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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:
2002-07-30 to 2002-09-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report date:
2003

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:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
not specified
GLP compliance:
yes (incl. QA statement)
Remarks:
2003-02-04
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Reaction mass of bis[2-(2-{[(prop-2-en-1-yloxy)carbonyl]oxy}ethoxy)ethyl] carbonate and diallyl 2,2'-oxydiethyl dicarbonate
EC Number:
700-483-4
Molecular formula:
not applicable
IUPAC Name:
Reaction mass of bis[2-(2-{[(prop-2-en-1-yloxy)carbonyl]oxy}ethoxy)ethyl] carbonate and diallyl 2,2'-oxydiethyl dicarbonate
Details on test material:
- Name of test material (as cited in study report): Carbonic acid, di(2-propenyl) ester, reaction products with 2,2'-oxybis-ethanol and 2,2-bis(hydroxymethyl)-l,3-propanediol.
CAS Nr: 145272282
- Molecular formula (if other than submission substance): C7H10O3
- Molecular weight (if other than submission substance): ~142
- Substance type: organic
- Physical state: colorless transparency liquid
- Stability under test conditions: stable at room temperature
- stability in solvent: stable in water, dimethylsulfoxide and acetone
- Storage condition of test material: at room temperature, shield from light

Method

Target gene:
Gens of Histidine Operons hisG46, hisC3076, hisC3052 and hisD6610 in Salmonella Typhimurium and gene of Tryptophan Operon (trpE) in E.coli.
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
DNA polymerase A deficient
Remarks:
rfa mutated, resulted in removal of the lipopolysaccharide coat down to the ketodeoxyoctanoate core, presumably making the organisms more permeable to large chemical agents.
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
DNA polymerase A deficient
Remarks:
carries the pKM101 plasmid to enhance error-prone repair
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
The maximum dose of the preliminary reverse mutation test was set at 5000 µg/plate and total six different dose levels with factor of 4 from the maximum dose (1250, 313, 78, 20 and 5 µg/plate). The maximum doses of the main reverse mutation test using Escherichia coli WP2 uvrA in the presence of metabolic activation was set at 5000 µg/plate for Escherichia coli WP2 uvrA and 1250 µg/plate for Salmonella typhimurium TA98, TA100, TA1535 and TA1537 and total six different dose levels with factor of 2 from the maximum dose. The maximum dose of the confirmatory reverse mutation test using Escherichia coli WP2 uvrA was set at 5000 µg/plate, for Salmonella typhimurium TA1535 at 313 µg/plate, and Salmonella typhimurium TA98, TA100 and TA1537 at 156 µg/plate and total six different dose levels with factor of 2 from the maximum dose (see Table 2 in "Remarks on results including tables and figures").
Controls
Untreated negative controls:
yes
Remarks:
solvent control
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
Lot.Nr ELQ2013, ELJ6826, 106F06681, ELE2329 and SEL1402 respectively Migrated to IUCLID6: furhter positive controls: 2-aminoanthracene, 9-aminoacridine, sodium azide, furylfuramide

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
Remarks:
the number of revertant colonies was comparable with the negative (vehicle) control
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 625 µg/plate or more in the presence of metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 µg/plate in the presence of metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

The test substance increased the number of the revertant colonies twofold or more compared with the negative control with reproducibility in Escherichia coli WP2 uvrA in the presence of metabolic activation (Table 1).

Table 1

Reverse mutation test using Salmonella typhimurium and Escherichia coli in the presence of metabolic activation

 

 

Exp. No. 11304

Number of revertant colonies per plate (mean)

Compound

Dose (µg/plate) _

Base pair substitution type

Frameshift type

TA100

TA1535

VP2 uvrA

TA98

TA1537

DMSO

 

127

 

7

 

27

 

27

 

8

 

 

128

( 128)

12

( 10)

35

( 31)

34

( 31)

7

( 8)

Test item

 

122

 

7

 

 

 

31

 

6

 

39

122

( 122)

10

( 9)

 

 

26

( 29)

4

( 5)

 

129

 

8

 

 

 

29

 

8

 

78

122

( 126)

7

( 8)

 

 

30

( 30)

8

( 8)

 

99

 

7

 

43

 

27

 

6

 

156

100

( 100)

6

( 7)

45

( 44)

23

( 25)

4

( 5)

 

96

 

4

 

50

 

24

 

4

 

313

83

( 90)

6

( 5)

61

( 56)

25

( 25)

4

( 4)

 

0 *

 

0 *

 

86

 

0 *

 

0 *

 

625

0 *

( 0)

0 *

( 0)

82

( 84)

0 *

( 0)

0 *

( 0)

 

0 *

 

0 *

 

95

 

0 *

 

0 *

 

1250

0 *

( 0)

0 *

( 0)

88

( 92)

0 *

( 0)

0 *

( 0)

 

 

 

 

 

39

 

 

 

 

 

2500

 

 

 

 

36

( 38)

 

 

 

 

 

 

 

 

 

0 *

 

 

 

 

 

5000

 

 

 

 

0 *

( 0)

 

 

 

 

B[a]P

 

877

 

 

 

 

 

295

 

64

 

5.0

1044

( 961)

 

 

 

 

244

( 270 )

68

( 66)

2AA

 

 

 

196

 

 

 

 

 

 

 

2.0

 

 

209

( 203 )

 

 

 

 

 

 

 

 

 

 

 

813

 

 

 

 

 

10.0

 

 

 

 

771

( 792 )

 

 

 

 

DMSO: dimethylsulfoxide
B[a ]P : benzo[a Jpyrene
2AA: 2-aminoanthracene
* : Toxic effect of the test substance was observed.

The test results were reproducible in the confirmatory test.

And also clear dose-related increase in the number of the revertant colonies was observed.On the other hand, the growth of the contaminant was not observed in the result of the sterility test. The comparison between the negative and positive control value, and the historical data, the negative control of Salmonella typhimurium TA100 in the presence of metabolic activation at the preliminary test was out of the range in the standard value based on the histrical data in our laboratory. But the negative and positive control value of this bacterial strain in case of absence of metabolic activation were within the range of the standard value. Therefore, it was judged that the genetic markers of and the sensitivity to mutagenic activity of this bacterial strain were appropriate. And the positive control value of this bacterial strain in case of presence of metabolic activation was within the range of the standard value. Therefore it was judged that metabolic activation worked appropriately. All of substances used as the positive controls increased the number of the revertant colonies twofold or more compared with the negative control in all bacterial strains. These results indicate that the test has been properly carried out. The environmental factor that might influence the reliability of this test was not detected. The precipitation of the test substance was not detected at any dose levels regardless of the presence or the absence of metabolic activation.

Applicant's summary and conclusion

Conclusions:
Interpretation of results: positive with metabolic activation in E.coli WP uvrA.
The mutagenic activity of the test substance is considered to be positive under the test conditions employed.
Executive summary:

The mutagenic activity of Carbonic acid, di(2-propenyl) ester, reaction products with 2,2'-oxybis-ethanol and 2,2-bis(hydroxymethyl)-l,3-propanediol was examined in the reverse mutation test by using bacterial strains that have two different patterns of mutation. One of the mutation is base-pair substitution of Salmonella typhimurium TA100, TA1535 and Escherichia coli WP2 uvrA, and another mutation is frameshift mutation of Salmonella typhuimurium TA98 and TA1537. The reverse mutation test was composed of the preliminary, main and confirmatory test in accordance with the test guideline applied, and the reproducibility of these test results was confirmed. The test was performed in pre-incubation methods in all bacterial strains in the presence and absence of metabolic activation. In this test, no statistical analysis was used to evaluate the test results. In the presence and absence of metabolic activation, the number of revertant colonies in each bacterial strain at all dose levels was compared with the negative control. The test substance was judged positive for the mutagenic activity when clear dose-related increase in the number of the revertant colonies and twofold or more increase in the number of the revertant colonies compared with the negative control was observed with reproducibility.

The results of the test are as follows :

1. The test substance increased the number of the revertant colonies twofold or more compared with the negative control in Escherichia coli WP2 uvrA in the presence of metabolic activation. Also, the dose-relativity was observed. And reproducibility of the test results was recognized.

2. The values of the negative controls and positive controls were appropriate in comparison with the historical data of our laboratory. Furthermore, all of the positive controls, such as 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide, sodium azide, 9-aminoacridine, benzo[a]pyrene and 2-aminoanthracene, increased the number of the revertant colonies twofold or more compared with the negative control in all bacterial strains, respectively. These results indicate that the test has been properly carried out.

3. From the foregoing results, it is concluded that the mutagenic activity of the test substance is judged positive under the test conditions employed.