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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:
key study
Study period:
8 Dec 1998 - 19 January 1999
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP-Guideline study, tested with the source substance Dipentaerythritol ester of nC5/iC9 acids. According to the ECHA guidance document "Practical guide 6: How to report read-across and categories (March 2010), the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report date:
1999

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: USA, EPA (TSCA) OPPTS harmonised guidelines
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
The Department of Health of the Government of the United Kingdom, UK
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
647028-25-9
Cas Number:
647028-25-9
IUPAC Name:
647028-25-9
Details on test material:
- Name of test material (as cited in study report): only trade name given
- Physical state: straw coloured liquid
- Analytical purity: 100%
- Storage condition of test material: room temperature in the dark

Method

Target gene:
"his operon" (for S. typhimurium strains) and "trp operon" (for E. coli strains)
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
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:
Preliminary toxicity study: 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation
First and second experiment: 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9: N-ethyl-N`-nitro-N-nitrosoguanidine (3, 5 and 2 µg/plate, respectively) for TA100, TA1535 and WP2uvrA; 9-Aminoacridine (80 µg/plate) for TA1537; 4-Nitroquinolone-1-oxide (0.2 µg/plate) for TA98.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: +S9: 2-Aminoanthracene (1 µg/plate) for TA100, (2 µg/plate) for TA1535 and TA1537, (10 µg/plate) for WP2uvrA; Benzo(a)pyrene (5 µg/plate) for TA98.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: inspection of the bacterial background lawn

OTHER: a preliminary toxicity test was carried out in the strains TA100 and WP2uvrA.
Evaluation criteria:
The test material may be considered positive in this test if the following criteria are met: The test material should have induced reproducible and statistically (Dunnett´s method of linear regression) significant increase in the revertant count in at least one strain of bacteria.
Statistics:
Mean values and standard deviation were calculated. Dunnett’s method of linear regression was used.

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, but tested up to precipitating concentrations
Remarks:
oily precipitate was observed at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
oily precipitate was observed at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: An oily precipitate was observed at 5000 µg/plate; however this did not prevent the scoring of revertant colonies.

RANGE-FINDING/SCREENING STUDIES: The dose range for the main test was determined in a preliminary toxicity assay in which the test material was tested at the following concentrations: 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate. The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA).

COMPARISON WITH HISTORICAL CONTROL DATA: All tester strain cultures exhibited a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls. Furthermore, positive control values were at least two times the respective vehicle control value for each strain. The historical control ranges are presented in Table 1 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

Table 1: Historical control ranges

History profile-Vehicle control values

 

-S9

TA 100

TA 98

TA 1535

TA 1538

TA 1537

WP2uvrA

TA 102

Extreme values

61-196

13-56

10-39

6-41

4-17

12-39

216-339

Mean

117 ± 24.7

28 ± 6.7

24 ± 5.2

21 ± 6.8

10 ± 2.0

20 ± 4.6

264 ± 29.8

                       History profile-Vehicle control values

+S9

TA 100

TA 98

TA 1535

TA 1538

TA 1537

WP2uvrA

TA 102

Extreme values

63-177

14-52

11-39

11-50

5-20

11-40

205-343

Mean± SD

120 ± 22.2

33 ± 7.1

18 ± 4.0

25 ± 6.2

11 ± 2.3

21 ± 4.8

283 ± 32.7

                       History profile-Positive control values

-S9

TA 100

TA 98

TA 1535

TA 1538

TA 1537

WP2uvrA

TA 102

Extreme values

277-1126

127-698

163-1005

190-799

161-1149

342-1209

540-1188

Mean± SD

613 ± 175.7

203 ± 57.2

466 ± 208.4

484 ± 146.0

812 ± 201.3

834 ± 197.2

785 ± 172.1

+S9

TA 100

TA 98

TA 1535

TA 1538

TA 1537

WP2uvrA

TA 102

Extreme values

412-1315

198-757

139-516

212-915

123-718

225-1089

511-1090

Mean± SD

949 ± 189.7

420 ± 112.5

291 ± 63.6

454 ± 131.0

266 ± 90.2

734 ± 210.5

705 ± 126.6

Table 2: 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 ± SD)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

