337.6± 47.5

194.6± 24.9

1515.0± 171.6

1100.6± 47.5

87.6± 17.6

9-AA = 9-aminoacridine

4-NP = 4-nitro-o-phenylenediamine

SA = sodium azide

2AA = 2-Aminoanthracene

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative