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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The mutagenic potential of Diethyl sulphate, compound with 2-(heptadec-8-enyl)-4,5- dihydro-1 H-imidazole-1-ethanol (1: 1 ) was examined in a guideline study according to OECD 471. Under the condition of the study, the test item is non-mutagenic to the  bacteria.

An in vitro Mammalian Chromosomal Aberration Test of Diethyl sulphate, compound with 2-(heptadec-8-enyl)-4,5-dihydro-1H-imidazole-1-ethanol (1:1) in CHO-K1 cells was performed as per OECD 473. The test item was found not to induce chromosomal aberration up to the tested concentration of 1.953125 µg/mL in the Phase I and Phase Il in CHO-K1 cells under the specified experimental condition.

The 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 476. The test item did not induce gene mutations in the CHO-K1 cell line, in the presence and absence of exogenous metabolic activation system (S9), respectively.

Link to relevant study records

Reference

Reference 1

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

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

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

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)

Target gene:

HPRT

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.

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.

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

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-06-21 to 2022-01-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

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)
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: test item was found to be soluble in dimethyl sulfoxide (200 mg/mL)
- Expiry date: 2023-04-23

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: CHO-K1 Cells were obtained from American Type Culture Collection (ATCC)

For cell lines:
- Absence of Mycoplasma contamination: yes
- Methods for maintenance in cell culture: maintained at 37 ± 2 °C in humidified atmosphere of 5 % C02 in air

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Aroclor 1254 - induced rat liver microsomal enzymes (S9 homogenate) purchased commercially from DRDO, Thane, India (Protein Content 38.9 mg/mL) was used in the assay (Stored at 80 °CC)
- method of preparation of S9 mix:
S9 mix (cofactors and liver homogenate) was prepared immediately prior to use in the experiment. The post-mitochondrial fraction (liver homogenate) was used at concentration of 1 % v/v (preliminary cytotoxicity assay and Phase I) in the S9 mix for final culture medium.

Cofactor solution contain following quantity of chemicals in 1000 mL of distilled water.
Cofactors for S9 Mix
Components/lngredients Per 1000 mL
Distilled water Make up to 1000 mL
D- Glucose-6-phosphate 1.6 g
Nicotinamide Adenine Dinucleotide Phosphate (NADP) 3.5 g
Magnesium chloride 1 .8 g
Potassium chloride 2.7 g
Sodium phosphate dibasic 12.8 g
Sodium phosphate monobasic 2.8 g

Test concentrations with justification for top dose:

- initial preliminary cytotoxicity test was performed at the concentrations of 62.5, 125, 250, 500, 1000 and 2000 µg/mL of culture medium, both in the presence (1 % v/v S9 Mix) and absence of metabolic activation system along with vehicle control.

- based on the preliminary cytotoxicity assay results, another cytotoxity assay (preliminary cytotoxicity assay (Il)) was performed at the concentrations of 0.488281, 0.976563, 1.953125, 3.90625, 7.8125, 15.625, 31.25 and 62.5 µg/mL, both in the absence and in the presence (1 % v/v S9 Mix) of metabolic activation system.

- phase I was performed at the concentrations of 0.244141, 0.488281, 0.976563, 1.953125 and 3.90625 µg/mL both in the absence and in the presence (1 % v/v S9 Mix) of metabolic activation system.

- phase Il was performed at the concentrations of 0.244141, 0.488281, 0.976563, 1.953125 and 3.90625 µg/mL along with vehicle and positive controls.

Vehicle / solvent:

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

- Justification for choice of solvent/vehicle: the solubility of the test item was determined before the start of the assay. Test item was found to be insoluble in distilled water (200 mg/mL), while it was found to be soluble in dimethyl sulfoxide (200 mg/mL). Hence, dimethyl sulfoxide was selected as a vehicle for this study.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

Presence of metabolic activation

Positive control substance:

cyclophosphamide

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

Absence of metabolic activation

Positive control substance:

mitomycin C

Details on test system and experimental conditions:

- Method of Formulation Preparation:
Test Item Dilution Preparation of Preliminary Cytotoxicity Assay
200 mg of test item was made up to 1 mL in DMSO to 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. Preliminary cytotoxicity assay was performed with concentration of 62.5, 125, 250, 500, 1000 and 2000 µg/mL of the test item in culture medium along with vehicle and negative controls.

- Test Item Dilution Preparation of Preliminary Cytotoxicity Assay (Il):
10 mg of test item was dissolved in 1 mL of DMSO to get a final concentration 10 mg/mL, Stock A. From this stock concentration, 625 µL was aspirated and added to 375 µL of DMSO to get a stock concentration of 6.25 mg/mL, Stock B. 0.5 mL of Stock B was added to 0.5 mL of DMSO to get stock concentration of 3.125 mg/mL. The subsequent serial dilutions were made to get concentrations of 1.5625, 0.78125, 0.390625 and 0.195313, 0.0488281 mg/mL with DMSO using a spacing factor of 2. Preliminary cytotoxicity assay (Il) was performed with concentrations of 0.488281, 0.976563, 1.953125, 3.90625, 7.8125, 15.625, 31.25 and 62.5 µg/mL of the test item in culture medium along with vehicle and negative controls.

- Test Item Dilution Preparation of Main study (Phase I and Phase Il):
For phase I and Il, 10 mg of test item was made up to 1 mL in DMSO to get final concentration 10 mg/mL. From this, 0.5 mL was added to 0.5 mL of DMSO to get stock concentration of 3.125 mg/mL. Similarly the stock concentrations of 1.5625, and 0.78125 mg/mL were achieved with DMSO using spacing factor of 2.
Phase I was performed with test item concentrations of 0.244141, 0.488281, 0.976563, 1.953125 and 3.90625 µg/mL both in the absence and in the presence of metabolic activation system along with vehicle, negative and positive controls.
Phase Il was performed with test item concentrations of of 0.244141, 0.488281, 0.976563, 1.953125 and 3.90625 µg/mL along with vehicle, negative and positive controls in the absence (continuous exposure) of metabolic activation system.

- Analysis:
300 metaphases per concentration was analysed under IOOX magnification for incidence of structural aberrations.

Evaluation criteria:

A test item can be classified as non-mutagenic if:
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend Test.

A test item can be classified as mutagenic if:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
- the increase is dose-related when evaluated with an appropriate trend test.

Statistics:

The percentage of cells with structural aberrations was considered as basic unit for statistical analysis. For main study statistical analysis was performed for occurrence of structural aberrations in chromosome and chromatid in treated cells to the results obtained for the vehicle control group, using one way Analysis of Variance.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: pH of the test item in media was checked at 0 hour and at 4 hour after incubation in C02 incubator.
Concentration (µg/mL) 0 hour 4 hour
7.31 7.40
62.5 7.39 7.46
125 7.41 7.62
250 7.30 7.42
500 7.40 7.59
1000 7.39 7.45
2000 7.40 7.49

- Data on osmolality:
Osmolality of the test item in media was checked at 0 hour and at 4 hour after incubation in C02 incubator.
Concentration (µg/mL) 0 hour 4 hour
460 457
62.5 436 434
125 437 434
250 437 435
500 437 433
1000 423 426
2000 421 417


- Water solubility: Test item was found to be insoluble in distilled water
- Precipitation and time of the determination: A volume of 50 µl test item solution was added to culture media to observe the precipitation.
Volume of test item Volume of medium (mL) Final concentration Precipitation results
Solution added (µL) (µg/mL)
50 4.95 62.5 no precipitation
50 4.95 125 no precipitation
50 4.95 250 no precipitation
50 4.95 500 no precipitation
50 4.95 1000 no precipitation
50 4.95 2000 no precipitation


Chromosome aberration test (CA) in mammalian cells:
- Results from cytotoxicity measurements:
Phase I was performed at the concentrations of 0.244141, 0.488281, 0.976563, 1.953125 and 3.90625 µg/mL both in the absence and in presence of metabolic activation system (short term treatment 3-4 hour) (1 % v/v S9 mix), while Phase Il was performed at 0.244141, 0.488281, 0.976563, 1.953125 and 3.90625 µg/mL in the absence of metabolic activation system (continuous treatment 24 hours), along with vehicle and positive controls.
In Phase I, the RICC (Relative Increase jn Cell Counts) observed was 98.57 (0.244141 µg/mL), 92.1 1 (0.488281 µg/mL), 84.23 (0.976563 µg/mL), 73.48 (1 .953125 µg/mL) and 71 .68 (3.90625 µg/mL) in the absence and 95.59 (0.244141 µg/mL), 92.20 (0.488281 µg/mL), 83.73 (0.976563 µg/mL), 74.58 (1 .953125 µg/mL) and 63.39 (3.90625 µg/mL) in the presence (1 % v/v S9 Mix) of metabolic activation system),

In Phase Il, the RICC (Relative Increase in Cell Counts) observed was 95.24 % (0.244141 µg/mL), 88.89 % (0.488281 µg/mL), 82.22 % (0.976563 µg/mL), 80.95 % (1 .953125 µg/mL) and 71 .43 % (3.90625 µg/mL).

Conclusions:

On the basis of the results of this study, it is concluded that Diethyl sulphate, compound with 2-(heptadec-8-enyl)-4,5-dihydro-1H-imidazole-1-ethanol (1:1) did not induce chromosomal aberration up to the tested concentration of 1.953125 µg/mL in the Phase I and Phase Il in CHO-K1 cells under the specified experimental condition.

Executive summary:

In vitro Mammalian Chromosomal Aberration Test of Diethyl sulphate, compound with 2-(heptadec-8-enyl)-4,5-dihydro-1H-imidazole-1-ethanol (1:1) in CHO-K1 cells was performed as per OECD guideline No. 473.

The test item was found to be insoluble in distilled water, while it was found to be soluble in dimethyl sulfoxide. During the evaluation of chromosomal abberations, three concentrations were scored as per the guideline and study plan, with the acceptable limit of 300 metaphases. No significant increase in the number of metaphase containing structural chromosome aberrations was observed up to tested concentration of 1.953125 µg/mL in the absence and in the presence of metabolic activation system in Phase I and Phase Il, when compared with the concurrent vehicle control.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-11-13 to 2018-01-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997-07-21

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: R-127, lot 170825
- Expiration date of the lot/batch: 2019-06-19

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Preliminary test: 5 mg / plate, 1.5 mg / plate, 0.46 mg / plate, 0.14 mg / plate, 0.04 mg / plate and 0.01 mg / plate
Main study with S9 mix: 0.14 mg / plate, 0.07 mg / plate, 0.035 mg / plate, 0.018 mg / plate, 0.009 mg / plate
Main study without s9 mix: 0.01 mg / plate, 0.005 mg / plate, 0.0025 mg / plate, 0.0013 mg / plate, 0.0007 mg / plate
Justification for top dose: results from preliminary test

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:
- Justification for choice of solvent/vehicle: solubility of the test item

Untreated negative controls:

yes

Remarks:

Solvent control

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-Aminoanthracene; ICR 191

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: over night
- Exposure duration: 48 h
- Expression time (cells in growth medium): 48 h

NUMBER OF REPLICATIONS: main study: 3

Evaluation criteria:

The t-test suggests that for the five strains, there is no significant increase (p < 0.05) in the number of revertant colonies in test substance comparing with its respective negative control with and without S9 metabolic activation, except that in strain TA 100, when S9 metabolic activation was present, significant increase (p = 0. 01) in revertant colony number was observed in test substance 0.018 mg / plate comparing with its negative control. However, as the increase of revertant colony number in this test substance is less than 2-fold comparing with its negative control, and remarkably less than its positive control, the increase is not considered as sign of mutagenicity.
The numbers of revertant colonies of the positive controls for the five strains are remarkably higher than their corresponding negative controls (> 2-fold) with and without S9 metabolic activation.

Statistics:

Student's t-test was used to assess whether there was any statistically significant difference between the number of revertant colonies of the test substance and their corresponding negative control.

Key result

Species / strain:

S. typhimurium TA 1535

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

Key result

Species / strain:

S. typhimurium TA 1537

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

Key result

Species / strain:

S. typhimurium TA 98

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

Key result

Species / strain:

S. typhimurium 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

Key result

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

Preliminary study:

No/ little growth of revertant colonies as well as absence of background lawn were observed on plates from 5 mg / plate to 0.04 mg / plate without S9 activation, which indicated cytotoxic effects. Therefore, based on the preliminary study, the dose levels selected for test substances without S9 activation in the main study were 0.01 mg / plate, 0.005 mg / plate, 0.0025 mg / plate, 0.0013 mg / plate and 0.0007 mg / plate, dose interval was 2.

No growth of revertant colonies as well as absence / moderately reduced of background lawn were observed on plates from 5 mg / plate to 0.14 mg / plate with S9 activation, which indicated cytotoxic effects. Therefore, based on the preliminary study, the dose levels selected for test substances with S9 activation in the main study were 0.14 mg / plate, 0.07 mg / plate, 0.035 mg / plate, 0.018 mg / plate and 0.009 mg / plate, dose interval was 2.

Conclusions:

Under the condition of the study, the test item - Diethyl sulphate, compound with 2-(heptadec-8-enyl)-4,5- dihydro-1 H-imidazole-1-ethanol (1: 1 ), is non-mutagenic to the bacteria.

Executive summary:

The mutagenic potential of Diethyl sulphate, compound with 2-(heptadec-8-enyl)-4,5- dihydro-1 H-imidazole-1-ethanol (1: 1 ) was examined in a guideline study according to OECD 471.

The test item exhibited cytotoxic effects to bacteria.

Under the condition of the study, the test item - Diethyl sulphate, compound with 2-(heptadec-8-enyl)-4,5- dihydro-1 H-imidazole-1-ethanol (1: 1 ), is non-mutagenic to the  bacteria.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The Mammalian bone marrow micronucleus test of test item Diethyl sulphate, compound with 2- (heptadec-8-enyl)-4,5-dihydra-1H-imidazole-1-ethanol (1:1) in mice was conducted according OECD 474. lt is concluded, that under the stated experimental conditions, the test item did not induce micronuclei formation up to the treated dose of 750 mg/kg b.w. Hence, the test item Diethyl sulphate, compound with 2-(heptadec-8-enyl)-4,5-dihydro-1H-imidazole-1-ethanol (1:1) is considered to be non-clastogenic at the dose of 750 mg/kg b.w. in the mammalian bone marrow micronucleus test in mice.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-06-22 to 2022-02-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted on 29th July, 2016

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian bone marrow chromosome aberration test

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)

Species:

mouse

Strain:

Swiss

Details on species / strain selection:

Mus musculus (Mouse)
Strain: Swiss albino

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Global Bioresearch Solution Pvt. Ltd., Pune, lndia.
- Age at study initiation: 07 - 08 weeks
- Weight at study initiation:
Male
minimum 26.19 g, maximum 31.54 g

Female
minimum 22.82 g, maximum 29.16 g

- Assigned to test groups randomly: Randomization was done based on recent b w. each. The animals were allocated to the different test groups manually using Microsoft excel calculations. Details of randomization were documented in the raw data.
- Housing: Maximum five male mice per polycarbonate cage (size 23 [cm] x 16 [cm], height 13 [cm]) were housed during the study in all groups. Sterilized corn cob (Batch No.: 221) procured from Krishna Corncob Industries, Aurangabad, was used as bedding material. lt was renewed as often as necessary to keep the animals dry and clean.
- Diet (e.g. ad libitum): A conventional laboratory pelleted diet was offered ad libitum for feeding
- Water (e.g. ad libitum): Aqua guard filtered drinking water in bottles was offered ad libitum
- Acclimation period: 5 days (dose range finding); 6 days (main study)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): room temperature was maintained between 21.00 °C - 22.50 °C (Dose Range Finding 1), 21.30 °C - 22.50 °C (Dose Range Finding II) and between 21.20 °C - 22.30 °C (Main Study).
- Humidity (%): relative humidity was kept between 52.00 - 2.30 % (Dose Range Finding 1) 53.50 - 62.50 % (Dose Range Finding II) and between 53.60 - 61.50 % (Main Study).
- Air changes (per hr): minimum 12 times per hour and filtered adequately.
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark

Route of administration:

oral: feed

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): Dose formulation was prepared freshly, shortly prior to dose administration twice at 24-hour-intervals
- Mixing appropriate amounts: To achieve the test item concentration, the test item was weighed 333, 500, and 750 mg, respectively on Day 0 and Day 1 of dosing for main study. Each test item concentration, to which vehicle (corn oil) was added gradually, and using a calibrated measuring cylinder volume was made up to 10 ml.
- Storage temperature of food: room temperature

Frequency of treatment:

The animals were treated with the test item or vehicle twice at 24-hour-intervals.

