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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:
09 - 23 May 1986
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1986
Report date:
1986

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
E.coli and/or TA 102 tester strain were not included in the study; cytotoxicity/precipitation was not determined.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
1,4:3,6-dianhydro-2,5-di-O-methyl-D-glucitol
EC Number:
226-159-8
EC Name:
1,4:3,6-dianhydro-2,5-di-O-methyl-D-glucitol
Cas Number:
5306-85-4
Molecular formula:
C8H14O4
IUPAC Name:
1,4:3,6-dianhydro-2,5-di-O-methyl-D-glucitol
Test material form:
other: liquid

Method

Target gene:
his operon
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: All TA strains carry a mutation of the uvr B gene coding for the DNA excision repair system (uvrB) and the deep rough mutation (rfa), TA100 and TA98 contain the R-factor plasmid (pkM101) to detect weak mutagens.
Species / strain / cell type:
S. typhimurium TA 1538
Additional strain / cell type characteristics:
other: All TA strains carry a mutation of the uvr B gene coding for the DNA excision repair system (uvrB) and the deep rough mutation (rfa), TA100 and TA98 contain the R-factor plasmid (pkM101) to detect weak mutagens.
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
- 1.6, 8, 40, 200, 1000 and 5000 µg/plate (with and without metabolic activation)
Experiment II
- 1.6, 8, 40, 200, 1000 and 5000 µg/plate (with and without metabolic activation)
Experiment III
- 1.6, 8, 40, 200, 1000 and 5000 µg/plate (with and without metabolic activation)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: Acridine Mutagen ICR191 (AM), 2-Aminoanthracene (2AA), Daunorubicin HCl (DR), 4-Nitro-o-phenylene diamine (4NPD), N-Methyl-N´-nitro-N-nitrosoguanidine (MNNG)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

NUMBER OF REPLICATIONS: 3 plates for each test concentration, 5 plates for negative controls, 2 plates for each positive control
Evaluation criteria:
A positive response in an experiment is achieved when a two-fold or greater increase in the mean number of revertant colonies per test plate (over and above that observed for the solvent control plates) is obtained at at least one dose level. A second criterion for a positive result is the observation of a statistically significant dose-related increase in the number of revertants. In either case, the observed effect should be reproducible.
Statistics:
Mean values (colony count/plate) and standard deviation were calculated. An assessment of the statistical significance was carried out using a one-tailed Student´s t-test. The corresponding probability for each dose level was determined from a t-table using the appropriate degrees of freedom.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
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:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: No information is given, if a range-finding study was performed. In addition, cytotoxicity was neither examined in a previous study nor during the course of the study.

COMPARISON WITH HISTORICAL CONTROL DATA: No information is available for historical control data.

Any other information on results incl. tables

Table 1: Test Results - Experiment I (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

TA 1535

TA 100

TA 1537

TA 1538

TA 98

0

19.0 ± 2.7

114.0 ± 16.2

10.0 ± 4.0

5.0 ± 1.9

15.2 ± 4.1

1.6

21.0 ± 1.7

101.7 ± 16.3

14.0 ± 0.0

6.7 ± 3.1

14.7 ± 3.5

8

20.7 ± 1.2

109.0 ± 3.6

12.0 ± 0.0

5.0 ± 2.0

16.7 ± 2.1

40

18.7 ± 1.5

96.0 ± 15.6

10.0 ± 1.4

6.7 ± 1.2

16.3 ± 3.5

200

18.7 ± 4.0

120.0 ± 16.1

C

5.7 ± 3.8

17.3 ± 4.6

1000

20.7 ± 2.1

108.7 ± 13.6

C

7.0 ± 2.6

17.3 ± 1.5

5000

26.0 ± 6.9

114.3 ± 12.7

18.3 ± 3.1

6.0 ± 1.0

17.0 ± 4.0

 

 

 

 

 

 

 

Positive controls, –S9

Name

MNNG

MNNG

AM

4NPD

DR

Concentrations

(μg/plate)

1

1

1

1

0.2

Mean No. of colonies/plate

(average of 2 ± SD)

59.0 ± 26.9

215.0 ± 2.8

90.0 ± 0.0

49.5 ± 0.7

298.0 ± 24.0

Concentrations

(μg/plate)

