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

Endpoint summary

Description of key information

The substance Diethyl sulphate, compound with 2-(heptadec-8-enyl)-4,5-dihydro-1H-imidazole-1-ethanol (1:1), was examined in a Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test according to OECD 422.
0, 13, 40 and 130 mg/kg bw daily oral doses were administered to Wistar rats. Based on the data, it can be concluded that the oral administration of test substance in male and female Wistar rats produced lesions of varied degrees in liver of both males and females. These lesions correlated only in G4 males with increased AST serum level and seem to be test item related in this dosage but cannot clearly interpreted as test item related in the lower dose groups of both sexes as several lesions were also found in the control groups. The lesions in testes and epididymis along with increased serum AST, calcium and phosphorous levels in males at 130 mg/kg body weight is considered test item related; whereas lesions in testes and epididymis along with increased serum calcium and phosphorous levels in males at 40 mg/kg body weight is considered to be test item related. However, no adverse pathological alterations were observed in the low dose group of male animals. In females no adverse pathological alterations can be interpreted as test item related except some more alterations in the liver in the highest dose group as compared to the control.

Based on the observed effects, following effect levels have been calculated:

• NOAEL for general toxicity in males: 13 mg/kg bw
• NOAEL for general toxicity in females: 40 mg/kg bw

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: oral, other

Remarks:

Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-06-24 to 2022-06-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- batch number of test material: 10419
- purity: > 93 %
- Expiry Date: 23.04.2023

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Dry and dark at ambient room temperature (20-30 ºC).
- Test item formulations were found to be homogeneous and stable up to 24 hour in vehicle corn oil.

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Animals were procured from a CPCSEA approved vendor [Cadila Pharmaceuticals Ltd., Ahmedabad, Gujarat (CPCSEA Registration No.: 161/PO/RcBiBt/S/99/CPCSEA)]
- Age at study initiation: Minimum 12 weeks at the start of oestrus cycle evaluation
- Weight at study initiation: Main study: Males: 264 to 391 g Females:184 to 250 g
- Fasting period before study:
- Housing: 2-3 rats/sex were housed in polycarbonate cages (size 37 [cm] x 21 [cm], height 20 [cm]). Cage rotation was carried out weekly during study period except during mating and during gestation and lactation only for females. Pregnant and lactating females with their offspring were caged individually.
- Diet: A conventional laboratory pelleted diet of batch no. 040521, 040721, 040921, 041021 and 041221 from approved supplier (Nutrivet Life Sciences, Pune) was offered ad libitum.
- Water: Aqua guard filtered drinking water in bottles was offered ad libitum.
- Acclimation period: 21 days (7 days of acclimatization and 14 days vaginal smear examination for estrous cycle evaluation) for main study prior to test item administration

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.10 - 22.50
- Humidity (%): 50.50 - 64.20
- Air changes (per hr): 12 times
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Active ingredient from sample of test concentration were extracted and diluted with suitable solvent and injected onto HPLC- PDA using following instrument parameters:
Instrument
brand: Shimadzu HPLC,
Model: Prominence - i LC-2030C 3D Plus

Mobile Phase
Pump A: Acetonitrile,
Pump B: Methanol (25: 75 %v/v)
Column: ODS Hypersil-C18 (250 mm x 4.6 mm x 5 µm)
Column Oven Temperature: 30 °C
Flow rate: 0.8 mL/min
Detector: PDA
Wavelength: 236 nm
Injection Volume: 5 µL
Retention Time: 2.294 minutes (approximately) (during specificity)

The selected analytical method was suitable for the analysis of the test item.

Duration of treatment / exposure:

- Animals of both sexes were dosed 14 days prior to mating.
- Male animals were administered the doses for 57 days, female animals were dosed for a minimum
of 50 days to maximum of 64 days.

Frequency of treatment:

daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

Group Treatment Dose (mg/kg bw) Concentration (mg/mL) No. of animals Males/Females
G1 Control 0 0 13/13
G2 Low 13 3.25 13/13
G3 Mid 40 10 13/13
G4 High 130 32.5 13/13
G1-R Control 0 0 5/5
recovery
G4-R High dose 130 32.5 5/5
recovery

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels 0, 13, 40 and 130 mg/kg body weight were selected for the main study based on the results of Dose Range Finding (DRF) study using the spacing factor 3.
- Fasting period before blood sampling for clinical biochemistry: Dams were fasted overnight prior to blood collection.
- Post-exposure recovery period: Recovery animals of both sexes were dosed for 50 days (till the day before first scheduled kill of females) during study period and kept 14 days after the first schedule sacrificed of dams without treatment to detect delayed occurrence, or persistence of or recovery from toxic effects.
- Dose range finding studies: A total of 40 Wistar rats (20 males and 20 females) were randomly allocated to five different dose groups, consisting of 4 animals/sex. The animals allocated to Group G1, G2, G3, G4 and G5 received daily 0, 12.5, 25, 50 and 250 mg/kg body weight of the test item respectively for 14 days. All animals were examined for estrous cycle during 14 days treatment. All rats were observed twice daily (morning and evening) for morbidity and mortality, throughout the acclimatization and study period. General clinical observations of rats of all the groups were made once a day, at around the same time each day and considering the peak period of anticipated effects after dosing. Males and females were weighed during randomization, on the first day of dosing, once weekly thereafter, and at termination. Feed consumption was measured once weekly. All animals were subjected to necropsy and detailed gross pathology evaluation. Animals were fasted overnight before necropsy. Animals were weighed, sacrificed using over dose of CO2 and examined externally. All orifices and the cranial, thoracic and visceral cavities was opened and examined macroscopically.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed twice daily (morning and evening) for morbidity and mortality throughout the acclimatization and study period.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: General clinical observations of animals of all groups were made once a day, preferably at the same time each day (approximately 1 hour post dosing).
Detailed clinical examinations were carried out once before the first treatment (to allow for within-subject comparisons) and weekly thereafter.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed during randomization, on the first day of dosing, at least weekly thereafter, and at termination. During pregnancy, females were weighed on days 0, 7, 14 and 20 and within 24 hours of parturition (day 0 or 1 post-partum), on day 7 and day 13 post-partum and before terminal sacrifice.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, during the pre-mating, pregnancy and lactation periods, feed consumption was monitored at least weekly. Feed consumption was not measured during mating period.
- Compound intake calculated: Yes, feed consumption per cage was calculated as follows: Feed consumption (g/day/animal) = [(Feed input–Feed leftover)/No. of animal x No. of days].

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during the last week of treatment
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: just prior to necropsy
- Animals fasted: Yes, overnight prior to blood collection
- How many animals: randomly selected from each group

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: just prior to necropsy
- Animals fasted: Yes, overnight
- How many animals: randomly selected from each group

PLASMA/SERUM HORMONES/LIPIDS: Yes
- Time of blood sample collection: just prior to necropsy
- Animals fasted: Yes, overnight prior to blood collection
- How many animals: randomly selected from each group

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Sensory reactivity to stimuli, assessment of grip strength, hind limb foot splay and motor activity assessment were conducted for five males and five females from control and treatment groups each, during the last week of treatment and that of recovery groups, in the last week of recovery period. Animals were subjected to examinations of various functional parameters which included: motor activity measurements using OPTO–VARIMEX 4 (Serial No.: 100180, manufactured by: Columbus Instruments, USA, supplied by: Gentech Marketing and Distribution, New Delhi, India), an automated animal activity measuring system; fore limb and hind limb grip strength, using grip strength meter (Serial No.: 0167-005L, manufactured by: Columbus Instruments, USA, supplied by: Gentech Marketing and Distribution, New Delhi, India); hind limb foot splay record and sensory reactivity measurements.
- Dose groups that were examined: all
- Battery of functions tested: Sensory reactivity, grip strength, hind limb foot splay and motor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, at scheduled date, all animals including pups were examined macroscopically for any pathological changes with emphasis on reproductive system. All adult animals of different groups were euthanized by over dose of CO2 asphyxiation followed by exsanguination. Pups at termination were euthanized by cervical dislocation following narcosis. The animals were examined externally in unopened condition. This was followed by opening of the carcasses and topographic macroscopic examination of organs. This included careful examination of the external surface of the body, all orifices, cranial, thoracic and visceral cavities and their contents. Simultaneously gross lesions examination was performed in accordance with the SOPs of the laboratory.

HISTOPATHOLOGY: Yes, All the collected organs/tissues of the rats of G1& G4, collected thyroid/parathyroid glands of pups and gross lesions were subjected to histopathological examination. All the tissues were trimmed, processed, embedded in paraffin wax. Sections were cut at a thickness of 3-5 micron and stained with hematoxylin and eosin stain. Testes and epididymides were observed as target organs upon preliminary microscopic evaluation of control and high dose rats. Hence, target organs from low dose (G2), mid dose (G3) and both recovery groups (G1R and G4R) were processed for further evaluation. Processed tissues were subjected to histopathological examination. The prepared slides were examined under microscope by the pathologist to note histopathological lesions, if any in different organs. The observed abnormalities were described according to morphological character, distribution, severity and included in raw data and report.

Statistics:

Raw data was analyzed using statistical software “Sigma Plot 11.0” (Supplied by Cranes Software International Ltd. Bangalore). The mean and standard deviation was calculated using the software and all data was summarized in tabular form. All continuous data (body weight, feed consumption, Functional Observational Battery parameters, hematology, clinical chemistry, absolute and relative organ weights, maternal and pup parameters etc.) were checked for normality using Shapiro Wilk test. All homogenous data was analyzed using ANOVA and data showing significance in their variances was subjected to Dunnett‟s t-test. All heterogeneous data was analyzed using F test and Student‟s t-test, Dunn‟s Test, Kruskal-Wallis, ANOVA on ranks.

Clinical signs:

no effects observed

Description (incidence and severity):

No apparent treatment related clinical signs were observed in males and females of all treatment groups throughout the treatment and recovery period except in 4 males of G4 (animal number 81, 87, 89 and 90) which showed lethargy from day 35 to 44. Detailed clinical examinations like home cage observation, handling observation and open field observation of all animals were observed to be normal during study period and this data was comparable among all the treatment groups hence no changes related to test item were observed.

Mortality:

no mortality observed

Description (incidence):

No mortality and no morbidity was observed in any of the groups of animals throughout the study period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights of all the treatment groups were comparable with respective controls whereas the percent body weight changes showed a significant decrease in G4 males as compared to respective controls and a statistically significant decrease during gestation and a decrease which is not statistically significant but may be considered of biological importance during lactation in G4 females which is attributed to the effect of the test item. In G2 males reduced body weight gain was recorded as compared to G1 in the last week of treatment and in G2 females but due to lack of consistency with time and the correlated parameter feed consumption it was considered to be incidental.
Among the recovery groups of males a significant decrease in body weight gain was recorded from day 15 till day 50 and thereafter post cessation of dosing the body weight gains were found comparable with the respective controls. Among G4R females of recovery groups the body weight gain showed an initial increase and thereafter it was found comparable with G1R females. Hence, the effect of body weight loss seemed to be reversible.
The statistically significant decrease in body weight change during gestation and a decrease which is not statistically significant but may be considered of biological importance during lactation in G4 females were recorded - this may be interpreted as evident adverse effects of the test item during pregnancy and lactation.
Hence, the test item may be referred to as maternal toxicant at this dose level of 130 mg/kg b.w. and seems to be reversible in females.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A significant reduction in feed consumption was recorded on day 8 and 15 in G4 males and females (pre-mating) and in G2 and G4 females on day 8 (pre-mating), day 7 and 14 during gestation period which was not found statistically significant but considered as biologically significant since the correlated parameter body weight gain also showed consistency with this observation in G4. Further correlations of feed intake and body weight change could not be interpreted. Among the recovery groups (G4R males and females) no test item related changes were recorded post cessation of dosing which showed possible reversibility of test item effects on body weight change and feed intake.
Statistically significant reduction in feed intake was recorded in G4R females from day 15 of dosing to 63 i.e. till the terminal sacrifice. However the recovery females did not show decreased body weight gain.
Hence, the changes in feed intake in females and males of the highest dose group correlates with the reduced body weight gain and is considered to be test item related but shows signs of reversibility.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No abnormality was observed in any of the treatment groups during opthalmological observation at performed before the terminal sacrifice.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

All haematological parameters in animals of different treated groups of both the sexes were comparable to their respective control groups.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Dose dependent increase in levels of serum calcium and phosphorus were noted in G3 and G4 males which explains the increase in forelimb grip strength, whereas a significant increase in serum AST levels was noted in G4 males. These effects are considered test item related. The clinical chemistry parameters were found to be comparable in high dose recovery groups as compared with their respective controls hence considered to be reversible.

Endocrine findings:

effects observed, treatment-related

Description (incidence and severity):

The hormone estimation revealed a possible gender specific endocrine disrupter activity of test item in all the treatment groups of females (adult females as well as day 13 female pups) thereby inducing significant alteration in the TSH levels in both adult females and day 13 female pups of all the groups (not dose dependent) and T4 and estradiol levels in G4 females.
Estrous cycle was evaluated for checking the regularity during treatment period and in cohabitation for confirmation of pregnancy. No significant changes in estrous cyclicity and pre-coital interval was recorded in any of the treatment groups as compared to control. Mating and fertility indices as well as the sex ratio of the pups did not reveal any effects.

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The mean and standard deviation of organ weights (absolute organ weight, organ weight relative to body weight) of either sex at the end of treatment period, showed statistically significant increase in relative organ weight of testes and heart in G4 males at 130mg/kg b.wt.)
Observed weight variations in heart were minor in nature hence considered as spontaneous.
However, variation in relative organ weight of testes were found significantly higher and can be correlated to the edema observed during microscopic observation in testes of G4 male as compared to control and hence was considered related to test item. Similar findings have been reported with other toxicants (Kumar and Nagar, 2014).

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A statistically significant decrease in grip strength of hind limb was recorded in G4 males and was explicable due to changes in an altered calcium metabolism and in turn affecting the neuromuscular activity which can be attributed to the test item. In the recovery groups, the grip strength was found comparable in females whereas an increase in forelimb grip strength was recorded in G4 males but since the biochemical parameters did not reveal any alterations in the serum calcium levels it may be considered incidental.

Neuropathological findings:

no effects observed

Description (incidence and severity):

The functional observation battery/neurobehavioral observation, sensory reactivity measurements as well as foot splay measurements were comparable over all groups and no treatment related changes were observed in animals of treatment and recovery groups in both the sexes.
The results of motor activity were found mainly comparable among all the treatment groups during treatment period but a significant reduction in CR (Clockwise rotation), DT (Distance travelled) and AT (Ambulatory time) in males and CCR (Counter clockwise rotation) in females of recovery groups G4 were significantly decreased as compared with their respective controls but due to lack of consistency and evidence of any alterations in the correlated parameters it was considered to be incidental.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Visceral examination of the rats of control and other treated groups showed various changes in liver viz. pale foci, whitish patches, enlargement etc. However these lesions were of no pathological significance when compared with control group.
It can be concluded that the oral administration of test substance in male and female Wistar rats at 130 mg/kg body weight (high dose), 40 mg/kg body weight (mid dose) and 13 mg/kg body weight (low dose) produced lesions of varied degrees in liver of both males and females. These lesions correlated only in G4 males with increased AST serum level and seem to be test item related in this dosage but cannot clearly interpreted as test item related in the lower dose groups of both sexes as several lesions were also found in the control groups. The lesions in testes and epididymis along with increased serum AST, calcium and phosphorous levels in males at 130 mg/kg body weight is considered test item related; whereas lesions in testes and epididymis along with increased serum calcium and phosphorous levels in males at 40 mg/kg body weight is considered to be test item related. However, no adverse pathological alterations were observed in the low dose group of male animals. In females no adverse pathological alterations can be interpreted as test item related except some more alterations in the liver in the highest dose group as compared to the control.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Number of rears, urine pools, and fecal bolus in animals of all the test groups of both the sexes did not show any treatment related changes as compared to the respective control groups.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

clinical biochemistry

food consumption and compound intake

organ weights and organ / body weight ratios

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

histopathology: neoplastic

other endocrine activity endpoints

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

other: serum

Organ:

testes

Treatment related:

yes

Dose response relationship:

yes

Conclusions:

Based on the experimental condition used in this study of Repeated Dose Oral Toxicity Study in combination with Reproduction/Developmental Toxicity of Diethyl sulphate, compound with 2-(heptadec-8-enyl)-4,5-dihydro-1H-imidazole-1-ethanol (1:1) (CAS no.: 68527-63-9) in Wistar Rats the following conclusions were drawn:
The NOAEL for General toxicity in males was considered to be 13 mg/kg body weight and to be 40 mg/kg body weight in females.

Executive summary:

The substance Diethyl sulphate, compound with 2-(heptadec-8-enyl)-4,5-dihydro-1H-imidazole-1-ethanol (1:1), was examined in a Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test according to OECD 422.
0, 13, 40 and 130 mg/kg bw daily oral doses were administered to Wistar rats.

Based on the data presented in this report, it can be concluded that the oral administration of test substance in male and female Wistar rats at 130 mg/kg body weight (high dose), 40 mg/kg body weight (mid dose) and 13 mg/kg body weight produced lesions of varied degrees in liver of both males and females. These lesions correlated only in G4 males with increased AST serum level and seem to be test item related in this dosage but cannot clearly interpreted as test item related in the lower dose groups of both sexes as several lesions were also found in the control groups. The lesions in testes, epididymis and liver, along with increased serum calcium and phosphorous levels, in males at 130 mg/kg body weight is considered to be test item related; the lesions in testes and epididymis along with increased serum calcium and phosphorous levels in males at 40 mg/kg body weight is considered to be test item related. However, no adverse pathological alterations were observed in the low dose group of male animals. In females no adverse pathological alterations can be interpreted as test item related except some alterations in the liver in the highest dose group when compared to the control group.

Based on these effects the following effect levels have been calculated:

• NOAEL for general toxicity in males: 13 mg/kg bw
• NOAEL for general toxicity in females: 40 mg/kg bw

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

13 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

guideline study reliable without restriction

Organ:

testes

Justification for classification or non-classification

There are conclusive and sufficient data available for classification of the test substance with regard to systemic effects following repeated dosing. Based on the data available, the test substance is classified as STOT Rep. Exp. 2 H373: 'May cause damage to the testis and liver through prolonged or repeated exposure' in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. (EC) 1272/2008.