Registration Dossier

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Diss Factsheets

Toxicological information

Toxicity to reproduction

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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 December 23, 2020 to March 4, 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 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
The formulation analysis at the end of treatment was done in Week 4, even if males were sacrificed on Week 5. The weight of females at arrival ranged between 198-213 grams. These deviations had no impact on the study.
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Amides, C8-18 (even numbered) and C18-unsatd., N-(hydroxyethyl)
EC Number:
931-330-1
Cas Number:
69227-24-3
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-(hydroxyethyl)
Test material form:
solid
Remarks:
Off-white to light yellow solid

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 for this species and strain at ERBC.
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 26 November 2020, the weight range for each sex was determined (200-218 g for males, 198-213 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 34 days 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. Nesting material was 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 and one female showing damaged eye (animal pretest no. 21) 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. 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. No replacements occurred after the first dose was administered.

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 100 mg/mL), according to stability data from ERBC study No. A4105. 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:
Mating was monogamous (one male to one female). Each female was placed with a single male, randomly selected, from the same group. Vaginal smears were taken 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. Animal no.X1620035 was separated after 14 days of cohabitation since mating was not detected.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical method was validated in ERBC Study no. A4105 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. A4105, 28-hour stability at room temperature and 8-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. A4105 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 4 (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 33/35 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, for a total of 50 to 63 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
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 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 the morning and in the afternoon each working day. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day.

- 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. From Day 1 to Day 3, clinical signs were done as follows: – 0.5-1 hour post-dose; – 2-2.5 hours post-dose; – 4-4.5 hours post-dose. From Day 4, observations were done 0.5-1 hour post-dose. From Day 9, due to the presence of post-dose reaction (salivation) the observations were done within 15 minutes from treatment. 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), all animals were 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 5 for males and on Day 12 post-partum for females with viable litters), all animals were selected from each group and the motor activity 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 gestation 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 during the pre-mating period starting from Day 1 of dosing. Individual food consumption for the 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:
Mating was monogamous (one male to one female). Each female was placed with a single male, randomly selected, from the same group. Vaginal smears were taken 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. Animal no.X1620035 was separated after 14 days of cohabitation since mating was not detected.

- Parturition check and duration of gestation
A parturition check was performed from Day 20 to Day 25 post coitum. 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.

- 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 using a computer generated random order). 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 and Aution Eleven AE4020, according to internal 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 retro-orbital sinus. 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) Vaginal smears were also taken from all females, before despatch to necropsy and the oestrous cycle phase recorded. Vaginal smears were also taken from all females, before despatch to necropsy and the oestrous cycle phase recorded.
Sperm parameters (parental animals):
The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed.
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:
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. Observation were performed once daily for all litters from Day 0 post partum. Pups found dead at birth were examined at necropsy (external and internal examination).
Pups killed or dying during the lactation period were weighed before the despatch to necropsy. 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 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, blood samples per sex (when possible) were taken for hormone determination/

-Anogenital distance (AGD):
The AGD of each pup was measured with a caliper on Day 1 post partum (as indicated in SOP No. ANI/366). The AGD was normalized 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.

- Blood collection and thyroid hormone determination (T4 and TSH) (delegated phase)
On Day 4 and 14 post partum, as part of the necropsy procedure, blood samples of approximately 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).

- Organ weights:
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 morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed. 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 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. All live pups sacrificed on Day 14 post-partum were examined for external abnormalities and sex confirmation by gonads inspection. 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 were weighed from one male and one female pup selected for blood collection of hormone determination and preserved in 10% neutral buffered formalin. The thyroid weight was determined after fixation.
Statistics:
Standard deviations were calculated as appropriate. For variables such as body weight, food consumption, clinical pathology parameters and organ weight, 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. Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test if n was more than 5. 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.
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, treatment-related
Description (incidence and severity):
Salivation was the only treatment-related clinical signs recorded in animals treated at 300 and 1000 mg/kg/day during the study, affecting both genders. Animals of the control group and those treated at 100 mg/kg/day did not show any sign during the whole treatment period. The number of animals affected by salivation as well as the duration of the sign increased with increasing dose, affecting all animals treated at 1000 mg/kg/day. This clinical sign, however, was considered not adverse since it was recorded only after each administration and not during the afternoon observations, at the end of each day. Furthermore, salivation was never recorded during the weekly detailed clinical signs, thus confirming its transitory nature. The subcutaneous mass in mammary area noted in one female (no. X1620061) receiving 1000 mg/kg/day was confirmed at macroscopic observations.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No mortality occurred throughout the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Group mean body weight or body weight gain was comparable between control and all tested dose levels, both on males and females.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No changes of toxicological significance were observed in food consumption during the study in either males or females. The slight but statistically significant decrease observed in females dosed at 1000 mg/kg/day at Day 20 post coitum, was considered as sporadic and of no toxicological significance, since it was recorded only on a single occasion.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No treatment-related changes were recorded. Similarly, for coagulation no treatment-related changes were recorded.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment related changes were recorded. Some females dosed at 1000 mg/kg/day showed increases of alkaline phosphatase (78%), alanine aminotransferase (42%) and aspartate aminotransferase (62%) and a decrease of triglycerides (41%). Males of the same group showed an increase of alanine aminotransferase (29%). The above changes were considered to be within the range of expected biological variation and therefore considered to be incidental.
Endocrine findings:
no effects observed
Description (incidence and severity):
No changes of toxicological relevance were observed.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes were observed. The statistically significant increase of diuresis recorded in males dosed at 1000 mg/kg/day (54%) was considered to be incidental due to the absence of other related changes.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test substance.
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.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test substance.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
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 3/4 cycles (mean value). The number of copulatory plugs (mean value) found in the cage were 3/4 in all groups. Animals, both in control and treated groups, mated after 3/4 days (mean pre-coital interval) of cohabitation. Copulatory and fertility indices for males and females were comparable between control and treated animals.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
- Fate of female: For one female treated at 100 mg/kg/day (X1620035), the identification of successful mating was not detected. The animal was separated from the male at the end of the 14 days of mating and it was found with viable litter in the cage after 20 days from the separation. One dam dosed at 300 mg/kg/day showed unilateral implantation on the right horn.

- Implantation, pre-birth loss data and gestation lenght of females: Gestation periods were similar in treated groups and controls. All dams gave birth between Days 22 and 23 post coitum. Corpora lutea, implantations and pre-implantation loss, live litter size and pre-natal loss (percentage) did not show dose-related or treatment-related differences.

- Litter data at birth, on Day 1, Day 4 and on Day 13 post partum of females and sex ratio of pups:
No significant differences were observed in litter data at birth, on Days 1, 4 and 13 post partum of treated groups, when compared to the control group. Sex ratios at birth and on Days 4 and 14 post partum did not show differences between groups.

Details on results (P0)

No mortality occurred during the study. Salivation was the only treatment-related clinical sign recorded in some males and females treated at 300 mg/kg/day and in all animals dosed at 1000 mg/kg/day, during the whole study. However, this clinical sign was considered not treatment-related since it was only recorded soon after each administration and not in the afternoon observations. This was also supported by the absence of salivation during the weekly detailed clinical observations, thus confirming its transitory nature. 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 item. 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 item.
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. All pregnant females had a comparable length of gestation period.
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:
1 000 mg/kg bw/day
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
urinalysis
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
reproductive function (oestrous cycle)
reproductive performance

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):
The main and not related to treatment clinical signs noted in control and/or treated pups were: cold to touch, small appearance and (apparently) no food intake. In addition, found dead and/or missing pups were also observed both in control and treated groups, with similar incidence.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
No mortality occurred throughout the study.
Body weight and weight changes:
not examined
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:
no effects observed
Description (incidence and severity):
No changes of toxicological relevance were observed.
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
The slight statistically significant increase noted in the anogenital distance of male pups of the low and high dose group with respect to the control group, was not considered relevant since it was with a positive trend (more “masculine”) and not associated with adverse effects (i.e.: feminisation) and were within the historical control data. No differences in the anogenital distance, performed on Day 1 post-partum, were seen between control and treated groups in female pups. No nipples were observed in male pups on Day 14 post-partum. Data were not tabulated but will be archived with all raw data.
Nipple retention in male pups:
effects observed, non-treatment-related
Description (incidence and severity):
The main and not related to treatment clinical signs noted in control and/or treated pups were: cold to touch, small appearance and (apparently) no food intake. In addition, found dead and/or missing pups were also observed both in control and treated groups, with similar incidence.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No differences were noted in thyroid weight between pups of the control and treated groups
Gross pathological findings:
not examined
Histopathological findings:
no effects observed
Description (incidence and severity):
- Decedent pups: No abnormalities or autolysed abdominal organs were observed in the decedent pups, both in control and treated groups, without any correlation with the dosage.
- Pups sacrificed on Days 4 (culled pups) and 14 post-partum: No relevant abnormalities were recorded in pups sacrificed on Days 4 and 14 post-partum. Only one pup from Dam no. X1620019 (control group) had left hindlimb swollen; however, this sign was not considered to be treatment related.
Other effects:
no effects observed
Description (incidence and severity):
Litter data at birth, on Day 1, Day 4 and on Day 13 post partum of females and sex ratio of pups: No significant differences were observed in litter data at birth, on Days 1, 4 and 13 post partum of treated groups, when compared to the control group. Sex ratios at birth and on Days 4 and 14 post partum did not show differences between groups.

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 during the lactation period. 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 Day 14 post partum.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
histopathology: non-neoplastic
other: see remarks

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 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 1000 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 with the test substance C8-18 and C18-unsatd. MEA in accordance with 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 1000 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 a 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 to 35 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 50 to 63 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 1000 mg/kg/day, during the study. However, this clinical sign was considered not treatment-related since it was only recorded soon after each administration and not in the afternoon observations. This was also supported by the absence of salivation during the weekly detailed clinical observations, thus confirming its transitory nature. 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 item. 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 item. 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 coitumLitter data and sex ratios were unaffected by treatment.No test item-related effects were seen in anogenital distance in pups of the treated groups, compared to controls. No nipples were found in male pups. Pre-weaning clinical signs of pups were comparable between treated and control groups.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 1000 mg/kg/day, as well as for growth and development of F1 pups until Day 14 post-partum (De Marzi, 2021).