Amides, C12-18(even-numbered) and C18(unsatd.), N-hydroxyethyl

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

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

Type of information:

read-across based on grouping of substances (category approach)

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

Justification for type of information:

Refer to section 13 of IUCLID for details on the category justification.

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

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.

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

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

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.

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: 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.

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.

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

Remarks on result:

other: no adverse effects observed

Key result

Critical effects observed:

no

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.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

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.

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:

Remarks on result:

other: no adverse effects observed

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

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 read across 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 coitum. Litter 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).

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A combined repeated dose toxicity study with the reproduction / developmental toxicity screening was conducted with the read across 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 coitum. Litter 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).

A combined repeated dose toxicity study with the reproduction / developmental toxicity screening was conducted in rats with the read across substance C16-18 MEA 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 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 33/34 days. Females were treated for 14 days prior to pairing, during pairing and throughout the gestation and lactation periods until Day 13 post-partum, up to at least 51 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, coagulation 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 treatment related mortality occurred throughout the study. Salivation was the only treatment-related clinical sign recorded in males and females treated at 1000 mg/kg/day, during the study. However, this clinical sign did not reveal any treatment related changes. 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. 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 (Liberati, 2022).  

Also, after discussion with ECHA in the frame of a Dossier Improvement Action Plan (DIAP), a testing proposal is submitted for the conduct of an extended one generation reproductive toxicity study (EOGRTS) according to OECD Guideline 443 with the read across substance C8-18 and C18-unsatd. DEA to further support the read across approach proposed for the FAA category members. 

 

Additional considerations 

Based on an in-depth analysis of the available information (see read-across justification in Section 13 of the IUCLID dataset), it is the hypothesis that a read-across within and across MEA, DEA and MIPA derived alkanolamides is scientifically plausible and justified. While there is at present no evidence for putting the read-across hypothesis in question, some limitations, predominantly related to the quality of individual endpoint studies (including quality of the test substance characterisations) and existing higher tier endpoint data gaps, are recognized. Accordingly, additional physico-chemical and toxicology data were generated in Tier 1 of a tiered testing programme to strengthen the toxicokinetic and toxicological link within and across the members of the DEA, MEA, and MIPA subcategories.

In Tier 1, a series of bridging studies according to OECD TG 421 and 422 were conducted with a representative short- and a long-chain substance of each subcategory (i.e., DEA, MEA, and MIPA). Additionally, taking advantage of the bridging studies samples, metabolomics analyses were conducted to enhance the quality and quantity of data from a biological perspective.

Overall, the results of the Tier 1 testing confirmed and supported the hypothesis of a similar toxicological profile within and across the different sub-categories. All investigated substances displayed in line with existing data a similar systemic toxicity profile with no observed repeated dose toxicity at the highest tested dose (i.e., NOAELs ≥ 700 mg/kg/day) and absence of reproductive or developmental toxicity. The absence of significant metabolome changes is in line with the Tier 1 in vivofindings, and thereby further confirming the read-across hypothesis that there is no significant difference in terms of type and strength of effects within and across the FAA subcategories.

In the present dossier update, proposed Tier 2 studies have been included with the aim to generate a complete set of higher toxicology data for a selected >1000 tpa substance (i.e., OECD TG 408/443/414 (rats)/414 (2nd species). The testing in Tier 2 will be conducted with C8-18 and C18-unsatd. DEA, the substance that is generally perceived to be the most critical from a toxicological point of view due to the proposed hazard classification of DEA. Additionally, a few minor non-significant metabolomic changes were noted for the investigated DEA-FAA substance (i.e., C16-18 and C18-unsatd. DEA), suggesting some type of biological activity, possibly explaining some findings in the 1000 mg/kg bw/day dose group in the dose-range finding study. These observations support the selection and recommendation to investigate a DEA-FAA substance as a 'worst case' for the FAA category in Tier 2.

The strategy and status overview are detailed in the document entitled‘ECHA-DIAP - FAA testing strategy summary status overview – Oct 22’, attached in Section 13 of the IUCLID dataset.

Effects on developmental toxicity

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

Not available

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

Refer to section 13 of IUCLID for details on the category justification.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Official Journal of European Community L 133, May 30, 1988; 87/302/EEC

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Official Journal of European Community L 180, March 01, 1991; 91/325/EEC

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Wiga D-Sulzfeld
- Age at study initiation: 8-10 wk
- Weight at study initiation: 209 g (mean)
- Housing: Single animal in Makrolon Type M3 cage (Ebeco) with standard softwood bedding
- Diet: Pelleted Altromin Maintenance Diet 1324, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 5 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 43-66
- Air changes (per h): 10-15
- Photoperiod (h dark/h light): 12/12

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test material was suspended in Arachidis oil, DAB 9 such that the required dose per kg body weight was contained in 5 mL.

VEHICLE
Concentration in vehicle: 0, 20, 60 and 200 mg/mL

Analytical verification of doses or concentrations:

no

Details on mating procedure:

Impregnation procedure: Purchased timed pregnant

Duration of treatment / exposure:

From Day 6 up to Day 15 post coitum

Frequency of treatment:

Once daily

Duration of test:

20 d

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:

30

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the results of toxicological examinations done before (Report No. 486 = TBD 830034, June 27, 1983) (details not reported)

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

BODY WEIGHT: Yes
- Time schedule for examination: On Day 0, 6, 16 and 20 post coitum

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: All maternal organs, with emphasis on the uterus and uterine contents

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Position of fetus in the uterus

Fetal examinations:

External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [approximately half per litter]
- Skeletal examinations: Yes: [approximately half per litter]
- Head examinations: Yes: [approximately half per litter]

(See Table 1 for exact number of fetuses examined)

Statistics:

The following statistical methods were used:
If the variables could be assumed to follow a normal distribution, the Dunnett-Test, based on a pooled variance, was applied for the comparison between the treated groups and the control group.
The Steel-Test was applied when the data could not be assumed to follow a normal distribution.
Fisher's Exact test for 2x2 tables was applied if the variables could be dichotomized without loss of information (Bonferroni-Holm-corrected).

Indices:

- Pre-implantation loss (%) = [(Number of corpora lutea - number of implantations)/number of corpora lutea] X 100
- Post-implantation loss (%) = [(Number of implantations - number of live fetuses)/number of implantations] X 100
- Sex ratio (%) = [(number of males/females)/number of fetuses] X 100

Historical control data:

None

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation and propulsion of the head in all dose groups. Additionally, the highest dose group showed a severe salivation. These symptoms were noted variable in the individual groups during the application period.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortality at any dose level.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No treatment-related effects on body weight gain were observed in the dams.

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

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Placenta and uterus weight: No significant differences between the control and the treatment groups.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic changes were observed in the survived dams except for one dam at 100 mg/kg/d bw, which showed greenish-brownish fluid in the uterine horn.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

None.

Number of abortions:

not examined

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Pre-implantation loss was not affected by the treatment. The post-implantation loss and total embryonic deaths were significantly increased in all treatment groups. However, these findings were considered to be incidental (non-treatment-related) because the values in the 100 mg/kg/d bw group were significantly greater than other two higher dose groups and in each group there was one single female with a high incidence of embryonic death.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

not examined

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

Apart from the control (1 dead foetus) and the 100 mg/kg bw/day groups (7 dead foetuses), all females had viable foetuses. The data for post-implantation loss, embryonic deaths and total foetuses showed some deviations, which were considered to be non-treatment-related.

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

not examined

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

gross pathology

mortality

organ weights and organ / body weight ratios

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The weights of live fetuses exhibited no significant differences on a litter and individual basis.

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

The data for post-implantation loss, embryonic deaths and total foetuses showed some deviations, which were considered to be non-treatment-related.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex ratio of the fetuses was not affected by the treatment.

Changes in litter size and weights:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

No macroscopic findings were observed at external examination of fetuses which were considered to be an effect of the treatment. 1 dead fetus in control and 7 dead fetuses (4 out of 7 partly mummified) in 100 mg/kg/d bw group were recorded. One fetus showed a stump tail at 300 mg/kg/d bw and paleness was observed in one fetus at 1000 mg/kg/d bw. These singular findings are normal observations in the animal strain used.

Skeletal malformations:

no effects observed

Description (incidence and severity):

(i) Retardations: No significant finding at 100 mg/kg/d bw. Two sternebrae were non-ossified in 19 and 29 fetuses (statistically significant) at 300 and 1000 mg/kg/d bw, respectively. Statistically significant increase in the number of fetuses with incomplete ossification of skull bones (17 fetuses) and decrease in the number of fetuses with incomplete ossification of 13th rib (0 fetus) was observed at 1000 mg/kg/d bw. The increased "incomplete ossified skull bones" was essentially due to only 2 dams. The other statistically significant differences were considered to be incidental because these retardation effects were not accompanied by weight retardation and were within the normal range of variation for this strain.
(ii) Variations: No variations in any group.
(iii) Malformations: One fetus with stump tail and missing vertebrae coccigycae at 300 mg/kg/d bw (not considered to be treatment-related).

Visceral malformations:

no effects observed

Description (incidence and severity):

No treatment-related abnormalities.

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

fetal/pup body weight changes

external malformations

skeletal malformations

visceral malformations

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Table 2. Summary of performance of mated females

Treatment dose (mg/kg/d)	0	100	300	1000
No. of mated females	30	30	30	30
No. of pregnant females	30	29	28	29
No. of females with premature litter	1	0	2	3
No. of mortalities	0	0	0	0
No. of females with live fetuses at termination	29	29*	26	26

* One dam out of these was not included because the weights of fetuses were not determined

Conclusions:

Under the study conditions, the NOAELs for parental toxicity and developmental toxicity were considered to be 1000 mg/kg bw/day.

Executive summary:

A study was conducted to evaluate the prenatal developmental toxicity of the read across substance, C12-18 and C18-unsatd. DEA, according to OECD Guideline 414, in compliance with GLP. The substance was administered to groups of 30 female rats by gavage at dose levels of 0, 100, 300 and 1000 mg/kg bw/day, once daily from Gestation Days (GD) 6 to 15 inclusive. Control animals were dosed with the vehicle alone (arachis oil, DAB 9). Clinical condition and reaction to treatment were recorded at least once daily. Body weights were reported on GD 0, 6, 16 and 20. All surviving females were sacrificed on GD 20 and the foetuses were removed by caesarean section. At necropsy, the females were examined macroscopically. Live foetuses were weighed, sexed and examined for visceral and skeletal abnormalities. No deaths or treatment-related changes in body weight gain and necropsy findings were observed in dams at any dose level. Treatment-related symptoms observed in all groups were salivation and propulsion of the head. The highest dose group showed severe salivation. Apart from the control (1 dead foetus) and the 100 mg/kg bw/day groups (7 dead foetuses), all females had viable foetuses. Pre-implantation loss and mean numbers of resorptions were not affected by treatment. The data for post-implantation loss, embryonic deaths and total foetuses showed some deviations, which were considered to be non-treatment-related. Mean placental and uterus weights were not affected by the treatment. Foetal sex ratio was comparable in all groups. No treatment-related foetal abnormalities were found at necropsy. The examined foetuses showed no treatment-related visceral and skeletal abnormalities/variations. One foetus at 300 mg/kg bw/day showed a stump tail and missing coccigycae vertebrae. Further, the data for skeletal ossifications showed some deviations in the two highest dose groups. However, all these effects were assessed to be non-treatment-related. Under the study conditions, the NOAELs for parental toxicity and developmental toxicity were considered to be 1000 mg/kg bw/day (Pitterman, 1994).

Reference 2

Endpoint:

developmental toxicity

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

supporting study

Study period:

November 2016 - June 2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

KL2 due to RA

Justification for type of information:

Refer to section 13 of IUCLID for details on the category justification.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Remarks:

no major deviations

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

SPF (Specific Pathogen Free) Sprague-Dawley - Crl: OFA (SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories France, Domaine des Oncins, 69210 L'ARBRESLE Cedex, France
- Age at study initiation: 10 to 11 weeks at the beginning of the treatment period
- Weight at study initiation: Between 224.4 and 326.7 g on the day of randomisation (d5pc) The weight variation of animals used was minimal (+/- 20% of the mean weight). About 10% more animals were ordered to allow selection of animals according to the criterion of body weight and they were used as spare animals in case of unforeseen events happen
- Fasting period before study:
- Housing: Animals were housed individually in cages of standard dimensions with sawdust bedding.
- Diet (e.g. ad libitum): RM3 (E)-SQC SDS/DIETEX feed (quality controlled/radiation sterilised) was available ad libitum except during the fasting experimental period. The certificate of analysis concerning this feed product is included in the report. The criteria for acceptable levels of contaminants in the feed supply were within the limits of the analytical specifications established by the diet manufacturer.
- Water (e.g. ad libitum): Drinking water was available ad libitum in polycarbonate bottles with a stainless steel nipple. A specimen of water is obtained approximately every 6 months and sent to Laboratoire de la Touraine - ZA n°1 du Papillon - Rue de l'Aviation - 37210 Parçay-Meslay - France, for analysis. The certificates of analysis are included in the report. The criteria for acceptable levels of contaminants in the water supplied were within the limits of the analytical specifications.
- Acclimation period:Animals arrived at CERB on day 1 of pregnancy (d1pc). Animals were supplied in several batches for logistical reasons. Each animal had five days in the laboratory animal house where the experiment took place before beginning of dosing. Only animals without any visible sign of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): The animals were placed in an air-conditioned (20-24°C) animal house
- Humidity (%): relative humidity between 45% and 65% (except during the cleaning slot). Between 28 Nov at 07.35 p.m. up to 29 Nov at 11.10 a.m., an abnormal decrease of hygrometry was noted in animal room.
- Air changes (per hr): non-recycled filtered air was changed approximately 10 times per hour
- Photoperiod (hrs dark/hrs light): The artificial day/night cycle involved 12 hours light and 12 hours darkness with light on at 7.30 a.m

IN-LIFE DATES: From: 25 November 2016 To: 22 December 2016

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

DIET PREPARATION no information available

VEHICLE
Corn oil will be used (Reference C8267)
- Lot/batch no. (if required): MKBS6944V and MKBW9504V, Expiry dates: 08 Oct 2020 and 06 Oct 2021 respectively
- Amount of vehicle (if gavage): 5 mL/kg body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentrations of test item in formulations were checked during the first and last week of the study. Each concentration level and the vehicle were checked. 1 mL samples of each test item dosing formulation were taken in duplicate by top, middle and bottom sampling. Similar samples from the vehicle were taken, from the middle of the formulation only. Only the middle samples were assayed. Samples were collected in glass ontainers and stored at room temperature. Labels on the containers were marked in waterproof ink with Testing Facility, Study Number, Name of the test item and Concentrations, Sampling Date, Sampling Time and Storage conditions. The sample labels also indicated whether the samples were taken from the top, middle (for vehicle) or bottom of the formulation container. The identity and concentration of the test item in the samples and the absence of the test item in the control formulation was determined by liquid chromatography with UV-detection within the validated stability period available at this moment i.e. one week after sampling Acceptance criteria of the formulation analysis were fixed usually +/- 15% to the nominal concentration. The analytical method is validated within the range 85-115% of the theoretical concentration for formulations between 19.765 mg/mL and 198.120 mg/mL, with a precision better than 10%. The samples in corn oil must be analysed within the stability period (i.e. 30 days at room temperature) and must be within the range 85-115% to meet the acceptance criteria.

Formulation analysis was performed on one formulation prepared during the first and the last week of the study. The concentrations tested were 20 mg/mL, 60 mg/mL and 200 mg/mL. The concentrations found were within the range of acceptance (15% of the intended concentration). The absence of test item was also confirmed in the vehicle samples. Therefore, these data confirmed that the formulations were properly prepared.

Details on mating procedure:

- Impregnation procedure: The breeding establishment will be responsible for mating. Females will be mated at the beginning of the morning. They will be inspected for the presence of a vaginal plug.

- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy (d0pc)

Duration of treatment / exposure:

The test item administered in pregnant rats from day 6 to day 19 of gestation

Frequency of treatment:

N-(2-hydroxypropyl) Oleamide or its vehicle will be administered once a day at approximately the same time (a maximum range of 4 hours between the start and the end of the daily treatment) at each chosen dose level

Duration of test:

21 days (days 0-20 post coitum)

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

300 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

20 mated females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Proposed doses are selected in agreement with the Sponsor. The choice is based on previous studies (Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test by oral route (gavage) in rats - OECD 422 - Study No. 39644 RSR). Moreover, the highest dose should reveal signs of toxicity and the lowest dose should represent a no-observed-adverse effects level.

Maternal examinations:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were observed in the home cage before the first dosing and at least once a day during the study except on d7 for 3/20 female of group 2. The time of observation during the treatment period was at 60 min post dose (+/- 30 min). Females showing signs of abortion or of premature delivery during the study, the day on which such findings seen were noted.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed on the following days:
• on d1 and d5 (day of randomisation)
• daily during treatment (from d6 to d19)
• on d20, day of necropsy, not fasted and not exsanguinated

FOOD CONSUMPTION : Yes
- Food consumption was measured and presented daily from d6 to d19

WATER CONSUMPTION : No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20: On day 20 of pregnancy, all surviving animals were killed by subtotal exsanguination following isoflurane inhalation. Females showing signs of abortion or of premature delivery during the study were killed on the day of such findings
- Organs examined: Animals showing signs of abortion or of premature delivery were necropsied as quickly as possible and specimens as required by the study plan obtained whenever practically possible. The main organs cited below were examined macroscopically. All animals were subjected to gross necropsy and their organs (brain, liver, stomach, small intestine (duodenum, jejunum, ileum), large intestine (caecum, colon, rectum), kidneys, spleen, adrenals, lungs, heart) were examined macroscopically. Uterus and ovaries from each female were macroscopically observed and fixed in an appropriate fixative. Gravid uteri were weighed before extraction of foetuses

OTHER:
All organs showing macroscopic signs of pathology and corresponding organs of control groups for comparison were fixed in an appropriate fixative. Remaining tissues were destroyed 6 months after issue of the draft release.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Number and uterine location of foetuses (live and dead): Yes

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: No data
- Skeletal examinations: Yes: [performed in the first instance only on control and high dose groups]
- Number of live or dead foetuses: Yes
- Individual foetal body weight (live and dead): Yes : [all per litter]
- Caudo-cranial measurement of live and dead foetuses: Yes : [all per litter]
- Gross evaluation and weight of placenta of all foetuses: Yes : [all per litter]
- Sexing of all foetuses: Yes : [all per litter]

Statistics:

See "Any other information on materials and methods incl. tables"
The validated computerised system used in this phase was the Xybion Path/Tox System, Version 4.2.2.

Indices:

Pre-implantation loss and post-implantation loss were calculated according to the following formula:
Pre-implantation loss (%):
((Number of corpora lutea - number of implantations) / Number of corpora lutea) x 100
Post-implantation loss (%):
((Number of implantations - number of live foetuses) / Number of implantations) x 100

Foetal or litter incidence was calculated according to the following formula:
Foetal or litter incidence (%):
(Total number of foetus or litter with a particular finding / Total number of foetus per group) x 100

Historical control data:

No data available

Clinical signs:

no effects observed

Description (incidence and severity):

There was an increased salivation in some females treated with test item at 300 mg/kg bw (in 1/20) on d13 and d14 and at 1000 mg/kg bw from d12 up to the end of the study (between 1 to 3/20). There was also chromodacryorrhoea in 1 or 2/20 different animals following the day of observation in females treated with test item at 300 mg/kg bw or at 1000 mg/kg bw. These signs were of low incidence and were not attributed to a toxicological effect of the test item. There was aggressiveness on d5 in 1/20 female treated with test item at 100 mg/kg bw. This sign was only observed on the first day of treatment and was not attributed to the test item.

Mortality:

no mortality observed

Description (incidence):

No mortality and no relevant clinical signs or signs of reaction to treatment were noted in treated females.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no change in body weight gain.

Food efficiency:

no effects observed

Description (incidence and severity):

There was a statistically significant isolated lower food consumption in females treated with test item at 100 mg/kg bw on d10 (-13%) and d18 ( -20%) or in females treated with test item at 1000 mg/kg bw on d6, d11 or d18 (between -14 and -18%). This lower food consumption is transient and attributable to the high variability in the control group. From d5pc, food consumption was lower for Female No. 1602737 treated with test item at 100 mg/kg bw.

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

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There was no change in uterus weight.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no macroscopic findings in mated females killed on d20pc. At the necropsy, there was a dark liquid in the vulva and vagina, a black abnormal content in stomach and enlarged spleen in Female No. 1602755 treated with test item at 300 mg/kg bw. In Female No. 1602730 treated with test item at 1000 mg/kg bw, there was dark liquid in the vagina, transparent and abnormal area in stomach, adhesion between lungs and thoracic cavity, adhesion between lungs, heart and diaphragm, cloudy liquid in the thoracic cavity and heart with firm area and granular aspect.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Number of abortions:

effects observed, non-treatment-related

Description (incidence and severity):

Signs of abortion (blood near genital orifice and in cage) were seen in two females, on d19 in one female treated with the intermediate dose (No. 1602755) and on d16 in one female treated with the highest dose (No. 1602730).

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

There was a higher percentage of pre and post implantation loss for Female No. 1602737 treated with test item at 100 mg/kg bw and Female No. 1602766 treated with test item at 300 mg/kg bw. For these females, there was no foetus, only resorptions were observed.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

In Female No. 1602755, there is 16 implantation sites, with 1 early resorption. In Female No. 1602730, there is 17 implantation sites, with 5 early and 3 late resorptions.

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

In Female No. 1602755, there is 16 implantation sites with 1 dead foetus. In Female No. 1602730, there is 17 implantation sites, with 9 dead foetus.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

On d20 of pregnancy (d20pc), all mated females were necropsied and all foetuses were examined.

Changes in number of pregnant:

no effects observed

Other effects:

not examined

Key result

Dose descriptor:

NOEL

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The mean body weight per litter and per group of live foetuses were indicated as well as the mean caudal-cranial measurement per litter and per group and the mean weight of the placenta of these foetuses per litter and per group.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not specified

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were isolated macroscopic findings seen in the control or treated groups at the same incidence such as point or area in head, limbs or back.

Skeletal malformations:

no effects observed

Description (incidence and severity):

The test item administered in pregnant rats from day 6 to day 19 of gestation did not induce any relevant changes in foetuses examined at skeletal examination.

Visceral malformations:

no effects observed

Description (incidence and severity):

The test item administered in pregnant rats from day 6 to day 19 of gestation did not induce any relevant changes in foetuses examined at visceral examination.

Details on embryotoxic / teratogenic effects:

There was no change in pre and post-implantation loss, in the number of corpora lutea, in the number of live and abnormal foetuses, in the number of normal and abnormal dead foetuses or in the early and late resorptions. There was no change in caudo-cranial measurement,foetus weight or proportion of male/female foetus. There was a statistically significant lower placenta weight in the group receiving test item at 100 mg/kg bw. This was low in amplitude (-6%) and was not attributed to a toxicological effect of test item.

Key result

Dose descriptor:

NOEL

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Table 7.8.2/2: Clinical signs – autonomic profile/miscellaneous - Group incidences

Clinicalsigns	Time	Vehicle	Test item100mg/kg	Test item300mg/kg	Test item1000mg/kg
Increased salivation	D5 D12 D13 D14 D15 D16 D17 D18 D19	0/20 0/20 0/20 0/20 0/20 0/20 0/20 0/20 0/20	0/20 0/20 0/20 0/20 0/20 0/20 0/20 0/20 0/20	0/20 0/20 1/20 1/20 0/20 0/20 0/20 0/20 0/19	0/20 1/20 0/20 2/20 1/20 1/19 3/19 2/19 2/19
Chromodacryorrhoea	D5 D9 D10 D11 D12 D14 D15 D17 D18 D19	0/20 0/20 0/20 0/20 0/20 0/20 1/20 0/20 0/20 0/20	0/20 0/20 0/20 0/20 0/20 0/20 0/20 0/20 0/20 0/20	0/20 1/20 1/20 1/20 1/20 0/20 0/20 1/20 1/20 0/19	0/20 0/20 1/20 0/20 0/20 1/20 2/20 1/19 1/19 1/19

Only times with clinical signs and showing differences with between groups are reported.

Results are expressed as the group incidence of animals showing the sign.

Corn oil

D: Day

P>0.05, when compared with the control group dosed with the vehicle, Fisher's test.

†: mortality occurred, no statistical analysis was performed.

Table 7.8.2/3: Body weight (mean table)

Treatment

D5

D6

D7

D8

D9

D10

D11

Vehicle

Mean SEM % N

260 6 NA 20

264 6 +2 20

267 6 +3 20

272 6 +5 20

277 6 +7 20

284 6 +9 20

288 6 +11 20

Test item 100mg/kg

Mean SEM % N P

262 5 NA 20 NS

267 6 +2 20 NS

270 6 +3 20 NS

275 6 +5 20 NS

279 5 +6 20 NS

286 6 +9 20 NS

289 6 +10 20 NS

Test item 300mg/kg

Mean SEM % N P

257 5 NA 20 NS

260 5 +1 20 NS

263 4 +2 20 NS

268 4 +4 20 NS

273 4 +6 20 NS

278 5 +8 20 NS

284 4 +11 20 NS

Test item 1000mg/kg

Mean SEM % N P

262 5 NA 20 NS

266 5 +2 20 NS

268 4 +2 20 NS

273 4 +4 20 NS

277 4 +6 20 NS

283 5 +8 20 NS

288 4 +10 20 NS

Threshold

18

18

18

18

18

18

18

Treatment

D12

D13

D14

D15

D16

D17

D18

D19

D20

Vehicle

Mean SEM % N

295 6 +13 20

300 6 +15 20

306 6 +18 20

313 6 +20 20

323 6 +24 20

337 6 +30 20

350 6 +35 20

363 6 +40 20

376 6 +45 20

Test item 100mg/kg

Mean SEM % N P

294 6 +12 20 NS

301 6 +15 20 NS

306 6 +17 20 NS

314 6 +20 20 NS

324 7 +24 20 NS

337 8 +29 20 NS

348 9 +33 20 NS

357 9 +36 20 NS

368 10 +40 20 NS

Test item 300mg/kg

Mean SEM % N P

289 5 +12 20 NS

294 4 +14 20 NS

300 4 +17 20 NS

308 4 +20 20 NS

318 4 +24 20 NS

333 4 +30 20 NS

342 5 +33 20 NS

355 6 +38 19 NS

367 6 +43 19 NS

Test item 1000mg/kg

Mean SEM % N P

293 4 +12 20 NS

299 4 +14 20 NS

306 5 +17 20 NS

311 5 +19 20 NS

320 5 +22 20 NS

336 5 +28 19 NS

348 5 +33 19 NS

359 5 +37 19 NS

370 6 +41 19 NS

Threshold

18

18

18

18

18

20

20

22

22

Results expressed in g

D: day

Vehicle: Corn oil

NS:P>0.05, when compared to control group

Analysis of variance for repeated measurements with Dunnett's test

NA: not applicable

%: variation expressed in percentage in relation to predose values

Table 7.8.2/4: Absolute weight of uterus (mean values)

Treatment		Uterus Weight (g)
Vehicle	Mean SEM N	71.6 2.5 20
Testitem 100mg/kg	Mean SEM N % P	70.4 5.5 20 -2 NS
Testitem 300mg/kg	Mean SEM N % P	67.6 4.0 20 -6 NS
Testitem 1000mg/kg	Mean SEM N % P	69.6 3.9 20 -3 NS
	Threshold	14.0

%: variation expressed in percentage in relation to control values

Vehicle: Corn oil

NS:P>0.05, when compared with control group

Analysis of variance with Dunnett's test if P <0.05

Table 7.8.2/5: Macroscopic observations - Group incidences

Organs	Observations	vehicle	Test item100mg/kg	Test item300mg/kg	Test item1000mg/kg
Placenta	Dark Total animals involved	0/256 0	0/259 0	14/243 14	0/246 0
Head	Punctate or point Area Total animals involved	1/256 2/256 3	0/259 2/259 2	1/243 1/243 2	1/246 2/246 3
Limbs	Punctate or point Area Total animals involved	1/256 2/256 3	2/259 3/259 5	1/243 5/243 6	1/246 4/246 5
Abdomen	Punctate or point Total animals involved	1/256 1	0/259 0	0/243 0	0/246 0
Tail	Area Twisted Total animals involved	1/256 0/256 1	0/259 0/259 0	0/243 1/243 1	0/246 0/246 0
Back	Punctate or point Area Total animals involved	14/256 0/256 14	10/259 5/259 15	11/243 0/243 11	11/246 2/246 13

Table 7.8.2/6: Caudo-cranial measurements and weights of live foetuses, weights of the placenta (mean values)

Treatment		Caudo-cranial measurement(mm)	Foetus weight(g)	Placenta weight(g)
Vehicle	Mean SEM N	35.2 0.2 256	3.77 0.03 256	0.567 0.006 255
Test item 100mg/kg	Mean SEM N % P	34.8 0.1 259 -1 NS	3.69 0.02 259 -2 NS	0.533 0.005 259 -6 ??
Test item 300mg/kg	Mean SEM N % P	34.7 0.3 243 -1 NS	3.70 0.04 243 -2 NS	0.557 0.006 242 -2 NS
Test item 1000mg/kg	Mean SEM N % P	35.2 0.1 246 0 NS	3.78 0.02 246 0 NS	0.569 0.005 246 0 NS
	Threshold	0.6	0.09	0.018

%: variation expressed in percentage in relation to control values

Vehicle: Corn oil

NS:P>0.05, ??:P<0.01, when compared with control group

Analysis of variance with Dunnett's test if P <0.05

Conclusions:

Under the study conditions, the NOAEL for embryo-foetal developmental toxicity was considered to be 1000 mg/kg bw/day.

Executive summary:

A study was conducted to evaluate the prenatal developmental toxicity of the read across substance, C18-unsatd. MIPA, according to OECD Guideline 414, in compliance with GLP. The substance diluted in corn oil was administered by gavage to groups of mated female Sprague-Dawley rats (20 mated females/dose) at the dose levels of 0, 100, 300, 1000 mg /kg bw/day from Days 6 to 19 after mating. The aim of the study was to investigate any possible adverse effects on the pregnant female rat and on the developing embryo and foetus following daily administration to pregnant rats by the oral route from implantation throughout organogenesis and until late gestation. Morbidity/mortality checks were performed twice daily. Clinical observations were performed before the first dosing and daily. Body weight was recorded on Day 5 then daily from Day 6 to Day 20. Food consumption was measured daily from Day 6 to Day 19. On Day 20 of pregnancy, all mated females were necropsied and all foetuses were examined. Foetuses were examined macroscopically and the following data were noted: number of live or dead foetuses, individual foetal body weight (live and dead), caudo-cranial measurement of live and dead foetuses, gross evaluation and weight of placenta of all foetuses, external morphological examination of these foetuses, sexing of all foetuses. The clinical signs (increased salivation and chromodacryorrhoea) observed were at low incidence and were not attributed to a toxicological effect of the read across substance. There was no change in pre and post-implantation loss, in the number of corpora lutea, in the number of live and abnormal foetuses, in the number of normal and abnormal dead foetuses or in the early and late resorptions. There was no change in caudo-cranial measurement, foetus weight or proportion of male/female foetus. There was a statistically significant lower placenta weight in the group receiving read across substance at 100 mg/kg bw/day. This was low in amplitude and was not attributed to a toxicological effect of read across substance. The read across substance administered in pregnant rats from Day 6 to Day 19 of gestation did not induce any relevant changes in foetuses examined at skeletal and visceral examination. Based on the above results, it could be concluded that the read across substance had no harmful effects on the prenatal development of the rat offspring at doses used in the present study. Under the study conditions, the NOAEL for embryo-foetal developmental toxicity was considered to be 1000 mg/kg bw/day (Mortier, 2018).

