Diallyl hexahydrophthalate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 November 2016 (Study plan) - 26 January 2017 (end of experimental period)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Unscheduled delay of reporting due to a QA organisational change within Test Facility Management. This change was considered to have had no affect on the study integrity or validity.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

yes

Remarks:

Unscheduled delay of reporting due to a QA organisational change within Test Facility Management. This change was considered to have had no affect on the study integrity or validity.

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

yes

Remarks:

Unscheduled delay of reporting due to a QA organisational change within Test Facility Management. This change was considered to have had no affect on the study integrity or validity.

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

The method described in the above mentioned guidelines conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Diallyl hexahydrophthalate

EC Number:

237-580-1

EC Name:

Diallyl hexahydrophthalate

Cas Number:

13846-31-6

Molecular formula:

C14H20O4

IUPAC Name:

1,2-bis(prop-2-en-1-yl) cyclohexane-1,2-dicarboxylate

Test material form:

liquid

Remarks:

Colourless

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Name: MDAC
- Chemical name: 1,2-Cyclohexanedicarboxylic acid, di-2-propenyl ester
- Other Name: Diallyl hexahydrophthalate
- CAS number: 13846-31-6
- Appearance: Colourless liquid

- Source and batch No.of test material: From the Sponsor (Osaka Soda Co., Ltd). Batch no. 40201
- Expiration date of the batch: 26 January 2019
- Purity test date: 99.2%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protected from light
- Stability under test conditions: Assumed stable for the duration of the study. Analytical determination of the test item concentration, stability and homogeneity was not performed because of the character and the short duration of the study.
- Solubility and stability of the test substance in the solvent/vehicle: Based on available information and the results of solubility testing, Dimethyl Sulphoxide (DMSO) was selected as vehicle (solvent) for this study. The appropriate vehicle and the behaviour of the test item formulations with the solution of top agar and phosphate buffer up to 5000 μg/tube were determined in a preliminary compatibility test.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: None.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test solutions were freshly prepared at the beginning of experiments by dilutiion of the stock solution with DMSO
- Final dilution of stock liquid: 100 mg/mL (5000 μg/plate), 31.62 mg/mL (1581 μg/plate), 10 mg/mL (500 μg/plate), 3.162 mg/mL (158.1 μg/plate), 1.0 mg/mL (50 μg/plate), 0.3162 mg/mL (15.81 μg/plate), 0.1 mg/mL (5 μg/plate), 0.03162 mg/mL (1.581 μg/plate), 0.01 mg/mL (0.5 μg/plate) 0.003162 mg/mL (0.1581 μg/plate).

In the initial mutation test, concentrations ranged from 100 mg/mL (5000 μg/plate) to 0.1 mg/mL (5 μg/plate) in all strains (+/- S9 mix)
In the confirmatory mutation test, concentrations ranged from 100 mg/mL (5000 μg/plate) to 0.003162 mg/mL (1.581 μg/plate) in all strains (+/- S9 mix)
In the complementary confirmatory mutation test, concentrations ranged from 10 mg/mL (500 μg/plate) to 0.003162 mg/mL (0.581 μg/plate) in strains TA100 (+/- S9 mix), TA1535 and TA1537 (- S9 mix).

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Cofactor-supplemented post-mitochondrial S9 fraction, prepared from the livers of phenobarbital/β-naphthoflavone-induced rats

Test concentrations with justification for top dose:

Test item concentrations:
In the initial mutation test, concentrations ranged from 100 mg/mL (5000 μg/plate) to 0.1 mg/mL (5 μg/plate) in all strains (+/- S9 mix)
In the confirmatory mutation test, concentrations ranged from 100 mg/mL (5000 μg/plate) to 0.003162 mg/mL (1.581 μg/plate) in all strains (+/- S9 mix)
In the complementary confirmatory mutation test, concentrations ranged from 10 mg/mL (500 μg/plate) to 0.003162 mg/mL (1.581 μg/plate) in strains TA100 (+/- S9 mix), TA1535 and TA1537 (- S9 mix).

Concentrations were selected on the basis of the Preliminary Compatibility Test and Preliminary Concentration Range Finding Test (Informatory Toxicity Test).Based on the results of the preliminary tests, 100 mg/mL stock solution was prepared to acheive the limit guideline dose of 5000 μg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (Supplier: Sigma-Aldrich Co. Batch No.: SZBG1310V, Expiry date: 25 April 2019)

- Justification for choice of solvent/vehicle: The solubility of the test item was examined using Distilled water, Dimethyl sulfoxide (DMSO), N,N-Dimethylformamide (DMF) and Acetone. The test item was insoluble in Distilled water at 100 mg/mL concentration. The test item was soluble at 100 mg/mL using DMSO, DMF and Acetone (clear solution was detected in each case). Due to the better biocompatibility, DMSO was selected as vehicle (solvent) for the study.

Two vehicle (solvent) control groups (DMSO and Distilled water) were used depending on the solubility of the test item (MDAC) and the solubility of strain specific positive control chemicals.

DMSO: (Supplier: Sigma-Aldrich Co. Batch No.: SZBG1310V, Expiry date: 25 April 2019)
Distilled water (Manufacturer: TEVA Pharmaceutical Industries Ltd, Batch No's: 7170914 / 7600914, Expiry date: 30 September 2017)

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 4-nitro-1,2-phenylene-diamine (NPD) and 2-aminoanthracene (2AA)

Remarks:

Selected on the basis of scientific literature, the experience of the Test Facility and the avaialbility of historical control data.

Details on test system and experimental conditions:

METHOD OF APPLICATION: The study included a Preliminary Compatibility Test, a Preliminary Concentration Range Finding Test (Informatory Toxicity Test) an Initial Mutation Test and a Confirmatory Mutation Tests. In the Preliminary Concentration Range Finding Test and Initial Mutation Test, the plate incorporation method was used. In the Confirmatory Mutation Test and Complementary Confirmatory Mutation Test the pre-incubation method was used.

Concentrations were selected on the basis of the Preliminary Compatibility Test and Preliminary Concentration Range Finding Test. In the Initial Mutation Test and Confirmatory Mutation Test different concentrations were used. Furthermore, a Complementary Confirmatory Mutation Test was also performed based on the results of the Confirmatory Mutation Test using a modified concentration range.

Initial Mutation Test concentrations: 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate
Confirmatory Mutation Test concentrations: 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate.

(In the Confirmatory Mutation Test, excessive cytotoxicity was observed in Salmonella typhimurium TA100 with and without metabolic activation and Salmonella typhimurium TA1535 and TA1537 without metabolic activation. As the number of analyzable doses did not meet the guidline requirements, an additional experiment (Complementary Confirmatory Mutation Test) was performed in these strains to complete the data).

Completmentary Confirmatory Mutation Test concentrations: 500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg test item/plate in Salmonella typhimurium TA100 with and without metabolic activation and Salmonella typhimurium TA1535 and TA1537 bacterial strains without metabolic activation.

DURATION
Experimental Method: The experimental methods were conducted according to the methods described Ames et al. [1975], Maron and Ames [1983], Kier et al. [1986], Venitt and Parry [1984], OECD Guideline No. 471 [1997], Commission Regulation (EC) No. 440/2008 [2008], EPA Guidelines, OPPTS 870.5100 [1998] [1996] and according to the relevant SOPs of CiToxLAB Hungary Ltd.

Preincubation period: The day before treatment, the frozen bacterial cultures were thawed at room temperature and 200 μL inoculum were used to inoculate each 50 mL sample of Nutrient Broth No. 2 for the overnight cultures in the assay. The cultures were incubated for 10-14 hours at 37oC in a Gyrotory Water Bath Shaker.

In the Preliminary Concentration Range Finding Test (Informatory Toxicity Test) the plate incorporation method was used. Based on the available information and the solubility and compatibility test, 100 mg/mL stock solution was prepared in DMSO, which was diluted in 6 steps by factors of 2, 2.5 and approximately √10. The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (TA98, TA100) were determined at MDAC concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate.

