(Z)-N-octadecyldocos-13-enamide

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

NOAEL (OECD 422, oral, rat): >= 1000 mg/kg bw/day

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 May - 09 Oct 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 2016

Deviations:

yes

Remarks:

no historical control data provided for several parameters in haematology (prothrombine time, although the results were described as "unusually high"), clinical chemistry, or reproductive performance

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

RccHan™;WIST

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS (UK) Ltd.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Males: 83 to 90 days old; Females: 98 to 104 days old.
- Weight at study initiation: Males: 326 to 355 g; Females: 196 to 239 g.
- Fasting period before study: no
- Housing: Prior to mating, the animals were housed in groups of up to 4/sex in polycarbonate cages with a stainless steel mesh lid and with softwood based bark-free fiber bedding. During mating, males and females were housed in a 1 : 1 ratio in grid bottomed cages with absorbent paper. After mating, males were housed in up to 4 animals while females were housed individually. Throughout the study, the cages were enriched with an aspen chew block (except during late gestation and lactation), a plastic shelter (except during pairing, late gestation and lactation) and with paper shavings from Day 20 after mating and throughout lactation.
- Diet: SDS VRF1 certified pelleted diet, ad libitum
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum
- Acclimation period:
Males: Six days before commencement of treatment;
Females: 20 days before commencement of treatment.

DETAILS OF FOOD AND WATER QUALITY:
Certificates of analysis for the diet were scrutinized and approved before any batch of diet was released for use. Certificates of analysis were routinely provided by the water supplier.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-70
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 26 May 2020 To: 19 July 2020

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of test item was ground in a mortar using a pestle and mixed with some vehicle to form a paste. Further amounts of vehicle were gradually added and mixed to produce a smooth, pourable suspension. The suspension was quantitatively transferred and diluted to volume and finally mixed using a high-shear homogenizer.
A series of suspensions at the required concentrations were prepared by dilution of individual weighings of the test item.
During preparation, formulations were stirred overnight using a magnetic stirrer.
Test item dosing formulations were prepared weekly and stored at ambient temperature (15 to 25°C).

VEHICLE
- Justification for use and choice of vehicle (if other than water):
Due to the low water solubility of the test item, propylene glycol was selected as the vehicle as it had proved suitable for dissolving the test substance and because there was an acceptable analytical method available for analysis of the test formulations.
- Concentration in vehicle: 16.66, 66.66 and 166.66 mg/mL
- Amount of vehicle: 6 mL/kg bw

Details on mating procedure:

- M/F ratio per cage: 1:1 within the same treatment groups.
- Length of cohabitation: Up to two weeks.
- Proof of pregnancy: Ejected copulation plugs in cage tray or sperm in vaginal smear referred to as Day 0 of gestation.
- After successful mating each pregnant female was caged individually.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each of the first and last preparation formulations were analysed for achieved concentration of the test item after extraction in and dilution in tetrahydrofuran (THF) followed by gas chromatographic analysis with flame ionisation detection (FID). Sample concentrations were determined with reference to external standards prepared in the concentration range of 20 µg/mL to 200 µg/mL. The mean concentrations were within 13% of the nominal concentration, confirming the accuracy of formulation. The difference from mean remained within 5%, confirming precise analysis. Procedural recoveries remained within the range established during the validation, confirming the continued accuracy of the analytical procedure. For the first and second preparation procedural recoveries were between 94.4% and 102.3%, and between 98.3% and 104.3%, respectively.
In addition, the homogeneity and stability of formulations during storage were confirmed as part of another study. It was demonstrated that formulations in the concentration range of 2 and 166.66 mg/mL were stable at ambient temperature (15 to 25°C) for 15 days or refrigerated (2 to 8°C) for 15 days.

Duration of treatment / exposure:

Males were treated daily for 2 weeks before pairing, up to necropsy after a minimum of 5 consecutive weeks.
Females that delivered were treated daily for 2 weeks before pairing, throughout pairing, gestation and until Day 12 of lactation.

Frequency of treatment:

daily, 7 days/week

Details on study schedule:

not applicable for an OECD 422 study

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

400 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels selected for investigation in this main combined repeated dose toxicity study with reproduction/developmental toxicity screening test (0, 100, 400 and 1000 mg/kg bw/day) were selected in conjunction with the sponsor and were based on the results of the 14-day repeated dose oral range-finding toxicity study (non-GLP) with the test item conducted at this laboratory. In that study, the same dose sequence was employed and there were no premature deaths and no signs observed in relation to the administration of dose. For males that received 1000 mg/kg bw/day, there was a suggestion of slightly reduced body weight gain from Days 11 to 15 when compared to males that received 400 mg/kg bw/day. For females that received 1000 mg/kg bw/day, there was a suggestion of slightly reduced body weight gain from Days 8 to 15 when compared to females that received 400 mg/kg bw/day. There was no effect of treatment on food intake and there were no macroscopic abnormalities detected at scheduled termination on Day 15.
Therefore, 1000 mg/kg bw/day was selected as the high dose level for the current study (1000 mg/kg bw/day represents the maximum feasible dose based on the viscosity of the test formulations) with 100 and 400 mg/kg bw/day selected as the low and intermediate dose levels, respectively, to assess any dose-response relationship.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily
- Cage side observations included: signs associated with dosing, clinical signs of ill-health and mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Prior to treatment, once per week during treatment, on Days 0, 6, 13 and 20 after mating and on Days 1, 6 and 12 of lactation.
- Detailed clinical observations included: Detailed physical examinations and arena observations.

BODY WEIGHT: Yes
- Time schedule for examinations in males: Prior to dosing on the day that treatment commenced (Day 1) and weekly thereafter.
- Time schedule for examinations in females: Prior to dosing on the day that treatment commenced (Day 1), weekly before pairing, on Days 0, 7, 14 and 20 after mating, in Day 1, 4, 7 and 13 of lactation and on the day of necropsy.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At termination, blood samples of approx. 0.5 mL were collected from the sublingual vein into tubes containing EDTA anticoagulant.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: No
- How many animals:
The 5 lowest numbered surviving males and the first 5 (control and low dose) or 6 (mid and high dose) lactating females with a surviving litter per group.
- Parameters examined: haematocrit (Hct), haemoglobin concentration (Hb), erythrocyte count (RBC), absolute reticulocyte count (Retic), mean cell haemoglobin (MCH), mean cell haemoglobin concentration (MCHC), mean cell volume (MCV), red cell distribution width (RDW), total leucocyte count (WBC), differential leucocyte count (neutrophils (N), lymphocytes (L), eosinophils (E), basophils (B), monocytes (M), large unstained cells (LUC), platelet count (Plt), prothrombin time (PT) and activated partial thromboplastin time (APTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At termination, blood samples of approx. 0.5 mL were collected from the sublingual vein into tubes containing lithium heparin as anticoagulant.
- Animals fasted: No
- Anaesthetic used for blood collection: Yes (isoflurane)
- How many animals: The 5 lowest numbered surviving males and the first 5 (control and low dose) or 6 (mid and high dose) lactating females with a surviving litter per group.
- Parameters examined: alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (Bili), bile acids (Bi Ac), urea, creatinine (Creat), glucose (Gluc), total cholesterol (Chol), triglycerides (Trig), sodium (Na), potassium (K), chloride (Cl), calcium (Ca), inorganic phosphorus (Phos), total protein (Total Prot), albumin (Alb) and albumin/globulin ratio (A/G Ratio).

THYROID HORMONE ANALYSIS: Yes
- Time of blood sample collection: At termination, blood samples were collected from surviving F0 males and F0 females from the sublingual vein.
- Anaesthetic used for blood collection: In adult animals, blood was collected under isoflurane anaesthesia.
- Animals fasted: No
- How many animals:
At termination: All surviving F0 males and all surviving F0 reproductive phase females (no samples obtained from females that failed to litter).
- Parameters examined: thyroxine (T4) and thyroid stimulating hormone (TSH).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations and dose groups examined:
Prior to dosing, on the 5 lowest numbered surviving males in each group during week 5 of treatment and on the first 5 lactating females per group at Day 7 – 9 of lactation.
- Battery of functions tested: sensory activity (approach response, pinna reflex, auditory startle reflex and tail pinch response), grip strength (forelimb and hindlimp grip strength) and motor activity

IMMUNOLOGY: No

Oestrous cyclicity (parental animals):

Oestrous cycles were evaluated by examination of vaginal smears. Dry smears were taken for 15 days before pairing using cotton swabs. Wet smears were taken for 14 days before treatment, after pairing until mating using pipette lavage and four days before scheduled termination.

Sperm parameters (parental animals):

A detailed qualitative examination of the testes was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the lumen. Any cell- or stage-specificity of testicular findings was noted.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number of pups (daily), sex ratio (on PND 1, 4, 7 and 13), stillbirths, live births, postnatal mortality, presence of gross anomalies, body weight (on PND 1, 4, 7, 11 and 13), physical or behavioural abnormalities, anogenital distance (AGD, on PND 1) and presence of nipples/areolae in male pups (on PND 13).

GROSS EXAMINATION OF DEAD PUPS:
Yes, where possible a macroscopic examination (external) with an assessment of stomach for milk content was performed for prematurely dead pups.

THYROID HORMONE ANALYSIS:
- Time schedule for collection of blood: On PND 4 and 13, blood was collected from F1 offspring by decapitation.
- Anaesthetic used for blood collection: No anaesthesia was used.
- Animals fasted: No
- How many animals:
On PND 4: Offspring from up to 2 females per litter.
On PND 13: 2 males and 2 females per litter (where possible).
- Parameters examined: thyroxine (T4) and thyroid stimulating hormone (TSH). T4 examination was given priority over TSH.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No

Postmortem examinations (parental animals):

SACRIFICE:
F0 Males were sacrificed after 5 weeks of treatment by carbon dioxide inhalation.
F0 females were sacrificed on lactation Day 13 by carbon dioxide inhalation.
F0 females that failed to produce a viable litter were sacrificed 25 days after mating by carbon dioxide inhalation.

GROSS PATHOLOGY: Yes
All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any of the organs and tissues (external and cut surface) mentioned below was recorded and the required tissue samples preserved in appropriate fixative.

ORGAN WEIGHTS:
The following organ weights were collected for the 5 lowest numbered surviving F0 males and F0 females with a surviving litter per group at scheduled termination and all F0 adult decedents: adrenals, brain, epididymides, heart, kidneys, liver, ovaries, prostrate, seminal vesicles with coagulating glands, spleen, testes and thymus.
The following organ weights were determined for the remaining F0 males and females per group: epididymides, ovaries, prostrate, seminal vesicles with coagulating glands, testes and uterus (including cervix and oviducts).
For bilateral organs, left and right organs were weighed together.

HISTOPATHOLOGY:
Tissue samples were dehydrated, embedded in paraffin wax and sectioned. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required. Sections were stained with hematoxylin and eosin; in addition samples of the testes were stained using a standard periodic acid/Schiff (PAS) method.
Histopathological examination of the following organs and tissues was performed for the five lowest numbered surviving males and females with a surviving litter per group at scheduled termination, and all F0 adult decedents:
Adrenals, brain (including cerebrum, cerebellum and pons), caecum, colon, duodenum, epididymides, eyes, heart (including auricular and ventricular regions), ileum, jejunum, kidneys, liver (sections from 2 lobes), lungs (section from 2 major lobes including bronchi), lymph nodes (left axillary and mesenteric), ovaries, Peyer’s patches, prostate, rectum, seminal vesicles with coagulating glands, skin with mammary glands (inguinal area), spinal cord (transverse and longitudinal sections at the cervical level), spleen, sternum (with marrow), stomach, testes, thymus, thyroids, trachea, urinary bladder and vagina. From the sciatic nerve and from the skeletal muscle only one sample was examined. From the uterus with cervix and oviducts samples were fixed, but only uterus and cervix were examined histopathologically.

In addition to the organs listed above, all abnormalities were investigated microscopically for all F0 males and females. Further, for the remaining F0 males and females per group the following organs were fixed, but not examined: epididymides, ovaries, prostrate, seminal vesicles with coagulating glands, testes, thyroids, uterus (including cervix and oviducts) and vagina.

Postmortem examinations (offspring):

SACRIFICE
- Offspring selected for thyroid hormone sampling on PND 4 and 13 was sacrificed by decapitation. Offspring not selected for thyroid hormone sampling was sacrificed on PND 13 by intraperitoneal injection of sodium pentobarbitone.

GROSS NECROPSY
- Gross necropsy consisted of external examination. Particular attention was paid to external genitals. Thyroid glands were preserved from one male and one female in each litter, where possible.

HISTOPATHOLOGY / ORGAN WEIGTHS: not performed

Statistics:

Please refer to the document "Statistical Analyis_OECD 422" under "Attached background material".

Reproductive indices:

Percentage mating (%) = (Number of animals mating / Animals paired) x 100;
Conception rate (%) = (Number of animals achieving pregnancy / Animals mated) x 100;
Fertility index (%) = (Number of animals achieving pregnancy / Animals paired) x 100;
Gestation index (%) = (Number of live litters born / Number of pregnant females) x 100;
Duration of gestation = Time elapsing between the detection of mating and commencement of parturition;
Pre-coital interval = Time between the first pairing and evidence of mating

Offspring viability indices:

- Post-implantation survival index (%) = (Total number of offspring born / Total number of uterine implantation sites) x 100;
- Live birth index (%) = (Number of live offspring on Day 1 after littering / Total number of offspring born) x 100;
- Viability index (%) = (Number of live offspring on Day 4 (before blood sampling) / Number of live offspring on Day 1 after littering) x 100;
- Lactation index (%) = (Number of live offspring on Day 13 after littering / Number of live offspring on Day 4 (after blood sampling)) x 100;
Offspring sex ratio:
Percentage males (%) = Number of males in litter / Total number of offspring in litter

Clinical signs:

no effects observed

Description (incidence and severity):

