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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:
experimental study
Adequacy of study:
key study
Study period:
1979-11-27 to 1980-01-16
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was not conducted under GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1980
Report date:
1980

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
no
Remarks:
The study was performed prior to the adoption of the GLP regulations
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Diallyl 2,2'-oxydiethyl dicarbonate
EC Number:
205-528-7
EC Name:
Diallyl 2,2'-oxydiethyl dicarbonate
Cas Number:
142-22-3
Molecular formula:
C12H18O7
IUPAC Name:
3-({[2-(2-{[(prop-2-en-1-yloxy)carbonyl]oxy}ethoxy)ethoxy]carbonyl}oxy)prop-1-ene
Details on test material:
- Name of test material (as cited in study report): T1562 (Diallyl Diglycol Carbonate)
- Date Sample Received: 1979-10-04
- Substance type: organic
- Physical state: liquid
- Analytical purity: 85% with 15% monomeric substances
- Impurities (identity and concentrations): data not available
- Composition of test material, percentage of components: data not available
- Isomers composition: data not available
- Purity test date: data not available
- Lot/batch No.: J-8600
- Expiration date of the lot/batch: data not available
- Stability under test conditions: data not available
- Storage condition of test material: in the dark at 4 ± 2°C.

Method

Target gene:
Gens of Histidine Operons (hisG46, hisC3076, hisD3052, hisG428 and hisD6610)
Species / strain
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA 1538, 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.
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
10 µl, 3 µl, 1 µl, 0.3 µl, 0.1 µl/ plate in the absence of metabolic activation and 0.3 µL, 0.1 µL, 0.03 µL, 0.01 µL, 0.003 µL/plate in the presence of metabolic activation. The dose is based on the full strength volume administered, which in this assay was 100 µl of 10%, 3%, 1%, 0.3%, 0.1%, 0.03%, 0.01%, 0.003% in acetone. The dilutions with acetone were prepared immediately prior to use.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: acetone is one of the suitable solvents for the test material.
Controls
Untreated negative controls:
yes
Remarks:
bacteria only
Negative solvent / vehicle controls:
yes
Remarks:
acetone alone in without metabolic activation assay, acetone plus S9 mix in with metabolic activation assay
True negative controls:
yes
Remarks:
sterility control: test material and S9 mix
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
further positive controls are: 9-aminoacridine (9-AA), 2-nitrofluorene (2-NF), 2-aminoanthracene (2-AA)
Details on test system and experimental conditions:
[See also "Any other informations on materials and methods incl. tables"]

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: without preincubation
- Exposure duration: 48 hours
- Expression time (cells in growth medium): 16-20 hours
- Selection time (if incubation with a selection agent): no data
- Fixation time (start of exposure up to fixation or harvest of cells): 48 hours

SELECTION AGENT (mutation assays): no data

SPINDLE INHIBITOR (cytogenetic assays): no data

STAIN (for cytogenetic assays): no data

NUMBER OF REPLICATIONS: triplicate

NUMBER OF CELLS EVALUATED: no data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: not reported

OTHER EXAMINATIONS: no
Evaluation criteria:
All sterility controls, including the five concentrations of the test compound and S9 mix, must be negative for bacterial growth. The average number of revertant colonies representing spontaneous mutations must be within the following acceptable range: TA1535, 6-40; TA1537, 3-16; TA1538, 6-35; TA100, 80-250, TA98, 10-60 (see Criteria for determination of a valid test in "Any other information on materials and methods incl. tables").
Statistics:
not reported

Results and discussion

Test results
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 1538, 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:
valid
Positive controls validity:
valid
Additional information on results:
no data
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: except that questionable mutagenicity activity in TA 98 with metabolic activation.

Any other information on results incl. tables

Table 1 shows the activity of T1562 in the Salmonella/ microsomal test in the absence of metabolic activation. The positive control for each of the five strains tested induced an average number of revertants/plate which was at least five times that found with the solvent control. All concentrations of T1562 tested failed to induce an average number of revertants/plate three times greater than that found in the solvent.

TABLE 1

Results of salmonella/microsomal assay without metabolic activation on T1562

Compound

Conc units/plate

Revertants per plate of bacterial tester strains

TA1535

TA1537

TA1538

TA100

TA98

Bacteria only (negative control)

100 µl

14.3±3.1

7.0 ± 1.0

8.3 ± 2.3

135.7 ± 13.7

34.0 ± 9.5

Acetone (solvent control)

100 µl

16.7 ± 1.5

9.0 ± 2.0

4.3 ± 1.2

126.3 ± 18.6

15.7 ± 5.1

Sod. azide (positive control)

3.0 µg

262.7* ± 7.4

NT

NT

1288.0* ± 158.6

NT

9-AA (positive control)

50.0µg

NT

562.0* ± .0

NT

NT

NT

2-NF (positive control)

5.0 µg

NT

NT

314.3* ± 11.9

NT

297.0* ± 11.1

T1562

0.1 µl

9.0 ± 3.6

7.7 ± 2.1

6.3 ± 4.0

122.3 ± 13.8

31.3 ± 9.8

0.3 µl

15.7 ± 5.1

5.0 ± 2.6

8.7 ± .6

124.0 ± 1.7

29.7 ± 7.0

1.0 µl

16.0 ± 4.6

8.0 ± 2.0

7.3 ± 1.2

92.7 ± 9.3

41.7 ± 12.4

3.0 µl

16.3 ± 4.2

5.3 ± 1.5

2.3 ± .6

97.3 ± 12.3

12.7 ± 5.5

10.0 µl

T

T

T

10.0 ± 5.0

8.0 ± .0

*= the  test  compound mean was at least  3.0 times  solvent control mean
NT= not tested
T= toxic values represent mean + 1 standard deviation of the number of colonies per plate at each experimental condition

Table 2 shows the activity of T1562 in the Salmonella/ microsomal test in the presence of metabolic activation. The positive control for each of the five strains tested induced an average number of revertants/plate which was at least five times that found with the solvent control. Concentrations of 0.003 µL/plate, 0.01 µL/plate and 0.03 µL/plate of T1562 in the TA98 strain did induce an average number of revertants/plate three times greater than that found in the solvent control. The sterility controls for the S-9 mix, positive controls, and the test article dilutions were all negative for bacterial contamination control.

TABLE 2

Results of salmonella/microsomal assay with metabolic activation on T1562

Compound

Conc units/plate

Revertants per plate of bacterial tester strains

TA1535

TA1537

TA1538

TA100

TA98

Bacteria only (negative control)

100 µl

10.0 ± 2.0

7.0 ± 1.0

8.3 ± 2.3

126.0 ± 3.0

34.0 ± 9.5

Acetone +S-9 mix (solvent control)

100 µl

11.7 ± 2.9

8.3 ± .6

12.3 ± 5.1

166.0 ± 6.6

31.7 ± 9.7

2-AA +S-9 (positive control)

2.5000 µg

137.7* ± 17.9

86.0* ± 2.6

244.0* ± 61.2

916.7* ± 163.1

628.0* ± 141.7

T1562+S-9 mix

0.0030 µl

12.7 ± 2.5

10.0 ± 1.7

14.3 ± 1.2

193.7 ± 16.2

113.0* ± 8.7

0.0100 µl

12.0 ± 1.0

8.3 ± 1.5

12.7 ± 2.5

157.3 ± 8.1

137.7* ± 20.4

0.0300 µl

12.0 ± 3.0

6.7 ± .6

14.0 ± 3.5

140.7 ± 3.8

147.0* ± 6.9

0.1000 µl

9.7 ± .6

5.7 ± 3.2

8.7 ± 1.5

98.3 ± 10.6

32.0 ± 1.7

0.3000 µl

5.0 ± 2.0

T

3.3 ± 1.5

87.0 ± 6.1

14.3 ± 1.2

* = THE TEST COMPOUND MEAN WAS AT LEAST 3.0 TIMES SOLVENT CONTROL MEAN
T= TOXIC VALUES REPRESENT MEAN + 1 STANDARD DEVIATION OF THE NUMBER OF COLONIES PER PLATE AT EACH EXPERIMENTAL CONDITION

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
ambiguous

The negative controls, positive controls, and sterility controls all fulfilled requirements for determination of a valid test.
Under the conditions employed in the assay described in this report, the data suggest that the test article exhibits no mutagenic activity in any strainswithout metabolic activation. In with metabolic activation questionable mutagenic activity was noted only in TA98 strain. The positive result in TA98 is questionable, since 1) the number of revertants induced was just at the level chosen for significance (3-fold increase), 2) a 10-fold increase in concentration of test material did not cause a dose-dependent increase in the number or revertants , 3) the positive control induced a significantly greater number of revertants than the test material, and 4) higher concentrations of test material that were not toxic (0.1 and 0.3%) did not increase the number of revertants with respect to control. 
Although the authors concluded that the test material was positive in this assay, the fact that mutagenicity was not noted in other strains with frame-shift mutations (i.e. TA1538 and TA1537), along with the questionable positive result with TA98 suggests that mutagenicity of the test compound is ambiguous.
Executive summary:

The purpose of this study was to employ the Salmonella/ microsomal assay to investigate the mutagenic potential of the test article that has been designated in this final report as T1562, otherwise, known as Diallyl Diglycol Carbonate.

Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 were exposed to the test substance at concentrations of 0, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3 or 10% in the presence and absence of metabolic activation. The cytotoxic concentration was 10%. In the presence of metabolic activation, concentrations of 0.003, 0.01 and 0.03% caused dose-dependent increases in revertants that were at least 3 times greater than that of control in the strain TA98 only. The positive controls responded appropriately. Carbonic acid, oxydiethylene diallyl ester was not mutagenic in this assay.

Although the authors concluded that the test material was positive in this assay, the fact that mutagenicity was not noted in other strains with frame-shift mutations (i.e. TA1538 and TA1537), along with the questionable positive result with TA98 suggests that mutagenicity of the test compound is ambiguous.