2

2

2

2

0.5

Mean No. of colonies/plate

(average of 2 ± SD)

854.0 ± 69.3

825.0 ± 21.2

125. 0 ± 12.7

100.5 ± 19.1

700.5 ± 3.5

Concentrations

(μg/plate)

5

5

-

5

1

Mean No. of colonies/plate

(average of 2 ± SD)

2285.0 ± 257.4

2160.5 ± 85.6

-

167.0 ± 25.5

1019.5 ± 64.3

+

0

14.0 ± 2.2

127.2 ± 18.9

15.4 ± 3.2

8.4 ± 3.8

16.0 ± 5.1

+

1.6

17.0 ± 3.6

132.3 ± 17.2

15.0 ± 2.0

7.0 ± 2.6

17.7 ± 5.7

+

8

15.3 ± 4.0

134.3 ± 7.4

15.0 ± 0.0

7.3 ± 1.5

20.7 ± 3.5

+

40

15.7 ± 4.6

132.3 ± 15.3

13.3 ± 3.5

7.3 ± 1.2

15.0 ± 3.6

+

200

22.3 ± 4.7

123.3 ± 11.6

9.0 ± 4.4

7.0 ± 1.0

16.0 ± 1.7

+

1000

16.3 ± 1.5

119.7 ± 8.5

18.5 ± 4.9

8.0 ± 1.7

15.3 ± 0.6

+

5000

18.3 ± 2.1

130.7 ± 1.2

8.0 ± 0.0

9.0 ± 4.0

13.0 ± 2.0

 

 

 

 

 

 

 

Positive controls, +S9

Name

2AA

2AA

2AA

2AA

2AA

Concentrations

(μg/plate)

0.5

0.2

0.5

0.2

0.2

Mean No. of colonies/plate

(average of 2 ± SD)

57.5 ± 2.1

282.0 ± 17.0

33.0 ± 0.0

4.5 ± 2.1

132.5 ± 19.1

Concentrations

(μg/plate)

1

0.5

1

0.5

0.5

Mean No. of colonies/plate

(average of 2 ± SD)

78.0 ± 5.7

479.0 ± 137.2

66.5 ± 16.3

4.0 ± 0.0

353.5 ± 12.0

Concentrations

(μg/plate)

2

1

2

1

1

Mean No. of colonies/plate

(average of 2 ± SD)

125.5 ± 26.2

953.0 ± 7.1

184.0 ± 1.4

6.0 ± 1.4

730.5 ± 91.2

2AA: 2-aminoanthracene

MNNG: N-methyl-N´-nitro-N-nitrosoguanidine

DR: Daunorubicin

AM: Acridine mutagen

4NPD: 4-nitro-o-phenylenediamine

C: Contaminated

Table 2: Test Results - Experiment II (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

TA 1535

TA 100

TA 1537

TA 1538

TA 98

0

13.0 ± 4.0

103.6 ± 20.4

10.5 ± 2.1

8.6 ± 3.4

12.8 ± 3.6

1.6

13.7 ± 4.6

107.0 ± 16.5

7.0 ± 1.0

12.0 ± 1.0

14.0 ± 3.0

8

12.3 ± 4.9

118.7 ± 9.1

6.5 ± 0.7

8.0 ± 1.0

11.7 ± 2.9

40

11.3 ± 3.5

121.3 ± 13.7

8.7 ± 0.6

7.7 ± 2.1

13.3 ± 1.5

200

14.0 ± 2.6

122.3 ± 4.5

5.0 ± 0.0

7.3 ± 3.1

11.3 ± 0.6

1000

11.7 ± 2.1

124.0 ± 26.6

6.7 ± 2.1

6.0 ± 1.7

14.3 ± 3.5

5000

12.0 ± 1.0

118.7 ± 16.1

6.5 ± 0.7

9.7 ± 3.2

13.7 ± 4.2

 

 

 

 

 

 

 

Positive controls, –S9

Name

MNNG

MNNG

AM

4NPD

DR

Concentrations

(μg/plate)

1

1

0.5

1

0.2

Mean No. of colonies/plate

(average of 2 ± SD)

133.0 ± 62.2

328.0 ± 89.1

49.0 ± 2.8

52.0 ± 15.6

323.5 ± 68.6

Concentrations

(μg/plate)

