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

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 28, 2020 to September 7, 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

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

Deviations:

yes

Remarks:

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

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

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

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

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

Sex:

male/female

Details on test animals or test system and environmental conditions:

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

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

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

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% aqueous solution of CMC

Details on exposure:

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

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

Details on mating procedure:

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

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

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

Duration of treatment / exposure:

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

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

Frequency of treatment:

Once daily, 7 days/week

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

vehicle

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

Low level

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Medium level

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High Level

No. of animals per sex per dose:

10 males/10 females/group

Control animals:

yes, concurrent vehicle

Details on study design:

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

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

Parental animals: Observations and examinations:

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

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

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

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

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

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

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

Oestrous cyclicity (parental animals):

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

Litter observations:

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

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

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

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

Postmortem examinations (parental animals):

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

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

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

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

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

Postmortem examinations (offspring):

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

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

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

Statistics:

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

Reproductive indices:

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

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

Offspring viability indices:

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

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

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

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

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

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight and body weight gain were unaffected by treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No treatment related effects were observed in food consumption.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

effects observed, non-treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

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

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

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

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

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

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

Reproductive performance:

no effects observed

Description (incidence and severity):

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

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

organ weights and organ / body weight ratios

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

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

Dermal irritation (if dermal study):

not examined

Mortality / viability:

not specified

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

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

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

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

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

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

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

no effects observed

Description (incidence and severity):

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

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

body weight and weight gain

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

developmental immunotoxicity

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

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

Executive summary:

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

Reference 2

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 March 10, 2021 to February 4, 2022

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.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted on 29 July 2016

Deviations:

yes

Remarks:

Females and males were supplied in the weight range of 178-235 and 201-222 g and not 175-200 and 200-225 g. Motor activity measurements were carried out Week 5 and not Week 4, as indicated in the Study Protocol. These deviations had no impact on the study

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Hsd: Sprague Dawley SD rats

Details on species / strain selection:

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

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Animal supply and acclimatization:
A total of 102 Hsd: Sprague Dawley SD rats (45 males and 57 virgin females), 7 to 8 weeks old and weighing 200 to 225 g for males and 175 to 200 g for females, were ordered from and supplied by Charles River Italia S.p.A., Calco (Lecco), Italy. After arrival, the weight range for each sex was determined and the animals were temporarily identified within the cage by means of a coloured mark on the tail. A health check was then performed by a veterinarian. An acclimatisation period of approximately 4 weeks was allowed before the start of treatment, during which time the health status of the animals was assessed by thorough observations.

- Animal husbandry:
The animals were housed in a limited access rodent facility. Animal room controls were set to maintain temperature and relative humidity at 22°C±2°C and 55%±15%, respectively; actual conditions were monitored, recorded and the records retained. There were approximately 15 to 20 air changes per hour and the rooms were lit by artificial light for 12 hours each day. From arrival to mating, animals were housed up to 5 of one sex to a cage, in polysulfone solid bottomed cages measuring 59.5×38×20 cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese). Nesting material was provided inside suitable bedding bags and changed at least twice a week. During mating, animals were housed one male to one female in clear polysulfone cages measuring 42.5×26.6×18.5 cm with a stainless steel mesh lid and floor (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese). Each cage tray held absorbent material which was inspected and changed daily. After mating, the males were re-caged as they were before mating. The females were transferred to individual solid bottomed cages for the gestation period, birth and lactation (measuring 42.5×26.6×18.5 cm). Nesting material was provided inside suitable bedding bags. In addition, suitable nesting material (Scobis 0 Mucedola) was provided as necessary and changed at least 2 times a week. Drinking water was supplied ad libitum to each cage via water bottles, except in the case of urinalysis investigations. A commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019 Settimo Milanese (MI), Italy) was offered ad libitum throughout the study. There was no information available to indicate that any non-nutrient substance likely to influence the effect of the test item was present in the drinking water or the diet. Records of analyses of water and diet are kept on file at ERBC. Dated and signed records of activities relating to the day to day running and maintenance of the study in the animal house were recorded.

- Allocation to groups:
On the day of allocation all animals were weighed. Animals at the extremes of the weight distribution were excluded to leave the required number of animals. Furthermore, female animals that exhibited anomalies in the oestrous cycle were not allocated to the main groups. The rats were allocated to the groups by computerised stratified randomisation to give approximately equal initial group mean body weights. 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. Any animal showing signs of ill health during the period between allocation and the start of treatment was subjected to pathological examination as considered appropriate and replaced with a surplus animal selected from the same batch.

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, 25 and 70 mg/mL), according to stability data from ERBC study No. A4125. 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.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical method was validated in ERBC Study no. A4125 in the range from 10 to 100 mg/mL. Linearity, accuracy and precision were within the limits stated in the validation study (r > 0.99; accuracy 80-120%; precision CV < 10%). In ERBC Study no. A4125, a 48 hour stability at room temperature and a 9 day stabiliy at 2-8°C were verified in the range from 10 to 100mg/mL. According to ERBC SOPs, suspensions were considered to be stable if concentration and homogeneity, after the defined period of storage, were still acceptable (80%-120% for concentration and CV < 10% for homogeneity). The proposed preparation procedure for the test item was checked in the range from 10 to 100mg/mL by chemical analysis (concentration and homogeneity) in ERBC Study no. A4125 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 onWeek 1 and 5 (last week with males and females) were analysed to check the homogeneity and concentration. Results of the analyses were within the acceptability limits stated in ERBC SOPs for suspensions (80-120% for concentration and CV < 10% for homogeneity). Chemical analysis was carried out by the Analytical Chemistry Department. The software used for this activity was Empower® 2 Build No. 2154.

Duration of treatment / exposure:

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

Females: Animals were dosed once a day, 7 days a week, for 14 days prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 13 post partum or the day before sacrifice (for a total of 43 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/weeks

Details on study schedule:

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

-Females: Animals were dosed once a day, 7 days a week, for 14 days prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 13 post partum or the day before sacrifice (for a total of 43 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.

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

700 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. Observations were performed at the same time interval each day (approximately 5 - 10 minutes and 1.5 - 2 hours post-dose), the interval was selected taking into consideration the presence of post-dose reactions. All observations were recorded for individual animals.

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

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

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

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

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

-Vaginal smears and oestrous cycle
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. Oestrous cycle was monitored by vaginal smears for 2 weeks before allocation. These data were not tabulated in the report. Vaginal smears were also taken from all females, before despatch to necropsy and the oestrous cycle phase recorded.

- 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.

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

- 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 parameters 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 prothrombin time. Clinical chemistry parameters assessed corresponds to : alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, gamma-glutamyltransferase, urea, creatinine, glucose, triglycerides, bile acids, total bilirubin, total cholesterol, total protein, albumin, globulin, A/G Ratio, sodium, potassium, calcium, chloride, inorganic phosphorus.

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

- Blood collection and thyroid hormone determination (T4 and TSH)
Blood collection for hormone determination was performed from all animals at termination.
Males
Blood samples (approximately 0.8 mL) for hormone determination were collected under isoflurane anaesthesia from the 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. Oestrous cycle was monitored by vaginal smears for 2 weeks before allocation. These data were not tabulated in this report, but will be archived with the raw data.

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

-Before despatch to necropsy:Vaginal smears were also taken from all females, before despatch to necropsy and the oestrous cycle phase recorded.

Sperm parameters (parental animals):

None

Litter observations:

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

-Pups identification, weight and observations: On Day 1, as soon as possible after parturition was considered complete (Day 0 post partum), all pups (live and dead) were counted, sexed and live pups were identified. Live pups were individually weighed on Days 1, 4 and 13 post partum. Observations 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 (by use of a random list generated by the computerised system) to yield, as nearly as possible, four pups per sex per litter. Partial adjustment (for example, 5 males and 3 females) was acceptable. On Day 4 post partum, at least one culled male and one culled female (where possible) were selected for hormone determination.

-Anogenital distance (AGD): The AGD of each pup was measured with a digital 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, the presence of nipples/areolae in each male pup was checked. Since no nipples were observed, these data were not reported (data archived with the other
raw data of the study).

-Blood collection for metabolomics analysis (External laboratory): 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.

-Organ weights
From all animals completing the scheduled test period, the organs were dissected free of fat and weighed. 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 analyses
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.

Postmortem examinations (offspring):

-Necropsy
All pups found dead in the cage or sacrificed for humane reasons were examined for external and internal abnormalities. All culled pups sacrificed at Day 4 post partum were subjected to an external examination. Sex was determined by internal gonads inspection. All live pups sacrificed on Day 14 post partum were examined for external abnormalities. Gonads were inspected from all pups in order to confirm the sex previously determined by external examination. All pups with abnormalities were retained in a 10% neutral buffered formalin.


- Nipple count retention on Day 14 post-partum:
The ventral region of male pups was checked for presence of nipples/areolae.

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

Statistics:

Standard deviations were calculated as appropriate. For 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 nonparametric 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 observed in animals of both sexes dosed at 250 (10 out of 10 males and 5 out of 10 females) and 700mg/kg/day (all males and females) with a dose-related frequency and incidence, from the first few days of the pre-mating phase up to termination. This treatment-related clinical sign is not considered to be adverse, since no effects on the health status of the affected animals were evident. No clinical signs were observed in animals dosed at 100mg/kg/day.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortality occurred throughout the study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weight and body weight gain for male and female animals were generally comparable between the treated and control groups, before and during mating, during gestation and post partum periods.
In a single occasion (Day 0 post coitum), females receiving 700mg/kg/day showed a very slight but statistically significant decrease in body weight (-5%) compared to the control group. This isolated change was followed by a regular growth of the animals. Due to its occasional occurrence and to its low magnitude, this change was not considered treatment related.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

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

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes were recorded.
A statistically significant decrease of monocytes was recorded between control and males dosed at 700mg/kg/day (49%). Values were within the range of historical control data and no other related finding was recorded, therefore this change was considered to be unrelated to treatment.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No changes were recorded.

Endocrine findings:

no effects observed

Description (incidence and severity):

No changes were observed.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No changes were observed.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

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

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

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

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic observations associated with the oral administration of test substance were present in the non glandular region of the stomach of high dose animals of both sexes. Forestomach: treatment-related changes were present in the non-glandular region of the stomach (forestomach) in 4/5 males and 4/5 females of the high dose group and consisted of mild to moderate epithelial hyperplasia (1). Epithelial hyperplasia was associated with hyperkeratosis (thickening of the stratum corneum). Chronic inflammation and oedema were observed in the submucosa in association with the hyperplasia, and, in two instances (animals nos. X1670071 and X1670077), there was also mucosal erosion/ulceration. No treatment-related changes were observed in the non glandular region of the stomach of the selected animals of both sexes from the mid-dose group. Any other microscopic observations other than that listed above had a comparable incidence in control and treated groups and/or are characteristically seen in untreated rats of the same age and were considered incidental and unrelated to treatment. There were no test item-related microscopic observations in the testis (stage aware evaluation on PAS-stained slides).

(1) Toxicologic Pathology, 2016; 29 (1 Suppl): 1S–124S - Thomas Nolte.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

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

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related anomalies were noted in the oestrous cycle and pre-coital interval of the treated females, when compared to controls. One female treated at 100mg/kg/day (no. X1670023), 2 treated at 250mg/kg/day (nos. X1670051 and X1670057) and 1 received 700mg/kg/day (no. X1670063) conceived after 6 days. The total number of oestrous cycles observed in all females before pairing (number of non sequential days in which the females were in oestrous) was of 4 times (mean value) in animals from low, mid-dose and controls, while it was 3 in animals of the high dose groups dosed at 700mg/kg/day). The number of copulation plugs was similar between control and treated groups. All females mated. However, 2 control females (nos. X1670011 and X1670017) and 1 from the low dose group (no. X1670021) were found not pregnant. Copulatory and fertility indices were considered to be unaffected by administration of the test substance.

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Two females of the control group (nos. X1670011 and X1670017 - Group 1) and one dosed at 100 mg/kg/day (no. X1670021 - Group 2) were found not pregnant. Unilateral implantation was observed in 1 female dosed at 700 mg/kg/day (no. X1670063 - Group 4). The number of females with live pups on Day 21 post partum was: 8 in the control group, 9 in the low dose group and 10 in the mid- and high dose groups (dosed at 0, 100, 250 and 700 mg/kg/day, respectively).
Copulatory and fertility indices were considered to be unaffected by administration of the test substance.
- Males: The copulatory and fertility indices were 90% for controls and 100% for low, mid- and high dose groups (dosed at 100, 300 and 750 mg/kg/day). One male of the high dose group (no. X16800
66) which cohabitated with 2 females, mated with the first female only (no. X1680065).
- Females: The copulatory and fertility indices were 90% for controls and high dose groups and 100% for low and mid-dose groups.

Salivation was observed in animals of both sexes dosed at 250 and 700mg/kg/day (all males and females) with a dose-related frequency and incidence, from the first few days of the pre-mating phase up to termination. These clinical signs were not considered to be adverse since no effects on the health status were evident in the affected animals. No clinical signs were observed in animals dosed at 100mg/kg/day. No treatment-related signs of neurotoxicity (weekly observations at removal from the cage and in an open arena, alterations in motor activity, grip strength and sensory reactivity to stimuli) were observed during the study in males and females. No differences in body weight and food consumption were observed in treated animals, compared to the control group. No treatment-related changes were observed in haematological (including coagulation), clinical chemistry parameters or urinalysis. No treatment-related changes were observed in thyroid hormones levels in parental male animals or in pups at Day 14 post partum determinations.
The number of females with live pups on Day 14 post partum was: 8 in the controls, 9 in the low dose and 10 in the mid and high dose treated groups (dosed at 0, 100, 250 and 100mg/kg/day). All females mated. However, 2 control females (nos. X1670011 and X1670017) and 1 from the low dose group (no. X1670021) were found not pregnant. No treatment-related anomalies were noted in the oestrous cycle of the treated females, when compared to controls. Copulatory and fertility indices did not show any treatment related differences among treated and control groups. No treatment-related changes were detected at post mortem examination (terminal body weight, organ weights and macroscopic observation) in treated animals, when compared with controls. Microscopic examination revealed treatment-related changes in the forestomach, the
non-glandular region of the stomach including epithelial hyperplasia associated with hyperkeratosis, chronic inflammation and oedema, mucosal erosion/ulceration in 8 out of 10 animals (4/sex) dosed at 700mg/kg/day. These findingswere associated with the squamous epithelium of the non-glandular stomach and were considered to be due to a local irritant effect of the test item. As humans lack a non-glandular stomach, the relevance of these treatment-related changes for humans can be challenged. Only when the degree of these changes is highly severe, the cell is subjected to irreversible modifications and the change
becomes adverse. Therefore, since the observed changes are not sufficiently severe to cause a degenerative damage of the organ they were not considered to be systemically adverse in this study.
There were no test item-related microscopic observations in the testis (stage aware evaluation on PAS-stained slides).

Key result

Dose descriptor:

NOAEL

Effect level:

700 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

body weight and weight gain

food consumption and compound intake

haematology

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

neuropathology

histopathology: non-neoplastic

reproductive function (oestrous cycle)

reproductive performance

other: Endocrine: Thyroid hormone

Key result

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related clinical signs were observed in pups during the 14-day observation period. Cold to the touch, apparently no food intake (milk) and small appearance, pallor were in general the mainly clinical signs noted in control and/or treated pups.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

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

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:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

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

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No nipples were observed in male pups.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

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

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Litter data at birth, on Day 1, Day 4 and onDay 13 post partum of females and sex ratio of pups: No differences which could be considered adverse were observed in total litter size, liveC litter size, mean pup loss, sex ratio and mean pup weights among the treated dams and the controls at birth and on Days 1, 4 and 13 post partum. Slight decreases in live litter size and mean litter weight were seen on Days 1 and 4 post partum in dams dosed at 100 and 700mg/kg/day when compared to controls. These were attributed to individual variation. Due to the low magnitude of the decreases and, in the absence of a clear dose-relationship, lack of statistical significance and in view of the comparison with the reference control data these decreased were not considered to be adverse.
Necropsy: Autolysed abdominal organs were generally observed in pups found dead at check carried out after birth and in pups which died during the lactation period. Since autolysis normally occurs when pups are found dead some time after death, it was not considered to be treatment-related.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

700 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

organ weights and organ / body weight ratios

other: Anogenital distance and nipple count, Necropsy, thyroid weight

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 700 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 700 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 C16-18 and C18-unstatd. DEA 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, 250 and 700 mg/kg/day). All doses were administered at a constant volume of 10 mL/kg body weight. 0.5% aqueous solution of carboxymethylcellulose was used as vehicle. According to the study design, males were treated for 14 days prior to pairing and during pairing with females until the day before necropsy, for a total of 33/34 days. Females were treated for 14 days prior to pairing, during pairing and throughout the gestation and lactation periods until Day 13 post-partum, for a total of up 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 from control and high dose groups. The stomach was also examined in 5 selected animals/sex of the mid-dose group. No mortality occurred throughout the study. Salivation was the only treatment-related clinical sign recorded in males and females treated at 250 and 700 mg/kg/day, during the study. However, this clinical sign was not considered to be adverse. 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 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 terms of implantation, pre-birth loss or gestation length between treated and control groups. All pregnant dams gave birth between Days 22 and 23 post coitum. Litter data and sex ratios were unaffected by treatment. No test substance-related effects were seen in anogenital distance in pups of the treated groups, compared to controls. No nipples were found in male pups. No differences were noted in thyroid weight between pups of the control and treated groups. No treatment-related findings were noted in pups which died or were sacrificed on Days 4 (culled pups) and 14 post partum.Changes were observed in the stomach of the majority of males and females dosed at 700 mg/kg/day at microscopic examination, due to a local irritant effect of the test substance but these signs were not considered to be severe. Under the study conditions, the NOAEL for general toxicity was considered to be 700 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 700 mg/kg/day, as well as for growth and development of F1 pups (Longobardi, 2022).

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

Reproductive/developmental toxicity screening test (OECD 421)

 

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

Combined repeated dose toxicity study with the reproduction / developmental toxicity screening (OECD 422)

A combined repeated dose toxicity study with the reproduction / developmental toxicity screening was conducted with the read across substance C16-18 and C18-unstatd. DEA 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, 250 and 700 mg/kg/day). All doses were administered at a constant volume of 10 mL/kg body weight. 0.5% aqueous solution of carboxymethylcellulose was used as vehicle. According to the study design, males were treated for 14 days prior to pairing and during pairing with females until the day before necropsy, for a total of 33/34 days. Females were treated for 14 days prior to pairing, during pairing and throughout the gestation and lactation periods until Day 13 post-partum, for a total of up 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 from control and high dose groups. The stomach was also examined in 5 selected animals/sex of the mid-dose group. No mortality occurred throughout the study. Salivation was the only treatment-related clinical sign recorded in males and females treated at 250 and 700 mg/kg/day, during the study. However, this clinical sign was not considered to be adverse. 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 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 terms of implantation, pre-birth loss or gestation length between treated and control groups. All pregnant dams gave birth between Days 22 and 23 post coitum. Litter data and sex ratios were unaffected by treatment. No test substance-related effects were seen in anogenital distance in pups of the treated groups, compared to controls. No nipples were found in male pups. No differences were noted in thyroid weight between pups of the control and treated groups. No treatment-related findings were noted in pups which died or were sacrificed on Days 4 (culled pups) and 14 post partum.Changes were observed in the stomach of the majority of males and females dosed at 700 mg/kg/day at microscopic examination, due to a local irritant effect of the test substance but these signs were not considered to be severe. Under the study conditions, the NOAEL for general toxicity was considered to be 700 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 700 mg/kg/day, as well as for growth and development of F1 pups (Longobardi, 2022).

