Diethyl sulphate, compound with 2-(heptadec-8-enyl)-4,5-dihydro-1H-imidazole-1-ethanol (1:1)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-06-28 to 2022-01-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Version / remarks:

adopted 29th July 2016

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Test material information

Constituent 1

Reference substance name:

Diethyl sulphate, compound with 2-(heptadec-8-enyl)-4,5-dihydro-1H-imidazole-1-ethanol (1:1)

EC Number:

271-272-8

EC Name:

Diethyl sulphate, compound with 2-(heptadec-8-enyl)-4,5-dihydro-1H-imidazole-1-ethanol (1:1)

Cas Number:

68527-63-9

Molecular formula:

C22H42N2O.C4H10O4S

IUPAC Name:

2-{2-[(8E)-heptadec-8-en-1-yl]-4,5-dihydro-1H-imidazol-1-yl}ethan-1-ol diethyl sulfate

Test material form:

liquid

Details on test material:

Test item formulations were found to be homogeneous and stable up to 24 hour in vehicle corn oil.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- batch number of test material: 10419
- Purity, including information on contaminants, isomers, etc.: > 93 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: dry and dark at ambient room temperature (20 – 30 °C)

Method

Target gene:

HPRT

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

CHO-K1 cell

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- concentration or volume of S9 mix and S9 in the final culture medium: 1% v/v

Test concentrations with justification for top dose:

The main study was performed with test item concentrations of 0.488281, 0.976563, 1.953125 and 3.90625 µg/ml in the absence and 0.976563 , 1.953125, 3.90625 and 7.8125 µg/ml in the presence of metabolic activation system along with vehicle, negative and positive controls.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulfoxide

- Justification for choice of solvent/vehicle: test item was found to be insoluble in distilled water, while it was found to be soluble in dimethyl sulfoxide. Hence, dimethyl sulfoxide was selected as vehicle for this study.

- Justification for percentage of solvent in the final culture medium: no precipitation was observed at the concentrations of 2000, 1000 and 500 µg/ml. Based on these results 2000 µg/ml was selected as a highest concentration for preliminary cytotoxicity assay.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

ethylmethanesulphonate

Details on test system and experimental conditions:

Media and Culture conditions
- appropriate culture medium and incubation conditions (culture vessels, humidified atmosphere of 5 % C0 2, and incubation temperature of 37± 2 °C) was used for maintaining the cells
- a-MEM medium (supplemented with nucleosides) with 10 % Fetal Bovine Serum, penicillin (100 U/ml) and streptomycin (100 µg/ml) for maintaining CHO-K1 cell lines was used

Maintenance of Cell line
- cell cultures cleansed of pre-existing mutant cells, e.g. by culturing in HAT medium (medium containing Hypoxanthine, Aminopterin, and Thymidine) were used for HPRT test

Test ltem Solubility
- solubility, precipitation, pH and osmolality of the test item in media was determined before the cytotoxicity test
- pH and Osmolality of the test item in media was checked at 0 hour and at 4 hour after incubation in CO2 incubator

Preliminary Cytotoxicity Study
- 200 mg of test item was made up to 1 ml in dimethyl aulfoxideto get a final concentration 200 mg/ml. From this stock concentration, 0.5 ml was aspirated and added to 0.5 ml of DMSO to get a stock concentration of 100 mg/ml. The subsequent serial dilutions were made to get concentrations of 50, 25, 12.5 and 6.25 mg/ml with dimethyl sulfoxide using a spacing factor of 2

Test ltem Dilution Preparation of Preliminary Cytotoxicity Assay (II)
- evaluation of cytotoxicity was done to define the concentrations to be used in the main experiment. Cytotoxicity was evaluated using RS, i.e. cloning efficiency (CE) of cells plated immediately after treatment adjusted by any lass of cells during treatment as compared with adjusted cloning efficiency in negative controls (assigned a survival of 100 %).

Evaluation criteria:

Criteria for a Positive Response
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
- the increase is concentration-related when evaluated with a trend test.
- any of the results are outside the distribution of the historical negative control data.
- when all the above criteria are met, the test item is then considered able to induce gene mutations in cultured mammalian cells in this test system (CHO-K1 cell line).

Criteria for a Negative Response
- none of the test concentrations exhibit a statistically significant increase compared with the concurrent negative control.
- there is no concentration-related increase when evaluated with a trend test.
- when the above criteria are met, the test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system (CHO-K1 cell line).

Statistics:

Non-parametric statistics was performed to assess a possible dose dependent increase of mutant frequencies using validated statistics software. The number of mutant colonies obtained for the groups treated with the test item was comparable to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both biological relevance and statistical significance was considered together.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In Preliminary Cytotoxicity Assay (1) cytotoxicity was observed at the lowest tested concentration of 62.5 µg/ml (relative survival (RS): 2.32 %) in the absence and (RS: 1.69 %) in the presence of metabolic activation system as compared to vehicle control

Applicant's summary and conclusion

Conclusions:

Diethyl sulphate, compound with 2-(heptadec-8-enyl)-4,5- dihydro-1H-imidazole-1-ethanol (1:1) did not induce gene mutations in the CHO-K1 cell line, in the presence and absence of exogenous metabolic activation system (S9), respectively.

Executive summary:

In vitro Mammalian Cell Gene Mutation Test (HPRT assay) for test item Diethyl sulphate, compound with 2-(heptadec-8-enyl)-4,5-dihydro-1H-imidazole-1-ethanol (1:1) in CHO-K1 cells has been performed as per OECD guideline No. 476 & B.17.

The test item was found to be insoluble in distilled water, while it was found to be soluble in dimethyl sulfoxide.

According to the results of the Cytotoxicity Assay II, the main study was performed at the concentrations of 0.488281, 0.976563, 1.953125 and 3.90625 µg/ml in the absence and 0.976563, 1.953125, 3.90625 and 7.8125 µg/ml in the presence (1 % v/v S9 Mix) of metabolic activation system. In Phase 1, the RS was 86.37 (0.488281 µg/ml ), 70.75 (0.976563 µg/ml), 75.69 (1.953125µg/ml) and 64.62 (3.90625µg/ml) in the absence and 89.30 (0.976563 µg/ml), 76.44 (1.953125 µg/ml ), 67.48 (3.90625 µg/ml ) and 61.74 (7.8125 µg/ml) in the presence (1 % v/v S9 Mix) of metabolic activation system.The observed mean mutant frequency was 8.44, 8.38, 8.93 and 9.66 at 0.488281, 0.976563, 1.953125 and 3.90625 µg/ml, respectively in absence and 8.10, 8.71, 9.03 and 9.52 at0.976563, 1.953125, 3.90625 and 7.8125 µg/ml, respectively in presence (1 % v/v S9 Mix), as compared to vehicle control.

On the basis of the results of this study Diethyl sulphate, compound with 2-(heptadec-8-enyl)-4,5- dihydro-1H-imidazole-1-ethanol (1:1) did not induce gene mutations in the CHO-K1 cell line, in the presence and absence of exogenous metabolic activation system (S9), respectively under the specified experimental condition.