Amides, C8-18 (even numbered) and C18-unsatd., N,N-bis(hydroxyethyl)

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 28, 2020 to September 7, 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

The following deviations occurred during the study: 1) at arrival, body weight of animals was 224 - 233 g for males and 199 - 202 g for females instead of 200 to 225 g for males and 175 to 200 g for females, as indicated in the Study Protocol. –2) an acclimatisation period of 53 days occurred before the start of treatment instead of approximately 3 weeks, as indicated in the Study Protocol. This was due to discussions with the Sponsor regarding dose selection, and after finalisation of the protocol, an additional two weeks were required for monitoring oestrous cycles. This deviation was not considered to have compromised the purpose or outcome of the study. There were no other deviations from the protocol.

Deviations:

yes

Remarks:

This deviation was not considered to have compromised the purpose or outcome of the study.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Amides, C8-18 (even numbered) and C18-unsatd., N,N-bis(hydroxyethyl)

EC Number:

931-329-6

Cas Number:

68155-07-7

Molecular formula:

The alkyl chain length of the amide ranges between 8 and 18 carbon atoms

IUPAC Name:

Amides, C8-18 (even numbered) and C18-unsatd., N,N-bis(hydroxyethyl)

Test material form:

liquid

Details on test material:

Appearance: yellowish liquid

Specific details on test material used for the study:

- Identity: Amides, C8-C18 (even numbered) and C18-unsatd.,N,N-bis(hydroxyethyl)
- Alternative name: Comperlan COD
- Batch no. 0021077031
- CAS no. 68155-07-7
- EC no. 931-329-6
- Appearance: yellowish liquid
- Storage conditions: Room temperature, protected from light

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The Sprague Dawley rat was the species and strain of choice because it is accepted by many regulatory authorities and there are ample experience and background data on this species and strain at ERBC.

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Animal supply and acclimatisation:
Age: 7 to 8 weeks old
Weight: 224-233 g (males) and 199 to 202 g (females)
Acclimatisation period: an acclimatization period of 53 days was allowed before the start of treatment, during which the health status of the animals was assessed by thorough observations.

- Animal husbandry:
The animals were housed in a limited access rodent facility.
Animal room controls: temperature of 22ºC ± 2ºC and relative humidity of 55% ± 15%.
There were approximately 15 to 20 air changes per hour and the rooms were lit by artificial light for 12 hours each day.
From arrival to pairing, animals were housed up to 5 of one sex to a cage, in polysulfone solid bottomed cages measuring 59.5×38×20 cm.
During mating, animals were housed one male to one female in clear polysulfone cages measuring 42.5×26.6×18.5 cm with a stainless steel mesh lid and floor. Each cage tray held absorbent material which was inspected and changed daily.
After mating, the males were re-caged as they were before mating. The females were transferred to individual solid bottomed cages for the gestation period, birth and lactation (measuring 42.5×26.6×18.5 cm).
Drinking water was supplied ad libitum to each cage via water bottles.
A commercially available laboratory rodent diet was offered ad libitum throughout the study.

- Allocation to groups:
On the day of allocation (7 days prior to the start of treatment), all animals were weighed. Animals at the extremes of the weight distribution were excluded to leave the required number of animals. Furthermore, female animals that exhibited anomalies in the oestrous cycle were not allocated to study. The rats were allocated to the groups by computerised stratified randomisation to give approximately equal initial group mean body weights. Individuals were uniquely identified within the study by sex, tattoo on the hind feet and ear notch and housed 5 of one sex per cage. The cages were identified by a label recording the study number, animal numbers and details of treatment. The arrangement of cages in batteries was such that cages from each treatment group were evenly distributed across the battery to minimise possible environmental effects.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% aqueous solution of CMC

Details on exposure:

- Preparation of dosing solutions:
The required amount of the test substance was suspended in the vehicle. The preparations were made daily or weekly, based on the stability data and on the needs of the study, at concentrations of 25, 50 and 100 mg/mL. Concentrations were calculated and expressed in terms of the test substance as supplied.
The test substance was administered orally by gavage at doses of 0, 250, 500 and 1000 mg/kg bw/day and at a dose volume of 10 mL/kg bw. Control animals were received the vehicle alone at the same dose volume. The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.

- Vehicle:
The 0.5% aqueous solution of carboxymethylcellulose was selected as vehicle based on the test substance properties.

Details on mating procedure:

Matings were monogamous (one male to one female). Animals were housed one male to one female in clear polysulfone cages. After mating, the males were re-caged as they were before mating. The females were transferred to individual solid bottomed cages for the gestation period, birth and lactation. Females were weighed weekly from allocation to positive identification of mating and on Days 0, 7, 14 and 20 post coitum. Vaginal smears were taken from the day after the start of pairing until positive identification of copulation (spermidentification, vaginal plug in situ or copulation plug found in the cage tray). The female was paired with the same male until positive identification of copulation occurred or 14 days elapsed.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of doses was performed in a separate study in order to validate both the analytical method and the preparation procedure and to verify the stability of the preparations.
The analytical method was validated from 1 to 100 mg/mL. Linearity, accuracy and precision were within the limits stated in Standard Operating Procedure (SOPs) for suspension (r >0.98; accuracy 85-115%; precision CV <10%). In the same study, 28-hour stability at room temperature and 8-day stability at 2-8°C were verified in the range from 1 to 100 mg/mL. The proposed preparation procedure for the test substance was checked in the range from 1 to 100 mg/mL by chemical analysis (concentration) to confirm that the method was suitable. Final results for all levels were within the acceptability limits stated in SOPs for concentration (85-115%) and homogeneity (CV <10%).
In the present study, samples of the preparations made on two occasions during the study (Day 1 and again towards the end of the study) were analysed to check the homogeneity and concentration. Chemical analysis was carried out using the software Empower® 2 Build No. 2154. Results of the analyses were within the acceptability limits stated in SOPs for suspensions (85-115% for concentration and CV <10% for homogeneity).

Duration of treatment / exposure:

- Males were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing, through the pairing period and thereafter until the day before necropsy (Day 29). Males were treated for a total of 28 days.

- Females were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post-partum periods until Day 13 post-partum (for at least 51 days). Non pregnant females and one that did not mate were dosed up to the day before necropsy.

Frequency of treatment:

Once daily, 7 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

vehicle

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

Low level

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Medium level

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High Level

No. of animals per sex per dose:

10 males/10 females/group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels of 250, 500 and 1000 mg/kg/day were selected based on information from a preliminary non-GLP study.

- Rationale for animal assignment: A total of 93 Sprague Dawley SD rats (43 males and 50 virgin females), 7 to 8 weeks old and weighing 200 to 225 g for males and 175 to 200 g for females were ordered.

Examinations

Parental animals: Observations and examinations:

- Mortality
All animals were checked early in each working day in the morning and in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day. This allowed post-mortem examinations to be carried out during the working period of that day.

1. Clinical signs
Before treatment and at least once daily during the study, each animal was observed, and any clinical signs were recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions (2-2.5 hour after treatment).

2. Body weight
Males were weighed weekly from allocation to termination. Females were weighed weekly from allocation to positive identification of mating and on Days 0, 7, 14 and 20 post-coitum. Dams were also weighed on Days 1, 4, 7, 13 post-partum and just before necropsy.

3. Food consumption
The weight of food consumed by each cage of males and females was recorded weekly during the pre-mating period starting from Day 1 of dosing. Individual food consumption for the females was measured on Days 7, 14 and 20 post coitum starting from Day 0 post-coitum and on Days 7 and 13 post-partum starting from Day 1 post-partum.

4. Mating
Matings were monogamous (one male to one female). Vaginal smears were taken from the day after the start of pairing until positive identification of copulation (spermidentification, vaginal plug in-situ or copulation plug found in the cage tray).
The female was paired with the same male until positive identification of copulation occurred or 14 days elapsed.

5. Parturition and gestation length
A parturition check was performed from Day 20 to Day 25 post-coitum. Two females (1 of mid-dose group and 1 of high dose group) which did not give birth 25 days after mating were sacrificed on Day 27 post coitum. These animals were found not pregnant at necropsy. In addition, one female of the high dose group did not mate during the 14 days of the mating period. This female was sacrificed thereafter and was found not pregnant.
Mating was not detected in one low dose female. However, this female gave birth and was sacrificed with its litter on Day 14 post-partum.
Gestation length was calculated as the time between the day of successful mating (Day 0 post coitum) and the day of birth when the parturition was defined complete (Day 0 post-partum). Due to the difficulties in the delivery, one female of the control group, was sacrificed for humane reasons.

6. Clinical pathology
Blood collection for thyroid hormone determination (T3, T4 and TSH):
In males, approximately 0.8 mL of blood samples were collected at termination under isoflurane anesthesia from the retro-orbital sinus.
In females, as a part of the necropsy procedure, approximately 0.8 mL of blood samples were withdrawn under isoflurane anesthesia from the abdominal vena cava from all parenteral female rats.
Samples were assayed to determine the serum levels of Total triiodothyronine (total T3), Total thyroxine (total T4) and Thyroid stimulating hormone (TSH) by RadioImmunoAssay (RIA).

Oestrous cyclicity (parental animals):

Oestrous cycle was monitored by vaginal smears in all stock females for 2 weeks before allocation in order to exclude from the study females with irregular cycle.
In females allocated to groups, vaginal smears were taken in the morning from Day 1 of treatment, up to positive identification of mating including not less than 2 weeks before the pairing. The vaginal smear data were examined to determine anomalies of the oestrous cycle and pre-coital interval.
Vaginal smears were also taken from all females, before despatch to necropsy with the exception of one control female sacrificed for humane reasons.

Litter observations:

1. Pups identification, weight and observation
As soon as possible after parturition was considered complete (Day 0 post-partum), all pups (live and dead) were counted, sexed and live pups were identified. Live pups were individually weighed on Days 1, 4 and 13 post-partum. Observations were performed once daily for all litters.

2. Anogenital distance (AGD)
The anogenital distance (AGD) of each pup was measured on Day 1 post-partum. The measure of AGD was normalized to the cube root of the pup’s body weight measured on Day 1 post-partum.

3. Nipple count
The presence of nipples/areolae in male pups was checked on Day 13 post-partum (a day before despatch to necropsy).

4. Clinical pathology
Blood collection for thyroid hormone determination (T3, T4 and TSH):
On Day 14 post-partum, blood samples (approximately 0.5 mL) were withdrawn under light ether anesthesia from the heart (by intracardiac puncture) from at least two pups (1 sample/sex, if possible) per litter.
Immunoanalysis:
Immunoanalysis was performed in samples from pups of Day 14 post-partum. Samples were assayed to determine the serum levels of Total triiodothyronine (total T3), Total thyroxine (total T4) and Thyroid stimulating hormone (TSH) by RadioImmunoAssay (RIA).

Postmortem examinations (parental animals):

1. Euthanasia
One female animal was sacrificed for humane reasons under carbon dioxide asphyxiation. Parental animals that had completed the scheduled test period, were killed by exsanguination under isoflurane anesthesia.
Surviving males were killed after the end of mating period on Day 29 of the study. Males were treated for a total of 28 days.
The females with live pups were killed on Day 14 post-partum. Females were dosed for a minimum of 51 consecutive days. The female showing no evidence of copulation was killed 25 days after the last day of the mating session. The non-pregnant females were killed on Day 27 post coitum.

2. Necropsy
The clinical history of adult animals was studied, and a detailed post-mortem examination was conducted (including examination of the external surface and orifices). Changes were noted, the requisite organs weighed, and the required tissue samples preserved in fixative and processed for histopathological examination.
Parental females were examined also for number of visible implantation sites (pregnant animals) and number of corpora lutea (pregnant animals). Uteri of apparently non-pregnant females were immersed in a 20% solution of ammonium sulphide to reveal evidence of implantation.

3. Organ weights
From all animals completing the scheduled test period, organs were dissected free of fat and weighed. The ratios of organ weight to body weight were calculated for each animal.
Organs: adrenal glands, epididymides, kidneys, liver, ovaries with oviducts, prostate gland, sciatic nerve, seminal vesicles, spleen, testes, thymus, thyroid and uterus-cervix.

4. Tissues fixed and preserved
Samples of all the tissues (all parental animals) were fixed and preserved in 10% neutral buffered formalin (except testes and epididymides which were fixed in modified Davidson’s fluid and preserved in 70% ethyl alcohol).
Tissues: adrenal glands, brain, clitoral gland, epididymides, kidneys, liver, mammary glands, ovaries with oviducts, parathyroid glands, pituitary glands, penis, prostate gland, sciatic nerve, seminal vesicles, spleen, testes, thymus, thyroid, uterus-cervix, and vagina.

5. Histopathological examination
Tissues: adrenal glands, clitoral gland, epididymides, kidneys, liver, mammary glands, ovaries with oviducts, seminal vesicles, testes, thyroid, uterus-cervix, and vagina.
After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometer thickness and stained with haematoxylin and eosin.
The histopathological examination was restricted:
- Tissues specified above from all males and females in the control and high dose groups killed at term.
- Tissues specified above from all animals killed or dying during the treatment period.
- All abnormalities in all groups.
In addition, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodi Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed.
The evaluation took into account the tubular stages of the spermatogenic cycle, in order to identify treatment-related effects, such as: missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen.

Postmortem examinations (offspring):

1. Euthanasia
Pups that had completed the scheduled test period (Day 4 or Day 14 post partum) and those sacrificed for humane reason (dams found dead or sacrificed) were euthanised by intraperitoneal injection of Sodium Thiopenthal.
Pups selected for blood collection for hormone determination were killed on the day of blood sampling.

2. Necropsy
All pups found dead in the cage or those sacrificed for humane reasons (dams found dead or sacrificed) were examined for external and internal abnormalities.
All culled pups sacrificed on Day 4 post-partum were subjected to an external examination.
Sex was determined by internal gonads inspection.
All live pups sacrificed on Day 14 post-partum were killed and examined for external abnormalities and sex confirmation by gonadal inspection.
Pups with abnormalities were retained in 10% neutral buffered formalin.

3. Organ weights
Pups at Day 14 post-partum: Thyroids were weighed from one male and one female pups selected for blood collection for hormone determination and preserved in 10% neutral buffered formalin.
The thyroid weights were determined after fixation.

Statistics:

Standard deviations were calculated as appropriate. For variables, e.g. body weight, food consumption and organ weights, the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.
The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the nonparametric version of the Williams test. The criterion for statistical significance was p<0.05.
The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.

Reproductive indices:

The following reproductive indices were calculated:
Males:
- Copulation Index (%) = (no. of males with confirmed mating)/(no. of males cohabitated) X 100
- Fertility Index (%) = (no. of males which induced pregnancy)/(no. of males cohabitated) X 100
Females:
- Copulatory Index (%) = (no. of females with confirmed mating)/(no. of females cohabitated) X 100
- Fertility Index (%) = (no. of pregnant females)/(no. of females cohabitated) X 100
- Pre-implantation loss (%) = (no. of corpora lutea - no. of implantations)/ no. of corpora lutea)/ X 100
- Pre-natal loss (%) = (no. of visible implantations – live litter size at birth)/(no. of visible implantations) X 100

Males and females:
- Precoital Interval = The number of nights paired prior to the detection of mating

Offspring viability indices:

- Pre-natal loss (%) = (no. of visible implantations – live litter size at birth)/(no. of visible implantations) X 100
- Pup loss on Day 0 post-partum (%) = (total litter size – live litter size)/(total litter size) X 100
- Post-natal loss on Day 13 post-partum (before culling) (%) = (live litter size at birth – live litter size at Day 4, before culling)/(live litter size at birth) X 100
- Post-natal loss on Day 13 post-partum, after culling) (%) = (live litter size on Day 4 (after culling) – liver litter size on Day 13)/(live litter size on Day 4, after culling) X 100
Sex ratios were calculated at birth, on Days 4 and 14 post-partum and were presented as the percentage of males per litter.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The most significant clinical signs observed during the treatment period in males receiving 1000 mg/kg/day were: ataxia, decreased activity and hunched posture. Other signs noted were staining at the urogenital region, piloerection and salivation. In all males receiving 250 and 500 mg/kg/day, salivation was generally observed during the treatment period with higher frequency in the mid-dose males.
The most significant clinical signs observed in females receiving 500 and 1000 mg/kg/day were: decreased activity, staining on the perigenital region, ataxia, hunched posture, piloerection and salivation during the pre-mating and mating phases. In addition, high dose females showed piloerection and salivation during the post coitum period. Salivation was still evident during the post partum period.
In females receiving 250 mg/kg/day (Group 2), the clinical sign observed was limited to salivation.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

A total of 3 animals died or were sacrificed during the course of the study: 1 female from the control group sacrificed on the day of parturition for humane reasons, one male from the mid-dose group that died on the day of necropsy immediately after the blood sampling and one female from the high dose group that was found dead on the day of delivery. No signs were seen in the male and the high dose female that could clearly establish the cause of death, while chronic inflammation and haemorrhage were noted in the control female possibly contributing to difficulty in delivery.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight and body weight gain were unaffected by treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No treatment related effects were observed in food consumption.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

effects observed, non-treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At the microscopic examination, treatment-related lesions were noted in the liver of high dose treated males and in the kidneys of high dose treated females as summarised below:

– Liver: minimal to mild centrilobular hepatocellular hypertrophy was seen in four males treated at the high dose. Hepatocyte hypertrophy could be associated with microsomal enzyme induction secondary to the exposure to the test substance and was distributed mainly in centrilobular areas; hepatocellular cytoplasm showed a pale, ground glass appearance and could be considered an adaptative change which correlated with an increased liver mean weight.

– Kidneys: mild to moderate nephropathy of kidneys, characterised by the presence of multi focal foci of tubule basophilia, infiltration by mononuclear inflammatory and hyaline casts, was observed in 2 of 9 high dose females, when compared to the controls. Such renal lesion was also reported in untreated Sprague Dawley rats.

No changes in spermatogenic cycles were seen in treated animals when compared to controls.

Histopathological findings: neoplastic:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Oestrous cycle was similar between the treated groups and control animals.

Reproductive performance:

no effects observed

Description (incidence and severity):

Reproductive parameters, litter data, pre- and post-natal loss and litter data were similar between the treated groups and control animals.
The reducted percentage in the fertility index was considered incidental considering that no alteration in the oestous cycle nor in the uterus were noted in females and no alterations were observed when evaluating the spermatogenic cycle in males of high dose.
Implantation, pre- and post-natal loss and gestation length were unaffected by treatment.
Litter data and sex ratios did not show any treatment-related effects.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

organ weights and organ / body weight ratios

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

Clinical signs in pups, did not reveal any treatment-related effects.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

not specified

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

Changes noted in anogenital distance were considered not related to treatment, since it was not associated with an adverse effect (i.e.: feminisation).

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No nipples were found in male pups at Day 13 of lactation.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Thyroid weight in pups did not show relevant differences between the treated groups and control.

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

no effects observed

Description (incidence and severity):

No relevant findings in thyroid hormones were recorded in pups on Day 14 post partum.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

ca. 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

body weight and weight gain

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

developmental immunotoxicity

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

Under the study conditions, the NOAEL (No Observed Adverse Effect Level) for reproductive and developmental toxicity was considered to be 1000 mg/kg/day.

Executive summary:

A reproductive/developmental toxicity screening test was performed in rats according to OECD Guideline 421, in compliance with GLP. The test substance was given to Sprague Dawley SD rats (10/sex/group) by oral administration (gavage) at dosages of 0 (control), 250, 500 and 1000 mg/kg bw/day. The vehicle was 0.5% aqueous solution of carboxymethylcellulose. The purpose of the study was to provide information on systemic effects in rats of both sexes, as well as any effects of the test substance on male and female reproductive performance, such as gonadal function, mating behaviour, conception, development of conceptuses, parturition and lactation of the offspring. Males were treated for 2 weeks prior to pairing and during pairing with females until the day before necropsy for a total duration of at least 4 weeks. Females were treated for 2 weeks prior to pairing and thereafter during pairing, gestation and lactation periods until Day 13. The non-pregnant female and the female sacrificed for humane reasons were dosed up to the day before necropsy. No treatment-related mortality was observed. Treatment-related clinical signs were noted in males and females of all dose groups. Bodyweight, body weight gain and food consumption were unaffected by the treatment. Oestrus cycle, reproductive parameters, litter data, pre- and post-natal loss and litter data were similar between the treated groups and control animals. Clinical signs in pups, external and internal examination and thyroid weight did not reveal any treatment-related effects. No nipples were found in male pups at Day 13 of lactation. Changes noted in the anogenital distance were considered not related to treatment. No relevant findings were seen in thyroid hormones measured in pups on Day 14 post-partum. At 1000 mg/kg bw/day, thyroid-stimulating hormone changes were observed in some males but were considered incidental since no other related changes were recorded. At 1000 mg/kg bw/day, changes in liver, kidney and adrenal weights, absolute and relative were noted in males. At macroscopic observation, the only relevant change observed following gross pathology examination was swollen liver in high dose treated males. At the microscopic examination, treatment-related lesions were noted in the liver of high dose treated males and in the kidneys of high dose treated females. The changes noted in the liver could be considered an adaptative change which correlated with an increased liver mean weight. The renal lesion observed was also reported in untreated Sprague Dawley rats. Under the study conditions, the NOAEL (No Observed Adverse Effect Level) for reproductive and developmental toxicity was considered to be 1000 mg/kg/day (Salvador, 2021).