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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:
experimental study
Adequacy of study:
key study
Study period:
11 Jul - 16 Aug 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

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:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Department of Health of the Government of the United Kingdom
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Ethylene diformate
EC Number:
211-077-7
EC Name:
Ethylene diformate
Cas Number:
629-15-2
Molecular formula:
C4H6O4
IUPAC Name:
ethylene diformate
Details on test material:
- Name of test material (as cited in study report): Ethylene Glycol Diformate
- Physical state: pale yellow liquid
- Analytical purity: minimum 80% (w/w)
- Lot/batch No.: 13082
- Expiration date of the lot/batch: 28 June 2013
- Storage condition of test material: room temperature in the dark

Method

Target gene:
his operon (for S. typhimurium strains)
trp operon (for E. coli strain)
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
- Type and identity of media: Oxoid Limited, Lot numbers 1078364 (08/16) and 1014570 (03/2016)
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Phenobarbitone/beta-Naphthoflavone.
Test concentrations with justification for top dose:
Preliminarty toxicity test: 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: DMSO
- Justification for choice of solvent/vehicle: the test item was fully soluble in DMSO at 50 mg/mL in solubility tests and DMSO was therefore selected as the vehicle.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG; 2, 3 and 5 µg/plate, -S9, WP2uvrA, TA100 and TA1535, respectively); 9-Aminoacridine (9AA; 80 µg/plate, -S9, TA1537); 4-Nitroquinoline-1-oxide (4NQO; 0.2 µg/plate, -S9, TA98)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene (2AA; 1, 2 and 10 µg/plate, +S9, TA100, TA1535 and TA1537, WP2uvrA, respectively); Benzo(a)pyrene (BP; 5 µg/plate, +S9, TA98)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar plate incorporation (first experiment), pre-incubation (second experiment)

DURATION
- Exposure duration: 48 h

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

NUMBER OF REPLICATIONS: triplicates each in two independent experiments
Evaluation criteria:
A result is considered positive, if any, one, or all of the following conditions are met: A dose-related increase in mutant frequency over the dose range tested (De Serres F J and Shelby M D (1979), Recommendations on data production and analysis using the Salmonella/microsome mutagenicity assay, Environmental Mutagenesis, 1, 87-92). A reproducible increase at one or more concentrations. Biological relevance against historical control ranges. Statistical analysis of data as determined by UKEMS (Mahon G A T et al., (1989) Analysis of data from microbial colony assays. In: Statistical Evaluation of Mutagenicity Test Data, UKEMS sub-committee on guidelines for mutagenicity testing, (Kirkland D J Ed.), Cambridge University PressReport, 26-65). Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met. Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgement about test item activity. Results of this type will be reported as equivocal.
Statistics:
Mean values and standard deviations were calculated.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH value was measured in Experiment 1 and 2 (7.58 and 7.56) and thus represented no problem to the Ames Test.

RANGE-FINDING/SCREENING STUDIES: In the preliminary toxicity test, the test item was tested at concentrations of 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation. The test was performed in the strains TA100 and WP2uvrA. After 48 h incubation at 37°C the plates were assessed for numbers of revertant colonies using a colony counter and examined for effects on the growth of the bacterial background lawn.The test item was non-toxic to the strains of bacteria used (TA100 and WP2uvrA).


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

Any other information on results incl. tables

Table 1: 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 ± Standard deviation)

Base-pair substitution type

Frameshift type

TA 100

TA1535

WP2uvrA

TA98

TA1537

0

137 ± 7.4

15 ± 2.6

30 ± 5.1

23 ± 3.6

10 ± 1.7

50

146 ± 1.5

17 ± 5.6

28 ± 7.6

22 ±3.8

12 ± 2.6

150

133 ± 27.8

17 ± 2.1

28 ± 2.1

25 ± 3.2

13 ± 3.1

500

125 ± 9.3

16 ± 1.5

28 ± 7.0

24 ± 2.5

11 ± 2.5

1500

144 ± 29.2

14 ± 4.0

31 ±7.0

19 ± 2.5

13 ± 1.0

5000

125 ± 10.4

21 ± 1.4

26 ± 3.0

24 ± 8.0

14 ± 3.8

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 ± SD)

742 ± 11.1

480 ± 28.2

787 ± 25.1

192 ± 5.5

1186 ± 123.7

+

0

136 ±12.2

18 ± 5.2

44 ± 2.1

30 ± 5.3

9 ± 2.1

+

50

126 ± 15.1

18 ± 7.5

37 ± 2.6

36 ± 10.1

10 ± 2.1

+

150

128 ± 7.0

22 ± 4.4

37 ± 5.1

22 ± 3.2

9 ± 2.1

+

500

112 ± 15.5

20 ± 5.9

32 ± 5.6

29 ± 3.2

12 ± 1.5

+

1500

115 ± 10.6

16 ± 4.0

31 ± 6.4

22 ± 2.5

9 ± 5.7

+

5000

125 ± 25.1

20 ± 9.0

42 ± 4.6

26 ± 12.4

9 ± 1.5

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 ± SD)

1349 ± 91.7

225 ± 7.2

387 ± 16.0

314 ± 39.0

148 ± 11.8

ENNG = N-ethyl-N-nitro-N-nitrosoguanidine

4NQO = 4-Nitroquinoline-1-oxide

2AA = 2-Aminoanthracene

BP = Benzo(a)pyrene

9AA 9-Aminoacridine

Table 2: Test Results of Experiment 2 (pre-incubation). 

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 100

TA1535

WP2uvrA

TA98

TA1537

0

99 ± 13.1

23 ± 5.8

22 ± 3.6

26 ± 5.6

14 ± 2.0

50

86 ± 8.1

17 ± 2.6

23 ± 3.1

23 ± 3.6

9 ± 4.5

150

75 ± 4.0

14 ± 5.0

24 ± 2.5

19 ± 1.5

15 ± 8.6

500

76 ± 4.0

17 ± 2.1

25 ± 6.0

21 ± 3.8

10 ± 4.0

1500

83 ± 9.2

19 ± 4.2

24 ± 4.0

16 ± 2.5

9 ± 1.5

5000

81 ± 6.7

15 ± 4.6

16 ± 2.5

17 ± 7.5

9 ± 2.6

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 ± SD)

453 ± 107.2

359 ± 77.4

673 ± 7.4

155 ± 3.5

1010 ± 197.7

 

 

TA 100

TA1535

WP2uvrA

TA98

TA1537

+

0

102 ± 25.2

13 ± 3.8

39 ± 2.3

34 ± 2.9

12 ± 5.6

+

50

91 ± 7.2

15 ± 1.2

36 ± 1.2

30 ± 1.2

11 ± 2.0

+

150

83 ± 21.0

13 ± 1.5

38 ± 0.6

33 ± 4.5

11 ± 4.2

+

500

87 ± 19.3

14 ± 6.4

36 ± 2.6

21 ± 1.0

16 ± 8.9

+

1500

80 ± 8.3

13 ± 2.9

30 ± 6.2

27 ± 4.4

14 ± 1.0

+

5000

96 ± 13.7

15 ± 2.0

35 ± 5.1

29 ± 10.2

16 ± 3.1

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 ± SD)

1206 ± 80.5

237 ± 6.7

300 ± 29.3

236 ± 4.5

205 ± 12.5

ENNG = N-ethyl-N-nitro-N-nitrosoguanidine

4NQO = 4-Nitroquinoline-1-oxide

2AA = 2-Aminoanthracene

BP = Benzo(a)pyrene

9AA 9-Aminoacridine

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative