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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:
key study
Study period:
1984-07-17 to 1984-07-27
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1984
Report date:
1984

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
fifth strain is missing in order to detect cross-linking mutagens
GLP compliance:
no
Remarks:
but QAU statement included
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Thiodiethylene bis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate]
EC Number:
255-392-8
EC Name:
Thiodiethylene bis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate]
Cas Number:
41484-35-9
Molecular formula:
C38H58O6S
IUPAC Name:
thiodiethylene bis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate]
Details on test material:
- Physical state: solid
- Analytical purity: > 99.5% (CIBA-GEIGY Ltd, Analyse Number: 5659, 25.10.1984)

Method

Target gene:
his operon
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
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:
20, 80, 320, 1280, and 5120 µg/0.1 mL with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see in "Any other informations on materials and methods"
Details on test system and experimental conditions:
METHOD OF APPLICATION: agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
The test substance is generally considered to be nonmutagenic if the colony count in relation to the negative control is not doubled at any concentration.
Statistics:
Arithmetic mean values and standard deviation were calculated.

Results and discussion

Test results
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:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At the concentrations of 320 µg/0.1 ml and above the substance precipitated in soft agar.

RANGE-FINDING/SCREENING STUDIES:
A preliminary toxicity test was carried out with concentrations ranging from 0.1 to 5000 µg/0.1 ml. Thereupon, the concentration of 5120 µg/0.1 ml was used as the highest in the mutagenicity test.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Plate incorportation assay - First experiment:

With or without S9-Mix

Test substance concentration

Mean number of revertant colonies per plate

(μg/plate)

(average of 3 plates ± Standard deviation)

 

Base-pair substitution type

 

Frameshift type

 

TA 100

TA1535

TA98

TA1537

0

121 ± 10

14 ± 10

32 ± 10

6 ± 1

20

103 ± 18

8 ± 3

32 ± 6

7 ± 1

80

128 ± 4

19 ± 2

23 ± 3

7 ± 3

320

118 ± 28

13 ± 5

24 ± 4

8 ± 5

1280

128 ± 23

11 ± 5

19 ± 2

6 ± 2

5120

107 ± 8

17 ± 1

17 ± 8

7 ± 2

Positive controls, –S9

Name

4NQO

SA

DR

9AA

Concentrations (μg/plate)

0.125

2.5

5

50

0.25

5

10

100

Mean No. of colonies/plate (average of 3 ± SD)

851 ± 81

1426 ± 105

366 ± 82

100 ± 22

1456 ± 141

1965 ± 181

841 ± 79

1531 ± 801

+

0

121 ± 6

11 ± 6

45 ± 3

17 ± 3

+

50

108 ± 10

11 ± 6

54 ± 10

16 ± 2

+

150

116 ± 6

13 ± 4

50 ± 6

18 ± 4

+

500

94 ± 9

14 ± 4

45 ± 3

15 ± 5

+

1500

101 ± 10

14 ± 7

53 ± 1

17 ± 3

+

5000

89 ± 13

13 ± 7

47 ± 5

17 ± 2

Positive controls, –S9

Name

 

CP

 

 

Concentrations (μg/plate)

 

250

 

 

Mean No. of colonies/plate (average of 3 ± SD)

 

547 ± 103

 

 

4NQO: 4-nitroquinoline-N-oxide

SA: sodium azide

DR: daunorubicin

9AA: 9(5)aminoacridine hydrochloride monohydrate

CP: cyclophosphamide

Table 2: Plate incorportation assay - Second experiment

With or without S9-Mix

Test substance concentration

Mean number of revertant colonies per plate

(μg/plate)

(average of 3 plates ± Standard deviation)

 

Base-pair substitution type

 

Frameshift type

 

TA 100

TA1535

TA98

TA1537

0

139 ± 10

12 ± 4

27 ± 6

5 ± 3

20

120 ± 23

16 ± 6

23 ± 5

10 ± 6

80

148 ± 19

14 ± 6

27 ± 6

9 ± 4

320

136 ± 14

15 ± 4

26 ± 7

12 ± 5

1280

126 ± 10

12 ± 7

23 ± 3

9 ± 4

5120

127 ± 9

12 ± 3

23 ± 2

11 ± 4

Positive controls, –S9

Name

4NQO

SA

DR

9AA

Concentrations (μg/plate)

0.125

2.5

5

50

0.25

5

10

100

Mean No. of colonies/plate (average of 3 ± SD)

992 ± 26

865 ± 65

806 ± 69

66 ± 21

1617 ± 139

1343 ± 61

1186 ± 188

973 ± 349

+

0

127 ± 17

15 ± 1

47 ± 9

10 ± 4

+

50

130 ± 10

13 ± 7

39 ± 4

10 ± 6

+

150

123 ± 24

17 ± 2

43 ± 5

13 ± 5

+

500

129 ± 9

12 ± 4

46 ± 15

9 ± 4

+

1500

114 ± 5

15 ± 2

33 ± 6

13 ± 5

+

5000

118 ± 20

12 ± 4

35 ± 8

6 ± 3

Positive controls, –S9

Name

 

CP

 

 

Concentrations (μg/plate)

 

250

 

 

Mean No. of colonies/plate (average of 3 ± SD)

 

369 ± 48

 

 

4NQO: 4-nitroquinoline-N-oxide

SA: sodium azide

DR: daunorubicin

9AA: 9(5)aminoacridine hydrochloride monohydrate

CP: cyclophosphamide

The slight increase in the number of back-mutant colonies observed in the second experiment without microsomal activation on strain TA 1537 at the concentrations of 20, 320 and 5120 µg/0.1 mL is attributed to fluctuations in the rate of spontaneously occurring back-mutants.

Applicant's summary and conclusion

Conclusions:
No evidence of the induction of point mutations by the test article or by the metabolites of the substance was detectable in the strains of S. typhimurium used in these experiments.
Executive summary:

In an Ames test similar to OECD guideline 471, the test substance was tested for mutagenic effects on histidine-auxotrophic mutants of Salmonella typhimurium, using the plate incorporation method. The investigations were performed on strains TA 98, TA 100, TA 1535 and TA 1537 with the following concentrations of the trial substance without and with microsomal activation: 20, 80, 320, 1280 and 5120 µg/0.1 mL. In order to confirm the results the experiments were repeated. In the experiments performed without and with microsomal activation, comparison of the number of back-mutant colonies in the controls and the cultures treated with the various concentrations of the test substance revealed no marked deviations. No evidence of the induction of point mutations by the test substance or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium used in these experiments.

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