2

2

1

2

0.5

Mean No. of colonies/plate

(average of 2 ± SD)

901.5 ± 252.4

1326.5 ± 26.2

70.0 ± 2.8

88.0 ± 2.8

770.0 ± 87.7

Concentrations

(μg/plate)

5

5

2

5

1

Mean No. of colonies/plate

(average of 2 ± SD)

1672.0 ± 32.5

1920.0 ± 83.4

127.0 ± 14.1

200.0 ± 46.7

809.5 ± 67.2

+

0

18.8 ± 5.1

125.4 ± 11.8

11.0 ± 5.0

10.2 ± 1.1

17.0 ± 1.9

+

1.6

18.7 ± 2.1

128.7 ± 4.5

11.3 ± 1.2

12.3 ± 5.7

18.0 ± 1.7

+

8

20.7 ± 3.1

142.0 ± 9.5

8.3 ± 4.2

13.0 ± 0.0

19.7 ± 4.7

+

40

15.0 ± 1.7

128.0 ± 21.0

10.0 ± 0.0

9.3 ± 1.5

17.0 ± 3.0

+

200

18.7 ± 3.1

124.3 ± 11.1

9.3 ± 1.5

9.0 ± 1.0

12.0 ± 1.0

+

1000

15.0 ± 1.0

127.7 ± 4.2

11.7 ± 2.5

9.3 ± 0.6

17.3 ± 3.8

+

5000

22.0 ± 4.0

135.3 ± 9.5

8.5 ± 2.1

10.7 ± 3.2

15.3 ± 3.5

 

 

 

 

 

 

 

Positive controls, +S9

Name

2AA

2AA

2AA

2AA

2AA

Concentrations

(μg/plate)

0.5

0.2

1

0.2

0.2

Mean No. of colonies/plate

(average of 2 ± SD)

48.0 ± 8.5

242.0 ± 35.4

51.0 ± 7.1

78.5 ± 2.1

78.0 ± 4.2

Concentrations

(μg/plate)

1

0.5

2

0.5

0.5

Mean No. of colonies/plate

(average of 2 ± SD)

70.5 ± 12.0

442.0 ± 60.8

271.5 ± 112.4

330.5 ± 103.9

183.5 ± 17.7

Concentrations

(μg/plate)

2

1

-

1

1

Mean No. of colonies/plate

(average of 2 ± SD)

114.5 ± 0.7

749.0 ± 282.8

-

487.5 ± 224.2

510.5 ± 38.9

2AA: 2-aminoanthracene

MNNG: N-methyl-N´-nitro-N-nitrosoguanidine

DR: Daunorubicin

AM: Acridine mutagen

4NPD: 4-nitro-o-phenylenediamine

Table 3: Test Results - Experiment III (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)

Frameshift type

TA 1537

(-S9-Mix)

TA 1538

(+ S9-Mix)

–/+

0

10.8 ± 1.9

7.0 ± 1.2

–/+

1.6

9.0 ± 1.7

9.0 ± 3.0

–/+

8

10.0 ± 4.6

6.7 ± 1.5

–/+

40

11.3 ± 2.1

9.7 ± 3.2

–/+

200

8.0 ± 4.4

8.0 ± 1.7

–/+

1000

12.7 ± 1.5

10.0 ± 2.6

–/+

5000

8.7 ± 4.0

11.0 ± 4.0

 

 

 

 

 

 

 

Positive controls, –S9

Name

AM

2AA

Concentrations

(μg/plate)

0.5

0.2

Mean No. of colonies/plate

(average of 2 ± SD)

67.0 ± 9.9

70.5 ± 2.1

Concentrations

(μg/plate)

1

0.5

Mean No. of colonies/plate

(average of 2 ± SD)

82.5 ± 6.4

141.0 ± 8.5

Concentrations

(μg/plate)

2

1

Mean No. of colonies/plate

(average of 2 ± SD)

123.0 ± 2.8

402.0 ± 1.4

AM: Acridine mutagen

2AA: 2 -aminoanthracene

Applicant's summary and conclusion

Conclusions:
Under the conditions of the conducted test the substance was not mutagenic in any of the five strains (TA 1535, TA 1537, TA 1538, TA 98 and TA 100) tested with and without metabolic activation.