Initial Mutation Test: A standard plate incorporation procedure was performed Bacteria (cultured in Nutrient Broth No.2 as described in Section 5.3.6.) were exposed to the test item both in the presence and absence of an appropriate metabolic activation system. Molten top agar was prepared and kept at 45°C. The equivalent number of minimal glucose agar plates (three/per concentration and for each control) was properly labelled. The test item and other components were prepared freshly and added to the overlay (45°C).

The content of the tubes:
Toop agar 2000 μL,
Vehicle (solvent) or test item solution (or reference controls) 50 μL,
Overnight culture of test strain 100 μL,
Phosphate buffer (pH 7.4) or S9 mix 500 μL.

This solution was mixed and poured on the surface of minimal agar plates. Foractivation studies, instead of phosphate buffer, 0.5 mL of the S9 mix was added to
each overlay tube. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative (solvent) and positive controls.
After preparation, the plates were incubated at 37°C for approximately 48 hours.

Procedure for Exposure in the Confirmatory Mutation Test and the Complementary Confirmatory Mutation Test: A pre-incubation procedure was performed as a Confirmatory Mutation Test since in the Initial Mutation Test no positive effect was observed. The same method was used in the Complementary Confirmatory Mutation Test.

For the pre-incubation method, bacteria (cultured in Nutrient Broth No.2) were exposed to the test item both in the presence and absence of an appropriate metabolic activation system. The equivalent number of minimal glucose agar plates was properly labelled. Molten top agar was prepared and kept at 45°C.
Before the overlaying, 50 μL of test item formulations or its vehicle (or positive reference controls or their solvent), 100 μL of the overnight culture of bacterial cells
and the 0.5 mL of S9 mix (activated test conditions) or phosphate buffer pH 7.4 (nonactivated test conditions) were added into the appropriate tubes to provide direct
contact between bacteria and the test item. These tubes (3 tubes per control and 3 tubes for each concentration level) were gently mixed and incubated for 20 min at 37ºC in a shaking incubator.

After the incubation period, 2 mL of molten top agar were added to the tubes, and then the content mixed and poured on the surface of minimal glucose agar plates. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative and positive controls. After preparation, the plates were incubated at 37°C for 48 hours.

- Exposure duration:
- Expression time (cells in growth medium): Initial mutation test and confirmatory test after preparation, plates were incubated at 37°C for 48 hours. In the complementary
confirmatory mutation test, and in the Confirmatory Mutation Test, the plates were incubated at 37°C for 72±1 hours.

NUMBER OF REPLICATIONS: 3 replicates/per plate concentration

DETERMINATION OF CYTOTOXICITY
- Method: Any inhibitory/cytotoxic effect was denoted by a reduced background lawn and/or a reduction in the number of revertant colonies.
An Inhibitory, cytotoxic effect of the test item was observed in the Preliminary Concentration Range Finding Test and in the main tests in all Salmonella typhimurium
strains with and without metabolic activation. The effect was excessive in Salmonella typhimurium TA100 with and without metabolic activation and Salmonella typhimurium TA1535 and TA1537 bacterial strains without metabolic activation in the Confirmatory Mutation Test, thus a complementary experiment was performed to
ensure validity. In the Complementary Confirmatory Mutation Test, the background inhibition was still observed in the examined strain without metabolic activation.

Rationale for test conditions:

This study followed the procedures as indicated by current internationally accepted guidelines and recommendations. In accordence with the guidencea, a pre-incubation procedure was performed as a Confirmatory Mutation Test since in the Initial Mutation Test (using the plate incorporation method) no positive effect was observed.

Evaluation criteria:

The study was considered valid if:
- the number of revertant colonies of the negative (solvent) and positive controls were in the historical control range in all strains of the main tests;
- at least five analyzable concentrations were presented in all strains of the main tests.

Criteria for a Positive Response:
A test item was considered mutagenic if:
- a dose–related increase in the number of revertants occurred and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic activation.

An increase was considered biologically relevant if:
- in all strains: the number of reversion was more than twice higher than the reversion rate of the negative (solvent) control.