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Sufficient information is available to evaluate this endpoint.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

A study was conducted to evaluate the prenatal developmental toxicity of the read across substance, C12-18 and C18-unsatd. DEA, according to OECD Guideline 414, in compliance with GLP. The substance was administered to groups of 30 female rats by gavage at dose levels of 0, 100, 300 and 1000 mg/kg bw/day, once daily from Gestation Days (GD) 6 to 15 inclusive. Control animals were dosed with the vehicle alone (arachis oil, DAB 9). Clinical condition and reaction to treatment were recorded at least once daily. Body weights were reported on GD 0, 6, 16 and 20. All surviving females were sacrificed on GD 20 and the foetuses were removed by caesarean section. At necropsy, the females were examined macroscopically. Live foetuses were weighed, sexed and examined for visceral and skeletal abnormalities. No deaths or treatment-related changes in body weight gain and necropsy findings were observed in dams at any dose level. Treatment-related symptoms observed in all groups were salivation and propulsion of the head. The highest dose group showed severe salivation. Apart from the control (1 dead foetus) and the 100 mg/kg bw/day groups (7 dead foetuses), all females had viable foetuses. Pre-implantation loss and mean numbers of resorptions were not affected by treatment. The data for post-implantation loss, embryonic deaths and total foetuses showed some deviations, which were considered to be non-treatment-related. Mean placental and uterus weights were not affected by the treatment. Foetal sex ratio was comparable in all groups. No treatment-related foetal abnormalities were found at necropsy. The examined foetuses showed no treatment-related visceral and skeletal abnormalities/variations. One foetus at 300 mg/kg bw/day showed a stump tail and missing coccigycae vertebrae. Further, the data for skeletal ossifications showed some deviations in the two highest dose groups. However, all these effects were assessed to be non-treatment-related. Under the study conditions, the NOAELs for parental toxicity and developmental toxicity were considered to be 1000 mg/kg bw/day (Pitterman, 1994).

A study was conducted to evaluate the prenatal developmental toxicity of the read across substance, C18-unsatd. MIPA, according to OECD Guideline 414, in compliance with GLP. The substance diluted in corn oil was administered by gavage to groups of mated female Sprague-Dawley rats (20 mated females/dose) at the dose levels of 0, 100, 300, 1000 mg /kg bw/day from Days 6 to 19 after mating. The aim of the study was to investigate any possible adverse effects on the pregnant female rat and on the developing embryo and foetus following daily administration to pregnant rats by the oral route from implantation throughout organogenesis and until late gestation. Morbidity/mortality checks were performed twice daily. Clinical observations were performed before the first dosing and daily. Body weight was recorded on Day 5 then daily from Day 6 to Day 20. Food consumption was measured daily from Day 6 to Day 19. On Day 20 of pregnancy, all mated females were necropsied and all foetuses were examined. Foetuses were examined macroscopically and the following data were noted: number of live or dead foetuses, individual foetal body weight (live and dead), caudo-cranial measurement of live and dead foetuses, gross evaluation and weight of placenta of all foetuses, external morphological examination of these foetuses, sexing of all foetuses. The clinical signs (increased salivation and chromodacryorrhoea) observed were at low incidence and were not attributed to a toxicological effect of the read across substance. There was no change in pre and post-implantation loss, in the number of corpora lutea, in the number of live and abnormal foetuses, in the number of normal and abnormal dead foetuses or in the early and late resorptions. There was no change in caudo-cranial measurement, foetus weight or proportion of male/female foetus. There was a statistically significant lower placenta weight in the group receiving read across substance at 100 mg/kg bw/day. This was low in amplitude and was not attributed to a toxicological effect of read across substance. The read across substance administered in pregnant rats from Day 6 to Day 19 of gestation did not induce any relevant changes in foetuses examined at skeletal and visceral examination. Based on the above results, it could be concluded that the read across substance had no harmful effects on the prenatal development of the rat offspring at doses used in the present study. Under the study conditions, the NOAEL for embryo-foetal developmental toxicity was considered to be 1000 mg/kg bw/day (Mortier, 2018). 

 

In addition, after discussion with ECHA in the frame of a Dossier Improvement Action Plan (DIAP), testing proposals are submitted for the conduct of prenatal developmental toxicity studies in rat and rabbit according to OECD Guideline 414 with the read across substance C8-18 and C18-unsatd. DEA to provide data on the substance and further support the read across approach proposed for the FAA category members.

Justification for classification or non-classification

The data available on the read across substances C12-18 and C18-unsatd. DEA, C8-18 and C18-unsatd. MEA, C16-18 MEA,  C8-18 and C18-unsatd. DEA, and C18-unsatd. MIPA suggest that the test substance is not a reproductive toxicant with regards to fertility or developmental effects. Therefore no classification is required according to EC (67/548/EEC) and CLP (EC 1272/2008) criteria.