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.

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 C8-18 and C18-unsatd. DEA.

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:

supporting study

Study period:

November 2016 - June 2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

KL2 due to read across

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 d15pc, 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

Clinical signs	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
Test item 100mg/kg	Mean SEM N % P	70.4 5.5 20 -2 NS
Test item 300mg/kg	Mean SEM N % P	67.6 4.0 20 -6 NS
Test item 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 (d20pc), 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 test 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 test substance at 100 mg/kg bw/day. This was low in amplitude and was not attributed to a toxicological effect of test substance. The test 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 test 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).

Reference 2

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:

other: No treatment-related effects observed on any of the parameters examined

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:

other: No treatment-related effects observed on any of the parameters examined

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). Based on the results of the read across study, a similar NOAEL values are expected for the test substance.

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study planned

Study period:

As of 2023 at the earliest. Depending on the results of the other planned studies (e.g. OECD TG 421, 408) and testing proposal acceptance.

Justification for type of information:

Justification for testing proposal:
TESTING PROPOSAL ON VERTEBRATE ANIMALS NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Amides, C8-18 (even numbered) and C18-unsatd., N,N-bis(hydroxyethyl (C8-18 and C18-unsatd. DEA), EC No. 931-329-6
CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION [please address all points below]:
- Available GLP studies : none
- Available non-GLP studies : none
- Historical human data : none
- (Q)SAR : not applicable - In vitro methods : not applicable
- Weight of evidence : not sufficient
- Grouping and read-across : not sufficient
- Substance-tailored exposure driven testing [if applicable] : none
- Approaches in addition to above [if applicable] : not applicable
- Other reasons [if applicable] : none
CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- Proposed adaptations were not considered sufficient to address this endpoint. This was discussed with ECHA in the frame of a Dossier Improvement Action Plan (DIAP).
FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
- Proposed study design: according to OECD Guideline 414 – rat, first species
Proposed test guideline: according to guideline OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
Planned study period: As of end 2023 at the earliest. Depending on on the results of the other planned studies (e.g. OECD TG 421, 408) and testing proposal acceptance.
Test material: Amides, C8-18 (even numbered) and C18-unsatd., N, N-bis(hydroxyethyl) (C8-18 and C18-unsatd. DEA); lot no: not specified,

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Species:

rat

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 data available.

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 (d20pc), 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 test 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 test substance at 100 mg/kg bw/day. This was low in amplitude and was not attributed to a toxicological effect of test substance. The test 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 test 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 C8-18 and C18-unsatd. DEA.

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 vivo findings, 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.

Justification for classification or non-classification

The available data suggests that the test substance is not a reproductive toxicant with regard to fertility or developmental effects. However, the registered compositions indicate that some of the test substances may contain ≥3% free DEA. This constituent has now been self-classified by its REACH registrants as Repr. 2 -H361: Suspected of damaging fertility or the unborn child(https://echa.europa.eu/registration-dossier/-/registered-dossier/15770/2/1/?documentUUID=b015c4bf-61f0-4b7b-b858-cfe6f686f088) according to CLP (EC 1272/2008) criteria.Therefore, based on the mixture classification rules, the test substance warrants no classification for reproductive or developmental toxicity when its free DEA content is <3% but classification as Repr. 2 -H361: Suspected of damaging fertility or the unborn child when the free DEA content is ≥3%