[No public or meaningful name is available]

Genetic toxicity: in vitro

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:

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 but passed a Quality Assurance audit.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1980

Report date:

1980

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

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

Test material information

Constituent 1

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:

diallyl 2,2'-oxydiethyl dicarbonate

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. For liquid materials, 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:

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'.

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.

Since the multi-constituent to be registered (EC 700-483-4) contains as main ingredient 'diallyl 2,2'oxydiethyl dicarbonate', the experimental data from this substance were used in a read-across approach.