Amides, C8-18(even-numbered) and C18(unsatd.), N-(2-hydroxypropyl)

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

Combined repeated dose toxicity study with the reproduction / developmental toxicity screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From February 04, 2021 to September 08, 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted on 29 July 2016

Deviations:

yes

Remarks:

Female animals were supplied in the weight range of 197-222 g and not 175-200 g, as indicated in the Study Protocol, while males were in the range of 211-246 g instead of 200-225 g. These deviations had no impact on 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-(2-hydroxypropyl)

EC Number:

931-596-9

Cas Number:

1335203-30-9

Molecular formula:

No molecular formula available for this UVCB.

IUPAC Name:

Amides, C8-18(even-numbered) and C18(unsatd.), N-(2-hydroxypropyl)

Test material form:

solid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Hsd: Sprague Dawley SD rats

Details on species / strain selection:

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

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Animal supply and acclimatization:
A total of 102 Hsd: Sprague Dawley SD rats (45 males and 57 virgin females), 7 to 8 weeks old and weighing 200 to 225 g for males and 175 to 200 g for females, were ordered from and supplied by Charles River Italia S.p.A., Calco (Lecco), Italy. After arrival, on 28 January 2021, the weight range for each sex was determined (211-246 g for males, 197-222 g for females) and the animals were temporarily identified within the cage by means of a coloured mark on the tail. A health check was then performed by a veterinarian. An acclimatisation period of approximately 4 weeks was allowed before the start of treatment, during which time 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 were set to maintain temperature and relative humidity at 22°C±2°C and 55%±15%, respectively; actual conditions were monitored, recorded and the records retained. 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 mating, animals were housed up to 5 of one sex to a cage, in polysulfone solid bottomed cages measuring 59.5×38×20 cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese). Nesting material was provided inside suitable bedding bags and changed at least twice a week. 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 (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese). 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). Nesting material was provided inside suitable bedding bags. In addition, suitable nesting material (Scobis 0 Mucedola) was provided as necessary and changed at least 2 times a week. Drinking water was supplied ad libitum to each cage via water bottles, except in the case of urinalysis investigations. A commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019 Settimo Milanese (MI), Italy) was offered ad libitum throughout the study. There was no information available to indicate that any non-nutrient substance likely to influence the effect of the test item was present in the drinking water or the diet. Records of analyses of water and diet are kept on file at ERBC. Dated and signed records of activities relating to the day to day running and maintenance of the study in the animal house were recorded.

- Allocation to groups:
On the day of allocation 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 the main groups. The rats were allocated to the groups by computerised stratified randomisation to give approximately equal initial group mean body weights. At allocation to the study, the body weight variation of animals did not exceed 20% of the mean weight of each sex. Individuals were uniquely identified within the study by sex, tattoo on the hind feet and ear notch and housed five per sex per cage. The cages were identified by a label and recording the study number, animal numbers and details of treatment. The arrangement of cages in batteries was such that cages from each group were distributed to minimise possible environmental effects and or contamination.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% CMC

Details on exposure:

The required amount of test substance was suspended in the vehicle. The formulations were prepared weekly or daily (concentrations of 10, 30 and 75 mg/mL), according to stability data from ERBC study No. A4126. Concentrations were calculated and expressed in terms of test substance as supplied. The test substance was administered orally by gavage at a dose volume of 10 mL/kg body weight. Control animals 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.

Details on mating procedure:

Pairing was monogamous (one male to one female) with the exception of 2 males (X1680050 and X1680066) which replaced 2 males died on Days 8 and 13 of pre-mating phase (nos. X1680048 and X1680078, respectively).
A vaginal smear was taken from all females from the day after the start of pairing until positive identification of copulation (sperm identification, vaginal plug in situ or copulation plugs found in the cage tray). The female was paired with the same male until positive identification occurred or 14 days had elapsed.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical method was validated in ERBC Study no. A4126 in the range from 10 to 100 mg/mL. Linearity, accuracy and precision were within the limits stated in ERBC validation protocol (r > 0.99; accuracy 80-120%; precision CV < 10%). In ERBC Study no. A4126, 28-hour stability at room temperature and 15-day stability at 2-8°C were verified in the range from 10 to 100 mg/mL. According to ERBC SOPs, suspensions are considered to be stable if concentration and homogeneity, after the defined period of storage, are still acceptable (80%-120% for concentration and CV < 10% for homogeneity). The proposed preparation procedure for the test substance was checked in the range from 10 to 100 mg/mL by chemical analysis (concentration and homogeneity) in ERBC Study no. A4126 to confirm that the method was suitable. Final results for all levels were within the acceptability limits stated in ERBC SOPs for concentration (80-120%) and homogeneity (CV < 10%). Samples of the preparations prepared on Weeks 1 and 5 (last week with males and females) were analysed to check the homogeneity and concentration. Results of the analyses were within the acceptability limits stated in ERBC SOPs for suspensions (80-120% for concentration and CV < 10% for homogeneity). Chemical analysis was carried out by the Analytical Chemistry Department. The software used for this activity was Empower® 2 Build No. 2154.

Duration of treatment / exposure:

- Males: Animals were dosed once a day, 7 days a week, for 14 days prior to pairing, through the pairing period and thereafter until the day before necropsy, for a total of 33/34 days. Dose volumes were adjusted once per week for each animal according to the last recorded body weight.

- Females: Animals were dosed once a day, 7 days a week, for 14 days prior to pairing and thereafter during pairing, post coitum and post-partum periods until Day 13 post-partum, or the day before sacrifice (for a total of 38 to 65 days). Dose volumes were adjusted once per week for each animal according to the last recorded body weight up to mating. During the gestation and lactation periods, dose volumes were calculated according to the last recorded body weight.

Frequency of treatment:

Once daily, 7 days/week

Details on study schedule:

Males: Animals were dosed once a day, 7 days a week, for 14 days prior to pairing, through the pairing period and thereafter until the day before necropsy or the day before sacrifice, up to a total of 33/34 days for surviving animals. Dose volumes were adjusted once per week for each animal according to the last recorded body weight.

Females: Animals were dosed once a day, 7 days a week, for 14 days prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 13 post partum or the day before sacrifice (for a total of 38 to 65 days). Dose volumes were adjusted once per week for each animal according to the last recorded body weight up to mating. During the gestation and lactation periods, dose volumes were calculated according to the last recorded body weight.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

750 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 rats per sex per dose

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

- Mortality:
Throughout the study, 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. Severely debilitated animals were observed carefully. One animal showing difficulty in parturition was sacrificed for humane reasons. A complete necropsy was performed in all cases.

- Clinical signs:
Once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs was recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions. Observations were performed at the same time interval each day (approximately 5 - 10 minutes and 1.30 - 2 hours post-dose), the interval was selected taking into consideration the presence of post-dose reactions. All observations were recorded for individual animals.

- Clinical Observations (Functional Observation Battery Tests):
Once before commencement of treatment and once a week thereafter, each animal was given a detailed clinical examination (ERBC SOP no. ANI/344). Each animal was removed from the home cage and observed in an open arena. The tests included observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern). Changes in fur, skin, eyes, mucous membranes, occurrences of secretions and excretions were also recorded. All observations were recorded for individual animals.

- Grip strength and sensory reactivity to stimuli:
Once during the study, towards the end of treatment (during Week 5 for males and Day 12 post partum for females with viable litters, where possible), 5 out of 10 males and 5 out of 10 females were randomly selected from each group for evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli) and for assessment of grip strength (ERBC SOP no. ANI/344). Measurements were performed using a computer-generated random order.

- Motor activity assessment (MA):
Once during the study, towards the end of treatment (during Week 4 for males and on Day 12 post partum for females with viable litters where possible), 5 males and 5 females were randomly selected from each group and the motor activity (MA) were measured (for approximately 5 minutes) by an automated activity recording device (ERBC SOP no. ANI/346). Measurements were performed using a computer-generated random order.

- Body weight
Parental animals: 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 weighed on Days 1, 4, 7, 13 post partum and just before to necropsy.

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

- Mating:
Pairing was monogamous (one male to one female) with the exception of 2 males (X1680050 and X1680066) which replaced 2 males died on Days 8 and 13 of pre-mating phase (nos. X1680048 and X1680078, respectively).
A vaginal smear was taken from all females from the day after the start of pairing until positive identification of copulation (sperm identification, vaginal plug in situ or copulation plugs found in the cage tray). The female was paired with the same male until positive identification occurred or 14 days had elapsed.

- Parturition check and duration of gestation
A parturition check was performed from Day 20 to Day 25 post coitum. Females which did not gave birth after 25 days of post coitum period were sacrificed shortly after. Gestation length was calculated as the time between the day of successful mating (Day 0 post coitum) and the day of birth. The day of birth (when parturition was complete) was defined as Day 0 post partum.

-Blood collection for metabolomics analysis:
At the end of treatment period, 1 mL was taken from all surviving males and females, under conditions of food deprivation. Blood samples were collected (from the retro-orbital sinus in the males, from the abdominal vena cava in the females) and immediately transferred with a 1 mL pipette tip into an Eppendorf tube containing 10 µL of 10% Titriplex III solution. The tube was immediately closed and gently mixed 5-6 times by inverting it. Blood was kept cool in ice water (approximately 4°C) during the collection period of up to 20 minutes. To separate plasma, the blood was centrifuged at 4°C, 20000 g for 2 minutes. Blood cells and plasma were separated; 0.5 mL of the supernatant plasma layer were pipetted with a 1 mL pipette tip into the second labelled Eppendorf tube and covered with a N2 atmosphere. Tubes were closed with the lid which was wrapped with Parafilm-M, and frozen at -80°C within at maximum 30 minutes after centrifugation, then stored at -80°C until despatch for additional investigations (metabolomics analysis) at an external laboratory. Results of the metabolomic analysis, the corresponding data and their interpretation are the responsibility of the Sponsor and are reported separately.

- Clinical pathology investigations
Blood collection was performed for hormone determination (0.8 mL) from all parental animals at termination under condition of food deprivation. Blood samples for haematology, clinical chemistry and coagulation were collected by random selection from 5 males and 5 females (females with viable litters) of each group, under condition of food deprivation. Following haematology and coagulation paramaters were assessed: haematocrit, haemoglobin, red blood cell count, reticulocyte count, mean red blood cell volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, white blood cell count, differential leucocyte count, platelets and prothrobin time. Clinical chemistry parameters assessed corresponds to : alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, gamma-glutamyltransferase, urea, creatinine, glucose, triglycerides, bile acids, total bilirubin, total cholesterol, total protein, albumin, globulin, A/G Ratio, sodium, potassium, calcium, chloride, inorganic phosphorus.

-Urinalysis (Only males randomly selected)
During the last week of treatment, individual overnight urine samples were also collected from the same animals selected for clinical pathology investigations (5 males/group, randomly selected). Before starting urine collection water bottles were removed from each cage and each animal received approximately 10 mL/kg of drinking water by gavage, in order to obtain urine samples suitable for analysis. The measurements performed on urine samples are as follows: appearance, volume (manually recorded), specific gravity, pH, protein, glucose, ketones, bilirubin, urobilinogen, blood.
These parameters were analysed by Menarini Aution Max AX 4280/Aution Eleven AE 4020/Aution Sticks 10EA or ATAGO Refractometer (for Specific gravity), according to in- ternal procedures. The sediment, obtained from centrifugation at approximately 3000 rpm for 10 minutes, was examined microscopically for: epithelial cells, leucocytes, erythrocytes, crystals, spermatozoa and precursors, other abnormal components.

- Blood collection and thyroid hormone determination (T4 and TSH)
Blood collection for hormone determination was performed from all animals at termination.
Males
Blood samples (approximately 0.8 mL) for hormone determination were collected under isoflurane anaesthesia from the abdominal vena cava . The order of collection was equalised between groups.
Females
As a part of the necropsy procedure, blood samples (approximately 0.8 mL) for hormone determination was withdrawn from the abdominal vena cava under isoflurane anaesthesia. The order of collection was equalised between groups.

- Immunoanalysis - Thyroid hormone determination (T4 and TSH)
Samples were assayed to determine the serum levels of Total thyroxine (total T4) and Thyroid stimulating hormone (TSH) by RadioImmunoAssay (RIA).

Oestrous cyclicity (parental animals):

- Before allocation and stock females:
All females ordered for the study were evaluated pre-exposure for oestrous cyclicity and animals that exhibit anomalies in the oestrous cycle were not allocated to the study. Oestrus cycle was monitored by vaginal smears for 2 weeks before allocation. These data were not tabulated in this report but will be archived with the raw data.

- Females allocated to groups: Vaginal smears were taken in the morning from Day 1 of dosing up to positive identification of mating. The vaginal smear data was examined to determine the following: 1. anomalies of the oestrous cycle 2. the pre-coital interval (i.e., the number of nights paired prior to the detection of mating)

-Before despatch to necropsy: Vaginal smears were also taken from all females, before despatch to necropsy and the oestrous cycle phase recorded. No vaginal smears were taken from females sacrificed for humane reasons.

Litter observations:

Litter data at birth, on Day 1, Day 4 and on Day 13 post-partum of females were recorded.

-Pups identification, weight and observations:
On Day 1, 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. Pups killed or dying during the lactation period were weighed before despatch to necropsy. Observation was performed once daily for all litters from Day 0 post partum. After culling, all pups were sacrificed with the dams on Day 14 post partum.

- Culling and pup selection for blood collection (serum hormone determination) at necropsy:
On Day 4 post partum, the size of each litter was adjusted by eliminating extra pups by random selection (by use of a random list generated by the computerised system) to yield, as nearly as possible, four pups per sex per litter. Partial adjustment (for example, 5 males and 3 females) was acceptable. On Day 4 post partum, at least one culled male and one culled female (where possible) were selected for hormone determination. No pups were eliminated when litter size will drop below the culling target (8 pups/litter).

-Anogenital distance (AGD):
The AGD of each pup was measured with a digital caliper on Day 1 post partum. The AGD was normalized to the cube root of body weight collected on Day 1 post partum.

- Nipple count:
On Day 13 post partum, all live male pups were observed for the presence of nipples/areolae. Since no nipples were observed, these data were not reported (data archived with the other raw data of the study).

- Blood collection and thyroid hormone determination (T4 and TSH) (delegated phase)
Pups on Days 4 and 14 post partum: On Day 4 and 14 post partum, as part of the necropsy procedure, blood samples of approx- imately 0.5 mL (by sex) were taken from each litter (1 sample for males and 1 sample for females, when possible). Blood samples were withdrawn under light ether anaesthesia from the heart (intracardiac puncture). The order of collection was equalised between groups.

Postmortem examinations (parental animals):

- 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.
Females: All females were examined also for the following: 1) number of visible implantation sites (pregnant animals) and 2) number of corpora lutea (pregnant animals). Uteri of females with no visible implantations were immersed in a 20% solution of ammonium sulphide to reveal evidence of implantation.

- Organ weights:
Parental animals: From all animals completing the scheduled test period, the following organs were dissected free of fat and weighed: abnormalities, adrenal glands, bone marrow, aecum, clitoral gland, colon, duodenum, epididymides, eyes, femur, heart, ileum, jejunum, kidneys, liver, lung, lymph nodes, mammary gland, nasal cavity, oessophagus, ovaries, parathyroid gland, pituitary gland, penis, prostate gland, rectum, sciatic nerve, seminal vesicles, spinal column, spinal cord, skeletal muscle, splee, stomatch, testes, thymus, thyroid gland, trachea, urinary bladder, uterus, vagina. The ratios of organ weight to body weight were calculated for each animal.

- Tissues fixed and preserved:
Samples of all the tissues (all parental animals) were fixed and preserved in 10% neutral buffered formalin (except eyes, optic nerves and Harderian glands, testes and epididymides which were fixed in modified Davidson’s fluid and preserved in 70% ethyl alcohol).

- Histopathological examination:
After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometer thickness and stained with haematoxylin and eosin. In addition, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS). The tubular stages of the spermatogenic cycle, was conducted 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. Identification of the stages of the spermatogenic cycle was carried out. The examination was restricted to 1) tissues from 5 males and 5 females randomly selected (animals evaluated for clinical pathology) in the control and high dose group killed at term and 2) all abnormalities in all groups.

Postmortem examinations (offspring):

- Necropsy:
All pups found dead in the cage or sacrificed for humane reasons were examined for external and internal abnormalities. All culled pups sacrificed at Day 4 post partum were subjected to an external examination. Sex was determined by internal gonads inspection.
Pups at Day 14 postpatum: All live pups sacrificed on Day 14 post partum were examined for external abnormalities.
Internal examination: Gonads were inspected from all pups in order to confirm the sex previously determined by external examination. All pups with abnormalities were retained in a 10% neutral buffered formalin.
Nipple count retention on Day 14 post-partum: The ventral region of male pups was checked for presence of nipples/areolae.

- Organ weights:
Pups at Day 14 post-partum: Thyroid was weighed from one male and one female pup selected for blood collection of hormone determination and preserved in 10% neutral buffered formalin for possible histopathological examination. The thyroid weight was determined after fixation.

Statistics:

Standard deviations were calculated as appropriate. For continuous variables 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 non- parametric version of the Williams test. The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.

Reproductive indices:

Group mean values were calculated for all parameters. The following reproductive indices were calculated for main groups animals:

- Males
Copulation Index (%) = (no.of males with confirmed mating / no.of males cohabitated) × 100
Fertility Index (%) = (no.of males which induced pregnancy / no.of males cohabitated) × 100

- Females
Copulatory Index (%) = (no.of females with confirmed mating / no.of females cohabitated) × 100
Fertility Index (%) = (no.of pregnant females/ no.of females cohabitated) × 100

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

Offspring viability indices:

Pre-implantation loss was calculated as a percentage from the formula: (no. of corpora lutea − no.of visible implantation / no. of corpora lutea) × 100
Pre-natal loss was calculated as a percentage from the formula: (no.of visible implantations − Live litter size at birth / no.of visible implantations) × 100
Post-natal loss at Day 0 post partum was calculated as a percentage from the formula: (Total litter size − Live litter size / Total litter size) × 100
Post-natal loss at Day 4 post partum (before culling) was calculated as a percentage from the formula: (Live litter size at birth − live litter size at Day 4 (before culling)/ Live litter size at birth) × 100
Post-natal loss at Day 13 post partum (after culling) was calculated as a percentage from the formula: (Live litter size on Day 4 (after culling) − Live litter size on Day 13 / Live litter size on Day 4 (after culling)) × 100
Anogenital distance in pups was presented as normalized to the cube root of body weight collected on Day 1 post partum.
Sex ratios was calculated at birth, on Day 4 and on Day 14 post partum and was presented as the percentage of males per litter.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Salivation was observed in 8 out of 10 males and 9 out of 10 females dosed at 300 and and in all males and females dosed at 750 mg/kg/day with a dose-related frequency (only occasionally in one individual females dosed at 100 mg/kg/day), from the first few days of the pre-mating phase up to termination.
Animals of the control group did not show any sign during the whole treatment period. Piloerection, pallor and swollen neck were also occasionally seen in 1 female dosed at 300 mg/kg/day (animal no. X1680059), while dyspnoea was observed in 1 male dosed at 750 mg/kg/day (animal no. X1680076). Due to the transient occurence and the low incidence, these signs were not considered treatment-related.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One male from Group 3 (dosed at 300 mg/kg/day, no. X1680048) and one male from Group 4 (dosed at 750 mg/kg/day, no. X1680078) were found dead on Days 9 and 13 of the pre- mating phase, respectively. In addition, 1 female from Group 2 (dosed at 100 mg/kg/day, no. X1680033) was sacrificed for humane reasons on Day 22 of the gestation phase. Altogether, these mortalities were not considered treatment-related.
Animal no. X1680048 (300 mg/kg/day) showed salivation and dyspnoea in the days be- fore death. Macroscopically, dark red colour and incomplete collapse of lungs and clear fluid in the thoracic cavity were noted. At microscopic observation, diffuse oedema and haemorrhage of the lungs were observed. Death was considered to be related to misgavage.
Animal no. X1680078 (750 mg/kg/day) showed salivation in the days before death. Macro- scopically, single dark red area of the calvaria and dark red fluid in the cranial and thoracic cavity were observed. At microscopic observation, sub meningeal haemorrhage of the brain, alveolar and interstitial haemorrhage of the lungs, were noted. Death was considered to be related to a traumatic impact of the head following an accidental fall from the cage.
Animal no. X1680033 (100 mg/kg/day), was sacrificed for humane reasons on Day 22 of the gestation phase for difficulty in parturition. Piloerection and pallor were observed prior to sacrifice. No abnormalities were detected at macroscopic observation. At microscopic observation, unilateral cortical hypertrophy was noted. On the basis of the prolonged time without completing the parturition, impaired parturition was considered to be the reason for sacrifice. The gross and microscopic evaluation did not allow to establish the cause of impared parturition. In the absence of a dose-relation and other treatment-related effects, the difficulty in parturition observed in this animal is not considered to be treatment-related.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weight and body weight gain for male and female animals were generally comparable between the treated and control groups, before and during mating, during gestation and post partum periods.
Before pairing, on Day 15, females receiving 750 mg/kg/day showed a decrease in body weight gain (-91%) compared to the control group, while statistically significant body weight losses were seen in the males dosed at 300 mg/kg/day on Day 15 of mating phase. These isolated changes were followed by a regular growth of the animals. Due to their occasional occurrence and/or in the absence of a dose-relation, these changes were not considered treatment-related.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was unaffected by treatment in both gender during the study.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes were recorded.
The statistically significant differences recorded between control and treated animals (mean corpuscular haemoglobin concentration in males, reticulocytes and platelets in females) were not dose-related and/or of minimal severity, therefore they were considered to be incidental.
No changes were recorded for coagulation.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes were recorded.
A statistically significant decrease of total bilirubin (48%) was recorded in males dosed at 750 mg/kg/day. The decrease of bilirubin has no biological/pathological significance, especially in rats where normal values are low (mean historical control data is 0.04 mg/dL) and the low limit of historical control data is 0 mg/dL.

Endocrine findings:

no effects observed

Description (incidence and severity):

Parental and pups on Day 14 post partum: No treatment related changes were observed in thyroid hormone.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Parental males: no changes were obersved.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Observation of animals at removal from the cage and in the open arena (neurotoxicity assessment) did not reveal changes attributable to the test item, compared to controls.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related alterations in motor activity, grip strength and sensory reactivity to stimuli were observed in any treatment group at the examination performed at the end of treatment.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no treatment-related microscopic observations at the end of the treatment period. Any microscopic observations had a comparable incidence in control and treated groups and/or are characteristically seen in untreated rats of the same age and were considered incidental and unrelated to treatment. There were no test item-related microscopic observations in the testis (stage aware evalu- ation on PAS-stained slides).

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Observation of animals at removal from the cage and in the open arena (neurotoxicity assessment) did not reveal changes attributable to the test item, compared to controls.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No treatment-related anomalies were noted in the oestrous cycle and pre-coital interval of the treated females, when compared to controls.
The total number of oestrous cycles observed in all females before pairing (number of non sequential days in which the females were in oestrous) were similar between control and treated groups and was of 3 times (mean value). The number of copulation plugs was similar between control and treated groups.
The copulatory and fertility indices were 90% for controls and 100% for low, mid- and high dose groups (dosed at 100, 300 and 750 mg/kg/day). One male of the high dose group (no. X1680066) which cohabitated with 2 females, mated with the first female only (no. X1680065). The copulatory and fertility indices were 90% for controls and high dose groups and 100% for low and mid-dose groups.
Copulatory and fertility indices were considered to be unaffected by administration of the test substance.
- Males: The copulatory and fertility indices were 90% for controls and 100% for low, mid- and high dose groups (dosed at 100, 300 and 750 mg/kg/day). One male of the high dose group (no. X1680066) which cohabitated with 2 females, mated with the first female only (no. X1680065).
- Females: The copulatory and fertility indices were 90% for controls and high dose groups and 100% for low and mid-dose groups.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

- Fate of females: The number of females with live pups on Day 14 post partum was: 9 in the control and high dose groups, 8 in the low dose, and 10 in the mid-dose treated groups (dosed at 0, 100, 300 and 750 mg/kg/day).

- Implantation, pre-birth loss data and gestation lenght of females: Mean gestation period was comparable between control and treated animals, being of 22/23 days. Corpora lutea, number of implantations sites and live litter size did not show dose-related or treatment-related differences, compared to controls.
Pre implantation loss (percentage) was similar between control and treated groups. Pre- natal loss (percentage) was increased in the 3 treated groups (with no dose-relation) when compared to controls, with no statistical significance. The relationship with the test item was considered to be unlikely, in the absence of a clear dose-relation and in view of the comparison with the reference control data.
No differences which could be considered adverse were observed in total litter size, live litter size, mean pup loss, sex ratio and mean pup weights among the treated dams and the controls at birth and on Days 1, 4 and 13 post partum.

- Litter data at birth, on Day 1, Day 4 and on Day 13 post partum of females and sex ratio of pups: The increase in mean post-natal loss observed in Group 3 females, compared to the control, was attributed to animal no. X1680037, which showed total litter loss on Day 2 post partum. The slight decrease in mean litter weight seen from Day 1 to Day 13 post partum in dams dosed at 750 mg/kg/day when compared to controls was mainly attributed to animal no. X1680075, with 2 pups only. No differences were observed in mean pup weights.

Details on results (P0)

Salivation was observed in 8 out of 10 males and 9 out of 10 females dosed at 300 and and in all males and females dosed at 750 mg/kg/day with a dose-related frequency (only occasionally in one individual females dosed at 100 mg/kg/day), from the first few days of the pre-mating phase up to termination. Animals of the control group did not show any sign during the whole treatment period. Piloerection, pallor and swollen neck were also occasionally seen in 1 female dosed at 300 mg/kg/day (animal no. X1680059), while dyspnoea was observed in 1 male dosed at 750 mg/kg/day (animal no. X1680076). Due to the transient occurence and the low incidence, these signs were not considered treatment-related. Mortality was observed but not treatment-related (attributed to misgavage and accidental trauma, respectively). Neurotoxicity assessment (removal from the home cage and observations in an open arena), motor activity, grip strength and sensory reactivity to stimuli did not reveal changes attributable to the test substance. Body weight and body weight gain of treated animals did not show differences throughout the study when compared to the control group. Body weight and body weight gain of treated animals did not show differences throughout the study when compared to the control group. The food consumption was comparable in all groups. No adverse findings were recorded in clinical pathology investigations (haematology including coagulation parameters and clinical chemistry). Hormone analysis did not show any relation to treatment. Urinalysis in male animals did not reveal changes attributable to the test substance.
Concerning the reproductive parameters, no relevant differences were found in terms of mating performance including the pre-coital interval (number of days paired to sperm positive day), copulatory evidence (positive identification of mating, i.e. the presence of sperm and/or copulation plug in situ or in the cage) or fertility index. Terminal body weight and organs weight did not show relevant differences between control and treated groups. At macroscopic and microscopic observations, no treatment-related changes were seen any in treated males and females, when compared to the controls.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 750 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

haematology

clinical biochemistry

urinalysis

organ weights and organ / body weight ratios

gross pathology

neuropathology

histopathology: non-neoplastic

reproductive function (oestrous cycle)

reproductive performance

other: Endocrine:Thyroid hormone

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related clinical signs were observed in pups during the 14-day observation period. Cold to the touch, apparently no food intake (milk) and small appearance, pallor, hair loss, were in general the mainly clinical signs noted in control and/or treated pups. Some pups were found dead or missing, possibly due to cannibalization after death. Incidence of these signs was comparable between treated and control groups.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

Some pups were found dead or missing, possibly due to cannibalization after death. Incidence of these signs was comparable between treated and control groups.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The slight decrease in mean litter weight seen from Day 1 to Day 13 post partum in dams dosed at 750 mg/kg/day when compared to controls was mainly attributed to animal no. X1680075, with 2 pups only. No differences were observed in mean pup weights.

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):

no effects observed

Description (incidence and severity):

No differences in the anogenital distance (value normalized to the cube root of body weight), performed on Day 1 post partum, were seen between control and treated groups both for male and female pups.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No nipples were observed in male pups.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No differences were observed in the weight of thyroid in treated pups, when compared to controls.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Autolysed abdominal organs were generally observed in pups found dead at check carried out after birth and in pups which died during the lactation period. Autolysis process, normally occurs when pups are found dead some time after death, therefore it is not considered to be treatment-related. No significant findings were seen in pups killed on Days 4 and 14 post partum.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Litter at birth, on Day 1, Day 4 and on Day 13 post partum of females and sex ratio of pups: No differences which could be considered adverse were observed in total litter size, live litter size, mean pup loss, sex ratio and mean pup weights among the treated dams and the controls at birth and on Days 1, 4 and 13 post partum. The increase in mean post-natal loss observed in Group 3 females, compared to the control, was attributed to animal no. X1680037, which showed total litter loss on Day 2 post partum. The slight decrease in mean litter weight seen from Day 1 to Day 13 post partum in dams dosed at 750 mg/kg/day when compared to controls was mainly attributed to animal no. X1680075, with 2 pups only. No differences were observed in mean pup weights.

Necropsy: Autolysed abdominal organs were generally observed in pups found dead at check carried out after birth and in pups which died during the lactation period. Autolysis process, normally occurs when pups are found dead some time after death, therefore it is not considered to be treatment-related. No significant findings were seen in pups killed on Days 4 and 14 post partum.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

Litter data and sex ratios were unaffected by treatment. Similar clinical signs were recorded in pups of the control and treated groups but these signs were non treatment related. No differences of toxicological relevance were seen between the control and treated pups in anogenital distance. Necropsy findings in decedent pups and in pups sacrificed on Days 4 and 14 post partum did not reveal any treatment-related effect, as well as in thyroid weight of pups sacrificed on Days 4 and 14 post partum.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

ca. 750 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

organ weights and organ / body weight ratios

gross pathology

other: Anogenital distance, Nipple count, sex ratios

Target system / organ toxicity (F1)

Key result

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 for general toxicity was considered to be 750 mg/kg/day for males and females. No effects of the test substance on male and female reproductive performance, such as gonadal function, mating behaviour, conception, parturition and lactation of the offspring until Day 14 post-partum were observed at any of the dose levels investigated. Therefore, the NOAEL for fertility and reproduction parameters of parental males and females was considered to be 750 mg/kg/day, as well as for growth and development of F1 pups until Day 14 post-partum.

Executive summary:

A combined repeated dose toxicity study with the reproduction / developmental toxicity screening was conducted in rats according to OECD Guideline 422, in compliance with GLP. Four groups of ten Sprague Dawley rats (males and females) were exposed by oral gavage to increasing concentrations of the test substance (0, 100, 300 and 750 mg/kg/day). All doses were administered at a constant volume of 10 mL/kg body weight. 0.5% aqueous solution of carboxymethylcellulose was used as vehicle. According to the study design, males were treated for 14 days prior to pairing and during pairing with females until the day before necropsy, for a total of 33/34 days. Females were treated for 14 days prior to pairing, during pairing and throughout the gestation and lactation periods until Day 13 post-partum, for a total of 38 to 65 days. The following investigations were performed: body weight, clinical signs (including neurotoxicity assessment, motor activity and sensory reactivity to stimuli), food consumption, clinical pathology investigations (haematology and clinical chemistry in five randomly selected animals/sex/group), macroscopic observations, organ weights. In addition, oestrous cycle evaluation of parental females (2 weeks before dosing, during pre-mating and mating phases, prior to necropsy), mating performance, thyroid hormone measurements and litter data were performed. For F1 pups, clinical signs, anogenital distance, external and/or internal examinations were recorded along with thyroid hormone levels in one randomly selected pup/sex/group at Day 14 post-partum. Routine histopathological examination was performed in control and high dose groups (five randomly selected animals/sex/group), which included identification of the stages of the spermatogenic cycle in five males. No mortality occurred throughout the study. Salivation was the only treatment-related clinical sign recorded in males and females treated at 300 and 750 mg/kg/day, during the study. However, this clinical sign was not considered to be adverse. Piloerection, pallor, swollen neck and dyspnoea were also occasionally noted in single female and male rat dosed at 300 and 750 mg/kg/day respectively. Due to the transient occurence and the low incidence, these signs were not considered treatment-related. Neurotoxicity assessment (removal from the home cage and observations in an open arena), motor activity, grip strength and sensory reactivity to stimuli did not reveal changes attributable to the test substance. Body weight and body weight gain of treated animals did not show differences throughout the study when compared to the control group. The food consumption was comparable in all groups. No adverse findings were recorded in clinical pathology investigations (haematology including coagulation parameters and clinical chemistry). Hormone analysis did not show any relation to treatment. Urinalysis in male animals did not reveal changes attributable to the test substance. Oestrous cycle, pre-coital intervals, copulatory index and fertility index did not show any differences that were considered to be related to treatment. No differences were observed in term of implantation, pre-birth loss data or gestation length of females between treated and control groups. All pregnant dams gave birth between Days 22 and 23 post coitum. Litter data and sex ratios were unaffected by treatment. No test substance-related effects were seen in anogenital distance in pups of the treated groups, compared to controls. No nipples were found in male pups. No differences were noted in thyroid weight between pups of the control and treated groups. No treatment-related findings were noted in pups which died or were sacrificed on Days 4 (culled pups) and 14 post partum. Under the study conditions, the NOAEL for general toxicity was considered to be 1000 mg/kg/day for males and females. No effects of the test substance on male and female reproductive performance, such as gonadal function, mating behaviour, conception, parturition and lactation of the offspring until Day 14 post-partum were observed at any of the dose levels investigated. Therefore, the NOAEL for fertility and reproduction parameters of parental males and females was considered to be 750 mg/kg/day, as well as for growth and development of F1 pups until Day 14 post-partum (Longobardi, 2022).