Post exposure period:

Bone marrow samples was collected -20 h following final treatment.

Dose / conc.:

333 mg/kg bw/day (actual dose received)

Remarks:

twice at 24 hours interval

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Remarks:

twice at 24 hours interval

Dose / conc.:

750 mg/kg bw/day (actual dose received)

Remarks:

twice at 24 hours interval

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

yes - with cyclophosphamide monohydrate

Tissues and cell types examined:

bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
- based on the results of range finding study
- according to the DRF Study II the highest dose was defined as 750 mg/kg due to observed clinical signs and because in higher doses of 1000 mg/kg and 1500mg/kg mortality was observed but no significant decrease of P/E ratio
- lowest assumed non-toxic dose was selected at 333 mg/kg as in DRF Study I no mortality or toxic clinical signs were observed at all used dose levels of up to 500 mg/kg


DETAILS OF SLIDE PREPARATION:
- cell suspension was centrifuged at 1500 rpm (for both DRF studies and main study) for 10 min and the supernatant was discarded
- small drop (~ 100 - 200 µL) of the resuspended cell pellet was smeared on a pre labelled slide in duplicates for each animal
- smear was allowed to dry completely. The smeared slides were fixed in absolute methanol for 5 mins and allowed to air dry for 15-20 min
- all the slides were stained with 5 % Giemsa stain for 30 mins followed by rinsing in water, drying and mounting with DPX

METHOD OF ANALYSIS:
- proportion of immature erythrocytes among total erythrocytes was determined for each animal by counting a total of at least 500 erythrocytes
- except for the DRF studies, all slides were independently coded and randomised before microscopic analysis
- around 4000 immature erythrocytes per animal were scored manually under 100X magnification for the incidence of micronucleated immature erythrocytes

Evaluation criteria:

A test item is considered clearly positive if:

a) at least one of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated immature erythrocytes compared with the concurrent negative control;
b) this increase is dose-related at least at one sampling time when evaluated with an appropriate trend test; and
c) any of these results are outside the distribution of the historical negative control data

A test item is considered clearly negative if, in all experimental conditions examined:

a) none of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated immature erythrocytes compared with the concurrent negative control;
b) there is no dose-related increase at any sampling time when evaluated by an appropriate trend test;
c) all results are inside the distribution of the historical negative control data, and bone marrow exposure to the test substance(s) occurred

Statistics:

Statistical analysis was performed for test item treated animals to the results obtained from the vehicle control group, using One Way Analysis of Variance and using "t"-test for results obtained from positive control group to the results obtained from the vehicle control group.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

DRF I:

- no mortality was observed in treatment groups at concentration 125, 250 and 500 mg/kg b.w.

- all animals in the vehicle control group displayed normal clinical signs

- no clinical symptoms were observed in treatment graups at concentration of 125, 250 and 500 mg/kg b.w. compared to the vehicle contral group

- reduction  in P/E ratio was observed  in both male and female at 125, 250 and 500 mg/kg body weight compared to the vehicle control

 

DRF II:
- based on the results of DRF 1, DRF II was performed with higher doses

- vehicle  contral (0 mg/kg  b.w.), low dose (DRF II G2 -  750 mg/kg b.w.), mid dose (DRF II G3 - 1000 mg/kg b. w.), and high dose (DRF II G4 - 1500 mg/kg b.w.)

- mortality was not observed in treatment  group at dose level 750 mg/kg b.w., whereas mortality  was observed in the treatment  groups  1000 mg/kg b.w. (50 % mortality in males and 100 % mortality in females) and 1500 mg/kg  b.w.  (100 % mortality in males and 50 % mortality in females)

- all the animals in the  vehicle control group displayed normal clinical signs

- in the high dose group (1500 mg/kg b.w.), clinical symptoms of lethargy and recumbency were observed

- in the low dose group clinical signs of lethargy were observed on day 1 of dosing, from which they recovered and displayed normal clinical signs before sacrifice

- dose dependent reduction in P/E ratio was observed in both male and  female  at 750, 1000 and 1500 mg/kg b.w. compared to the vehicle contral group

- with regards to the clinical signs and P/E reduction, the highest dose of main study was defined at 750mg/kg b.w. The doses and sex were selected for main study based on outcome of DRF I and II as reference.

 

Main study:

- conducted using five groups (five male animals per group): vehicle control group, three treatment groups and a positive control group

- vehicle control group  was administered orally with corn oil

- treatment groups were administered at the dose levels of 333, 500 and 750 mg /kg b.w., via oral raute twice at 24 hours interval

- positive control group was administered with 50 mg/kg b.w. of cyclophosphamide monohydrate via intraperitoneal route

- concurrently, one additional group (five male animals) was treated with the highest dose (750 mg/kg b.w.) for plasma analysis for demonstration of test item exposure to the bone marrow

 

- in the main experiment, no mortality was observed in any of the control or treatment groups

- clinical symptoms of lethargy were observed in all doses groups after treatment but recovered before the next dose and sacrifice

- a significant, but not dose dependent decrease of P/E ratio was observed in all treatment groups. Therefore, it is concluded that regarding these endpoints toxicity to the bone marrow was observed in the treatment groups, treated up to  the  dose level of 750  mg /kg  b.w., when compared to concurrent vehicle control group

- no statistically significant increase in the number of micronuclei (% MNPCE) was observed in male animals treated up to the dose level 750 mg/kg b.w. when compared to the concurrent vehicle control group

- statistically significant increase in number of micronuclei (% MNPCE) was observed in the positive control group (treated with 50 mg of cyclophosphamide monohydrate/kg b.w.) when compared to concurrent vehicle control group

 

Conclusions:

lt is concluded, that under the stated experimental conditions, the test item did not induce micronuclei formation up to the treated dose of 750 mg/kg b.w. Hence, the test item Diethyl sulphate, compound with 2-(heptadec-8-enyl)-4,5-dihydro-1H-imidazole-1-ethanol (1:1) is considered to be non-clastogenic at the dose of 750 mg/kg b.w. in the mammalian bone marrow micronucleus test in mice.

Executive summary:

Mammalian bone marrow micronucleus test of test item Diethyl sulphate, compound with 2- (heptadec-8-enyl)-4,5-dihydra-1H-imidazole-1-ethanol (1:1)  in mice was conducted per OECD No. 474, and B.12.

The main experiment was conducted using five groups (five male animals per group): vehicle control group, three treatment groups and a positive control group. The vehicle control group  was administered orally with corn oil. The treatment groups were administered at the dose levels of 333, 500 and 750 mg /kg b.w., via oral route twice at 24 hours interval. The positive control group was administered with 50 mg/kg b.w. of cyclophosphamide monohydrate via intraperitoneal route. Concurrently, one additional group (five male animals) was treated with the highest dose (750 mg/kg b.w.) for plasma analysis for demonstration of test item exposure to the bone marrow.

In the main experiment, no mortality was observed in any of the control or treatment groups. No statistically significant increase in the number of micronuclei (% MNPCE) was observed in male animals treated up to the dose level 750 mg/kg b.w. when compared to the concurrent vehicle control group.

lt is concluded, that under the stated experimental conditions, test item Diethyl sulphate, compound with 2-(heptadec-8-enyl)-4,5-dihydro-1H-imidazole-1-ethanol (1:1) did not induce micronuclei formation up to the tested dose level of 750 mg/kg b.w. Hence, the test item Diethyl sulphate, compound with 2-(heptadec-8-enyl)-4,5-dihydro-1H-imidazole-1-ethanol (1:1) is considered to be non-­clastogenic in the mammalian bone marrow micronucleus test in mice up to the tested dose level of 750 mg/kg b.w.

 

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Under the conditions of the studies, the test item - Diethyl sulphate, compound with 2-(heptadec-8-enyl)-4,5- dihydro-1 H-imidazole-1-ethanol (1: 1) shows neither genetic toxicity in vitro nor in vivo.