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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
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1994
Report date:
1994
Reference Type:
secondary source
Title:
Unnamed
Year:
2001

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 1997
Deviations:
yes
Remarks:
S. typhimurium strain TA 102 or an E. coli strain not included, only 2-aminoacridine used as the positive control with metabolic activation and the S-9 mix was not characterised with a mutagen requiring metabolic activation
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

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 Aroclor 1254
Test concentrations with justification for top dose:
Experiment I and II: 1.0, 5.0, 10, 50 and 100 µL/plate (all strains), with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: the test substance was miscible to 1mL/mL in acetone and corn oil. The stock solution of the test substance was prepared in acetone, therefore acetone was selected as the vehicle
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide (1 µg/plate, -S9, TA 1535, TA 100); 9-aminoacridine (50 μg/plate, -S9, TA 1537); 2-nitrofluorene (5 μg/plate, -S9, TA 98, TA 1538); 2-aminoanthracene (1.25 μg/plate, +S9,TA 1535, TA 1537, TA 98, TA 100, TA 1538)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

NUMBER OF REPLICATIONS: triplicates in each of two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (thinning of bacterial background lawn); reduction in the number of revertant colonies per plate

OTHER:
In a preliminary range-finding test, the cytogenicity of the test substance was assessed. TA 100 was treated with 0.05, 0.1, 0.5, 1.0, 5.0, 10, 50 and 100 µL/plate, with and without metabolic activation. The genotypes of the tester strains were confirmed by testing for histidine requirement and rfa-mutation, and TA and TA 100 were confirmed positive for the pKM101 plasmid. The viabilty of the tester strain was confirmed. The sterility of the S9-mix was checked before the beginning and the end of each experiment and was found to be satisfactory.
Evaluation criteria:
Evaluation criteria:
A response is considered positive if at least one strain has a dose that produces a mean reversion frequency that is 2 times or more greater than the mean reversion frequency of the corresponding solvent control plates and the response is dose dependent. The degree of toxicity will be considered in relation to the results. A response is considered equivocal if it does not fulfill the criteria of either negative or positive response and/or the study director does not consider the reponse to be either positive or negative.
Statistics:
Mean values and standard deviations were calculated for the number of revertants per plate.

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:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: the test substance was soluble up to 1 mL/mL in acetone and corn oil
- Precipitation: slight precipitation was observed in the range-finding assay from 50 µL/plate (TA 100) with and without metabolic activation and in the main assays with and withour metabiolic activation.

RANGE-FINDING/SCREENING STUDIES: cell viability was comparable between control and treated strains up to the highest concentration level of 100 µL/plate. Slight precipitation was observed in the range-finding assay from 50 µL/plate (TA 100) with and without metabolic activation.

Any other information on results incl. tables

Table 1: Experiment 1

EXPERIMENT 1 (plate incorporation test)

S9-Mix

Without

 

Test item (µL/plate)

TA 98

TA 100

TA 1535

TA 1537

TA 1538

NC

26 ± 5.9

80 ± 4.0

21 ± 4.0

12 ± 3.6

10 ± .6

1.0

24 ± 2.3

80 ± 3.8

21 ± 1.5

13 ± 3.1

10 ± 3.8

5.0

24 ± 1.5

78 ± 1.5

19 ± 3.8

11 ± 2.9

9 ± 4.0

10

29 ± 5.9

69 ± 2.0

16 ± 3.5

11 ± 3.1

10 ± 2.6

50

27 ± 3.6*

85 ± 4.9*

16 ± 0.6*

9 ± 2.6*

10 ± 2.1*

100

23 ± 3.5*

82 ± 12.8*

20 ± 0.6*

9 ± 3.0*

9 ± 4.0*

2-NF

775 ± 71.5

-

-

-

857 ± 60.2

SA

-

476 ± 4.7

456 ± 16.7

-

-

9-AA

-

-

-

86 ± 10.8

-

S9-Mix

With

 

 

 

 

 

 

 

Test item (µL/plate)

TA 98

TA 100

TA 1535

TA 1537

TA 1538

NC

26 ± 6.0

57 ± 4.0

11 ± 1.7

13 ± 1.2

14 ± 1.7

1.0

31 ± 6.7

61 ± 0.6

13 ± 1.5

13 ± 2.3

14 ± 1.5

5.0

34 ± 1.2

75 ± 6.0

14 ± 4.0

11 ± 3.1

16 ± 7.2

10

32 ± 2.5

63 ± 4.4

16 ± 1.2

11 ± 2.6

11 ± 5.0

50

29 ± 4.0*

67 ± 2.0*

15 ± 0.6*

10 ± 3.6*

11 ± 4.9*

100

27 ± 3.6*

61 ± 1.7*

15 ± 1.5*

11 ± 2.1*

12 ± 0.6*

2AA

665 ± 43.9

716 ± 68.0

366 ± 1.5

138 ± 1.0

720 ± 114.1

*slight precipitate

NC = Vehicle Control, acetone

2-NF: 2-nitrofluorene

SA: sodium azide

9AA:9-aminoacridine

2AA: 2-aminoanthracene

 

Table 2: Experiment 2

EXPERIMENT 2 (plate incorporation test)

S9-Mix

Without

 

Test item (µL/plate)

TA 98

TA 100

TA 1535

TA 1537

TA 1538

NC

27 ± 2.6

66 ± 2.1

20 ± 0.6

11 ± 3.1

11 ± 1.2

1.0

24 ± 1.5

70 ± 2.6

19 ± 2.9

12 ± 2.9

11 ± 2.1

5.0

25 ± 3.1

71 ± 2.5

19 ± 2.1

13 ± 5.5

12 ± 2.0

10

28 ± 3.0

56 ± 2.1

19 ± 4.6

12 ± 2.5

12 ± 2.5

50

26 ± 4.7*

63 ± 5.5*

23 ± 1.2*

11 ± 1.2*

11 ± 1.7*

100

21 ± 3.2*

61 ± 3.8*

21 ± 2.6*

10 ± 2.5*

9 ± 0.6*

2-NF

781 ± 73.2

-

-

-

919 ± 97.6

SA

-

486 ± 31.3

462 ± 14.6

-

-

9-AA

-

-

-

100 ± 6.0

-

S9-Mix

With

 

 

 

 

 

 

 

Test item (µL/plate)

TA 98

TA 100

TA 1535

TA 1537

TA 1538

NC

33 ± 6.5

57 ± 6.6

17 ± 1.5

14 ± 1.5

13 ± 1.0

1.0

36 ± 4.0

57 ± 3.1

16 ± 5.5

12 ± 2.5

12 ± 0.6

5.0

36 ± 2.6

57 ± 8.9

14 ± 4.0

12 ± 2.0

12 ± 2.3

10

33 ± 3.1

58 ± 11.7

14 ± 1.2

12 ± 0.6

12 ± 3.0

50

37 ± 3.8*

58 ± 10.1*

15 ± 1.0*

10 ± 2.5*

15 ± 4.9*

100

33 ± 7.0*

60 ± 9.0*

12 ± 2.1*

13 ± 3.2*

13 ± 0.6*

2AA

699 ± 50.8

802 ± 97.2

344 ± 5.9

122 ± 8.1

673 ± 68.5

*slight precipitate

NC = Vehicle Control, acetone

2-NF: 2-nitrofluorene

SA: sodium azide

9AA:9-aminoacridine

2AA: 2-aminoanthracene

 

 

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative