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

Genetic toxicity: in vivo

Administrative data

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

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 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted on 29th July, 2016

Test guideline 2

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

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)

Test animals

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

Administration / exposure

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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

twice at 24 hours interval

Doses / concentrations 2

Dose / conc.:

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

Remarks:

twice at 24 hours interval

Doses / concentrations 3

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

Examinations

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.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

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

 

Applicant's summary and conclusion

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.