Criteria for a Negative Response:
The test item was considered to have shown no mutagenic activity in this study if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Statistics:

The mean number of revertants per plate, the standard deviation and the mutation factor (the mean number of revertants on the test item plate / mean number of revertants on the vehicle control plate) values were calculated for each concentration level of the test item and for the controls using Microsoft ExcelTM software.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- No confounding effects (i.e. precipitate) were observed throughout the initial, confirmatory or complementary confirmatory mutation test.

RANGE-FINDING/SCREENING STUDIES: A preliminary concentration range finding using the plate incorporation method was undertaken prior to the main testing. This test was performed using Salmonella typhimurium TA98 and TA100 strains in the presence and absence of metabolic activation system (±S9 mix) with appropriate untreated, negative (vehicle/solvent) and positive controls. Each sample (including the controls) was tested in triplicate. The tested concentrations were 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)

Negative reference control data - DMSO
without metabolic activation (-S9 Mix) with metabolic activation (+S9 Mix)
TA98 TA100 TA1535 TA1537 E.coli TA98 TA100 TA1535 TA1537 E.coli
Mean 21.5 98.6 11.6 7.1 32.6 29.1 109.4 11.3 8.7 39.0
St.Dev 5.8 22.3 4.9 3.3 10.1 7.1 21.8 3.6 3.6 10.1
Range 6-55 40-217 1-43 1-25 7-81 11-67 53-229 2-27 1-29 9-85
n 1183 1173 1179 1185 1182 1182 1170 1178 1185 1176

n=number of cases
Negative reference control data - Distilled Water
without metabolic activation (-S9 Mix) with metabolic activation (+S9 Mix)
TA98 TA100 TA1535 TA1537 E.coli TA98 TA100 TA1535 TA1537 E.coli
Mean 23.3 103.0 11.5 7.5 34.7 30.9 112.1 11.1 9.1 41.0
St.Dev 6.3 24.2 4.9 3.2 10.3 7.3 23.0 3.5 3.4 10.2
Range 11-45 45-215 2-47 2-24 12-84 10-53 64-222 3-39 1-20 17-91
n 201 1092 1101 201 1122 201 1101 1107 201 1116

n=number of cases
Untreated control data
without metabolic activation (-S9 Mix) with metabolic activation (+S9 Mix)
TA98 TA100 TA1535 TA1537 E.coli TA98 TA100 TA1535 TA1537 E.coli
Mean 22.7 103.4 11.4 6.9 33.7 30.0 111.3 11.3 8.7 39.8
St.Dev 5.8 22.9 5.0 3.2 10.3 6.8 20.6 3.7 3.6 10.2
Range 9-46 54-210 1-46 1-24 11-82 10-56 65-204 1-35 1-28 16-89
n 1108 1093 1101 1107 1104 1108 1095 1100 1110 1101

n=number of cases
Positive reference control data
without metabolic activation (-S9 Mix) with metabolic activation (+S9 Mix)
TA98 TA100 TA1535 TA1537 E.coli TA98 TA100 TA1535 TA1537 E.coli
Mean 351.9 1263.9 1184.3 466.2 1054.1 2425.9 2445.4 238.0 223.1 267.3
St.Dev 124.9 211.5 217.0 186.0 144.8 346.2 316.9 150.5 61.0 123.4
Range 152-2336 880-2120 208-2440 149-2104 516-1708 312-4918 1192-5240 101-2216 117-838 125-2512
n 1108 1095 1101 1107 1107 1108 1095 1104 1110 1101

n=number of cases

Applicant's summary and conclusion

Conclusions:

Experiments used histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a metabolic activation system. In the Initial Mutation Test and Confirmatory Mutation Test, none of the observed revertant colony numbers were above the respective biological threshold value, with no dose-related trends or indication of any treatment related effect. In all test item treated groups, the numbers of revertant colonies did not exceed biological parameters when compared to the vehicle control and were within the normal biological variability of the test system.

The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies. At least five analyzable concentrations were presented in all strains of the main tests, the examined concentration range was considered to be adequate and the study was considered valid.

Under the experimental conditions the test item (MDAC) did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. The test item, MDAC, had no mutagenic activity in the applied bacterium tester strains under the test conditions used inb this study.