0

81 ± 11.0

16 ± 1.5

25 ± 3.5

19 ± 2.5

7 ± 3.8

50

77 ± 2.1

14 ± 0.6

22 ± 0.6

24 ± 0.6

9 ± 3.6

150

80 ± 12.2

14 ± 1.0

22 ± 0.6

20 ± 6.7

10 ± 3.8

500

83 ± 10.8

13 ± 2.3

23 ± 2.9

21 ± 6.0

7 ± 2.6

1500

76 ± 1.5

18 ± 5.2

18 ± 2.6

22 ± 1.2

7 ± 3.2

5000

90 ± 11.8 P

18 ± 8.2 P

24 ± 4.0 P

28 ± 4.9 P

5 ± 0.6 P

Positive controls, –S9

Name

ENNG

ENNG

ENNG

4NQO

9AA

Concentrations

(μg/plate)

3

5

2

0.2

80

Mean No. of colonies/plate

(average of 3)

323 ± 11.7

283 ± 10.0

503 ± 24.2

166 ± 5.3

1027 ± 358.2

 

 

TA100

TA1535

WP2uvrA

TA98

TA1537

+

0

105 ± 7.8

11 ± 1.2

28 ± 3.8

30 ± 3.1

20 ± 3.6

+

50

101 ± 7.0

12 ± 0.6

27 ± 3.8

29 ± 6.6

20 ± 3.2

+

150

102 ± 5.5

16 ± 4.0

32 ± 1.5

27 ± 4.0

23 ± 2.0

+

500

93 ± 16.6

14 ± 4.0

28 ± 6.6

30 ± 6.7

20 ± 1.2

+

1500

86 ± 3.6

15 ± 3.5

26 ± 4.0

31 ± 3.1

17 ± 6.5

+

5000

93 ± 7.8 P

12 ± 1.2 P

24 ± 1.5 P

33 ± 5.7 P

15 ± 2.6 P

Positive controls, +S9

Name

2AA

2AA

2AA

BP

2AA

Concentrations

(μg/plate)

1

2

10

5

2

Mean No. of colonies/plate

(average of 3)

920 ± 153.0

198 ± 9.3

555 ± 27.8

551 ± 132.0

196 ± 6.5

Table 3: 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 ± SD)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

0

96 ± 4.5

18 ± 4.2

26 ± 3.8

18 ± 5.3

11 ± 6.5

50

97 ± 18.6

17 ± 4.0

29 ± 6.5

16 ± 3.6

12 ± 0.6

150

100 ± 9.5

20 ± 9.0

28 ±0.0

16 ± 3.8

11 ± 1.7

500

101 ± 8.4

18 ± 6.2

33 ± 6.0

19 ± 4.4

12 ± 1.2

1500

87 ± 6.4

17 ± 7.1

29 ± 7.1

22 ± 0.0

12 ± 1.2

5000

93 ± 1.0 P

19 ± 3.1 P

33 ± 5.1 P

17 ± 3.2 P

15 ± 6.0 P

Positive controls, –S9

Name

ENNG

ENNG

ENNG

4NQO

9AA

Concentrations

(μg/plate)

3

5

2

0.2

80

Mean No. of colonies/plate

(average of 3)

880 ± 31.5

257 ± 34.4

1191 ± 59.3

166 ± 4.6

1084 ± 263.4

 

 

TA100

TA1535

WP2uvrA

TA98

TA1537

+

0

85 ± 4.0

14 ± 3.5

35 ± 1.2

31 ± 5.0

21 ± 1.5

+

50

91 ± 7.5

19 ± 0.7

35 ± 10.1

30 ± 8.0

24 ± 1.5

+

150

84 ± 9.5

14 ± 1.0

34 ± 6.1

29 ± 10.7

22 ± 3.5

+

500

101 ± 14.3

10 ± 0.6

42 ± 0.6

28 ± 4.5

22 ± 2.1

+

1500

94 ± 8.7

14 ± 0.6

36 ± 5.3

31 ± 4.7

21 ± 1.0

+

5000

101 ± 6.7 P

14 ± 3.2 P

34 ± 7.0 P

30 ± 5.5 P

22 ± 1.2 P

Positive controls, +S9

Name

2AA

2AA

2AA

BP

2AA

Concentrations

(μg/plate)

1

2

10

5

2

Mean No. of colonies/plate

(average of 3)

865 ± 43.2

246 ± 7.5

1051 ± 90.0

611 ± 51.5

182 ± 7.0

ENNG = N-Ethyl-N´-nitro-N-nitrosoguanidine

4NQO = 4-Nitroquinoline-1-oxide

9AA = 9-Aminoacridine

BP = Benzo(a)pyrene

2AA = 2-Aminoanthracence

P = precipitate

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative