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

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Based on the experimental condition used in the 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:

• Mating and fertility indices as well as the sex ratio of the pups did not reveal any effects. 


• No significant changes in estrous cyclicity and pre-coital interval were recorded in any of the treatment groups as compared to control.
  Mean pre-coital interval showed an increase in G4 as compared to G1 whereas the mating index (%) and fertility index (%) did not seem to be affected by the test item in the absence of clear dose dependency.


• The mean gestational length did not show any significant change but the number of females parturiting on day 21 showed a dose dependent decrease which may be considered as a possible effect of test item.


• There was a statistically significant reduction in litter size in G4 females accompanied by a significant reduction in implantation sites in all treatment groups and the number of live birth from day 0/1 until day 4 in G4 pups was decreased significantly in the highest dose group.


• A reduction in body weight gain of pups from day 4 to day 13 (high dose group pups) post culling is considered to be attributed to the maternal and developmental (growth) toxicity of the test item at a dose level of 130 mg/kg body weight.


• 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.

As most sensible parameters the significant decrease in litter size and in implantation sites was considered as test item induced reproductive toxicity in the high dose group.

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

• NOAEL for reproductive toxicity in males is considered to be 13 mg/kg body weight.


• LOAEL for reproductive toxicity in female is considered to be 13 mg/kg body weight.


• NOAEL for maternal toxicity is considered to be 40 mg/kg body weight.


• NOAEL for developmental parameters of offspring´s is considered to be 40 mg/kg body weight.


• NOAEL for the endocrine disrupter activity in adult females and female pups of the test item could not be established from this study.


• NOAEL for the endocrine disrupter activity in adult males and male pups of the test item is considered to be 130mg/kg body weight.

 

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

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, 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:

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

Details on mating procedure:

- Length of cohabitation: until pregnancy occurs or two weeks elapsed
- Proof of pregnancy: Females showing sperm positive smear were separated and housed individually.
• estrous cycle
• sperm in vaginal smear referred to as day 0 of pregnancy

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
Make : 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)

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.
- Culling was performed on day 4 post partum randomly keeping the litter size of 4 males and 4 females wherever possible, the surplus pups were sacrificed on day 4 post partum. The selected pups were sacrificed on day 13 post partum.
- Dams were sacrificed on day 14 post-partum. Dams were fasted overnight prior to blood collection. Non-pregnant females were sacrificed on 56th day of the study.
- Dosing was continued in both sexes during the mating period, continued throughout pregnancy (gestation), and lactation up to the day before scheduled kill (day 14 post partum).
- 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.

Frequency of treatment:

daily

Dose / conc.:

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

Remarks:

Dose Range Finding (DRF) Study

Dose / conc.:

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

Remarks:

Dose Range Finding (DRF) Study

Dose / conc.:

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

Remarks:

Dose Range Finding (DRF) Study

Dose / conc.:

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

Remarks:

Dose Range Finding (DRF) Study

Dose / conc.:

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

Remarks:

Dose Range Finding (DRF) Study

Dose / conc.:

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

Remarks:

Main study

Dose / conc.:

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

Remarks:

Main study

Dose / conc.:

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

Remarks:

Main study

Dose / conc.:

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

Remarks:

Main study

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.

Parental animals: Observations and examinations:

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].

Oestrous cyclicity (parental animals):

Estrous cycle was monitored daily from beginning of the treatment period until evidence of mating. When taking vaginal smear, care was taken to avoid disturbance to vaginal mucosa.

Sperm parameters (parental animals):

Parameters examined in male parental generations: testis weight, epididymis weight.

Litter observations:

Each litter was examined after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than corresponding control pups), and the presence of gross abnormalities. After parturition for live pups counting, marking and sex identification was performed within 24 hours of parturition (day 0 or 1 post-partum) and on day 4 and day 13 post-partum. The pups were observed for any abnormal behaviour.
The ano-genital distance (AGD) of each pup was measured within 24 hours of parturition (day 0 or 1 post-partum) on day 4 and on day 13 post-partum. The number of nipples/areolae in male pups was counted on post natal day (PND) 13.

Postmortem examinations (parental animals):

SACRIFICE
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.

GROSS NECROPSY
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 / ORGAN WEIGHTS
Organs of all males and females, randomly selected from each group were trimmed of adherent tissue/fat and weighed, prior to preservation in fixative. Testes and epididymides of all adult males were weighed. Organs were kept in normal saline till they were weighed.
All the collected organs/tissues of the rats of G1& G4 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.

Postmortem examinations (offspring):

SACRIFICE
At scheduled date, all animals including pups were examined macroscopically for any pathological changes with emphasis on reproductive system. Pups at termination were euthanized by cervical dislocation following narcosis.

GROSS NECROPSY
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 / ORGAN WEIGHTS
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.
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.

Reproductive indices:

litter size
No. of implantation sites
No. of post-implantation loss
Post-implanation Loss in %
Mean Pre-coital Interval
gestational length
gestation day
pups weight
post-natal loss

Offspring viability indices:

Pups Survival Index (%)
Mean Pups Body Weight
Sex Ratio
Gross Observation
Mean Pups Normalised Ano-genital Distance

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.

Dermal irritation (if dermal study):

not examined

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 whichshowed 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.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

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.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

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.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No significant changes in estrous cyclicity and pre-coital interval were recorded in any of the treatment groups as compared to control.
One animal of G5 (animal no. 39) showed an increase in estrous cycle duration from typical 4-5 days to 7 days on treatment at a dose level of 250 mg/kg body weight as compared to the control during this period. This disturbance may be considered as tendency of cycle extension/disturbance induced by test item.

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

sperm retention at stage VII (male: G4: 6/13)
epididymides-reduced sperm (male: G4: 3/13)

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Mating and fertility indices as well as the sex ratio of the pups did not reveal any effects. Mean pre-coital interval showed an increase in G4 as compared to G1 whereas the mating index (%) and fertility index (%) did not seem to be affected by the test item in the absence of clear dose dependency.
There was a significant decrease in litter size (no. of live birth) and in number of implantation sites on post partum/lactation day 0/1 in G4 as compared to G1 which is considered to be sensitive parameters for endocrine disrupter properties (ECETOC Monograph no. 31, 2002).
A significant reduction in the number of implantation sites was recorded in all the treatment groups indicating a possible reproductive toxicity induced by the test item but since the significant reduction in litter size was recorded only in G4, the test item is considered to severely affect the reproduction at a dose level of 130 mg/kg body weight and in the absence of significant reduction in litter size, the reduction in implantation sites which does not show dose dependency as well, may be considered incidental at 40 and 13 mg/kg body weight.

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

food consumption and compound intake

clinical biochemistry

organ weights and organ / body weight ratios

reproductive function (sperm measures)

reproductive performance

Key result

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

reproductive performance

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: endocrine disrupter activity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

Organ:

uterus

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

A reduction in body weight gain of pups from day 4 to day 13 (G4 group pups) post culling is considered to be attributed to the maternal and developmental (growth) toxicity of the test item at a dose level of 130 mg/kg body weight.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

No treatment related changes in hormone levels was recorded in adult males and day 13 male pups.
A significant reduction even if not strictly dose dependent in the TSH levels of day 13 female pups of all the treated groups was recorded and a similar trend of TSH levels was observed in the adult females over all dose groups along with a significant decrease in T4 levels of G4 females.
Estradiol levels of all the treatment groups showed a decrease as compared to control, in G2 and G3 significantly, in G4 not significantly but also decreased compared to the control rats. These results were not strictly dose dependent but due to the consistency of the results in both female pups as well as adult females (TSH levels) and attributing it to the known alkylating property of test item (Diethyl Sulphate [MAK value documentation], volume 20, 2003) and under the light of the fact that epigenetic changes, such as DNA methylation and/or acetylation and histone modifications, appear to be involved in mechanisms related to endocrine disruption (Lauretta et al., 2019) the test item is considered to show endocrine disrupter property in female sex.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

No treatment related changes were observed in normalized ano-genital distance (AGD) of pups.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

There was a significant increase in thyroid/parathyroid absolute weight of male pups of G2 and G3 and female pups of G3 in comparison with respective controls. Observed weight variations in heart were minor in nature and thyroid/parathyroid weights did not show any dose dependency or related changes in hormone levels hence considered as inconsistent and incidental.
The developmental parameters of the pups were comparable among all the groups except the body weight gain from day 4 to day 13 in G4 and are considered test item related since the maternal toxicity induced by the compound affected the development of pups.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Pups observed during terminal sacrifice during study did not reveal any lesion of pathological significance related to test item.

Histopathological findings:

no effects observed

Description (incidence and severity):

Histopathological evaluation of thyroid/parathyroid glands collected at day 13 from pups as mentioned in study plan did not showed any microscopic alterations related to test item.

Other effects:

no effects observed

Description (incidence and severity):

The sex ratio of the pups did not reveal any effects.

The developmental parameters of the pups were comparable among all the groups except the body weight gain from day 4 to day 13 in G4 and are considered test item related since the maternal toxicity induced by the compound affected the development of pups.
Furthermore a correlation with T4 and TSH and the increase in thyroid weight in males pups of G2 and G3 and female pups of G3 is not clear as there was no alteration of hormone levels in males and no clear dose dependency observed.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

40 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

other: developmental parameters

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to other toxic effects:

reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Conclusions:

Based on the experimental condition used in this study and the findings 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 with 14 days recovery, where in 0, 13, 40 and 130 mg/kg bw, daily oral doses were tested following conclusions were drawn:
- NOAEL for general toxicity in males is considered to be 13 mg/kg bw
- NOAEL for reproductive toxicity in males is considered to be 13 mg/kg bw
- NOAEL for general toxicity in females is considered to be 40 mg/kg bw and
- LOAEL for reproductive toxicity in female is considered to be 13 mg/kg bw
- NOAEL for maternal toxicity is considered to be 40 mg/kg bw.
- NOAEL for developmental parameters of offspring‟s is considered to be 40 mg/kg bw
- NOAEL for the endocrine disrupter activity in adult females and female pups of the test item could not be established from this study.
- NOAEL for the endocrine disrupter activity in adult males and male pups of the test item is considered to be 130mg/kg bw

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 observed effects, following effect levels have been calculated:

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


• NOAEL for reproductive toxicity in males: 13 mg/kg bw


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


• LOAEL for reproductive toxicity in females: 13 mg/kg bw


• NOAEL for maternal toxicity: 40 mg/kg bw


• NOAEL for developmental parameters of offspring‟s: 40 mg/kg bw


• NOAEL for the endocrine disrupter activity in adult males and male pups: 130mg/kg bw

 

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

13 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Guideline study, reliable without restriction

Effects on developmental toxicity

Description of key information

Based on the experimental condition used in the 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:

• There was a statistically significant reduction in litter size in G4 females accompanied by a significant reduction in implantation sites in all treatment groups and the number of live birth from day 0/1 until day 4 in G4 pups was decreased significantly in the highest dose group.


• A reduction in body weight gain of pups from day 4 to day 13 (G4 group pups) post culling is considered to be attributed to the maternal and developmental (growth) toxicity of the test item at a dose level of 130 mg/kg body weight. 


• The normalized ano-genital distances and nipple retention of the pups were comparable among all dose groups.

As most sensible parameters the significant decrease in litter size and in implantation sites was considered as test item induced reproductive toxicity in the high dose group.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

40 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Guideline study, reliable without restriction

Justification for classification or non-classification

The data from the available study provide an initial hazard assessment with a preliminary self-classification: Repr. 2, H361f ‘Suspected of damaging fertility’ in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. (EC) 1272/2008.