There were no treatment related clinical signs and no signs associated with dose administration for males and females throughout the study.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female that received 400 mg/kg bw/day was killed on Day 1 of lactation due to total litter loss. This female had given birth to one live female pup which was subsequently killed for welfare reasons. Macroscopic examination revealed three implantation sites in the uterus, and the mammary tissue was inactive. In the absence of similar instances of litter loss in the 1000 mg/kg bw/day group, this isolated litter loss at 400 mg/kg bw/day was considered incidental and unrelated to treatment.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There was considered to be no effect of treatment at any dose level investigated on the group mean body weight gain of males throughout the study, or of females during the 2-week pre-pairing, gestation or lactation periods. Occasional differences from control attained statistical significance, however these differences were small and showed no consistency between the sexes, and were therefore attributed to normal biological variation.
For details please refer to Table no. 1 under “Any other information on results incl. tables”.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no effect of treatment on group mean food intake for males from Day 1 to 36 of treatment and females from Day 1 to 15 of treatment (prior to pairing), Day 0 to 20 of gestation or Day 1 to 13 of lactation at 100, 400 or 1000 mg/kg bw/day.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Haematological investigations conducted at scheduled termination did not reveal any evidence of an adverse effect of treatment.
In all groups of treated males, a non-dose-dependent increase in total white blood cell concentrations was apparent when compared to controls, attributable to increases in neutrophil, monocyte and large unstained cell concentrations; several of these differences attained statistical significance. Similar increases in white blood cell concentrations were not apparent in treated females.
Females given 1000 mg/kg bw/day showed a statistically significant decrease in mean cell volume (MCV), although in the absence of any other associated erythrocytic changes. Prothrombin times (PT) were also statistically significantly shortened for females given 1000 mg/kg bw/day when compared to controls, however this difference was attributable to an atypically long PT time for one control female and therefore this apparent difference in PT time was considered incidental.
Without histopathological correlates for the haematological changes, these minor changes were considered to be of no toxicological significance.
For details please refer to Table no. 2 under “Any other information on results incl. tables”.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Biochemical analysis of the plasma at scheduled termination did not reveal any adverse effects of treatment.
Dose dependent reductions in glucose concentrations were evident for males that received 400 or 1000 mg/kg bw/day when compared to the controls with statistical significance attained. In addition, males in all treated groups showed a marked and statistically significant, but non dose dependent, increase in chloride concentration, with potassium concentrations also statistically significantly high in males given 400 or 1000 mg/kg bw/day, and statistically significantly increased sodium concentrations in males given 1000 mg/kg bw/day. Similar changes in glucose and electrolyte concentrations were not apparent in treated females.
In females, total protein concentrations were statistically significantly low in all treated groups when compared to the controls. These values did not exhibit a dose relationship. These minor changes were considered to be of no toxicological significance.
For details please refer to Table no. 3 under “Any other information on results incl. tables”.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Motor activity:
Group mean total activity scores for males given 1000 mg/kg bw/day were statistically significantly low when compared to controls both in terms of low beam breaks (ambulatory activity) and high beam breaks (rearing activity). Review of the individual 6-minute recording periods revealed a general trend towards lower activity counts throughout the 1-hour assessment period.
For females, locomotor activity was considered unaffected at all dose levels investigated. Group mean activity scores showed some inter-group variation and attained an isolated and statistically significant reduction for high beam breaks at the 12-minute interval in the 1000 mg/kg bw/day group, however in the absence of any effects on total beam breaks, this isolated reduction was considered incidental.
For details please refer to Table no. 4 under “Any other information on results incl. tables”.
Sensory reactivity observations and grip strength:
Sensory reactivity values and forelimb and hindlimb grip strength values for males and females that received 100, 400 or 1000 mg/kg bw/day were similar to the controls.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At the terminal sacrifice (after 5 weeks of treatment for males, and on Day 13 of lactation for females), test substance-related microscopic findings were observed in the liver, mesenteric lymph node and jejunum.
In the liver, minimal or slight multifocal macrophage infiltrate was observed in all males and females administered 1000 mg/kg bw/day.
In the mesenteric lymph node, dose-related minimal to moderate increased cellularity of foamy macrophages was observed in all males and females administered 400 or 1000 mg/kg bw/day, and in one male administered 100 mg/kg bw/day.
In the jejunum, dose-related minimal or slight increased foamy macrophages was observed in the lamina propria of the mucosa in all males and the majority of females administered 1000 mg/kg bw/day, and in the majority of males and one female administered 400 mg/kg bw/day.
These findings were considered to represent accumulation of the test substance in macrophages after absorption in the intestines with subsequent distribution to the liver and draining mesenteric lymph nodes. These findings were not considered adverse due to having no associated overt inflammatory, degenerative or hyperplastic processes.
All other microscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls), and/or their severity was as expected for Han Wistar (RccHan™;WIST) rats; therefore, they were not considered to be treatment-related.
There were no histopathological correlates for the haematological or biochemical changes observed (see above). Similarly, there were no histopathological correlates for the slightly increased testes weights in all groups of treated males or for the slightly increased spleen weights in females given 1000 mg/kg bw/day.
For details please refer to Table no. 6 under “Any other information on results incl. tables”.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Thyroid hormone analysis:
The mean serum T4 concentrations in samples obtained from F0 adult male animals were unaffected by treatment with the test material.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Estrous cycles for females that received 100, 400 or 1000 mg/kg bw/day were similar to that of the controls before and during treatment. There was no effect of treatment on the stage of oestrus at termination.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

At histopathological examination the testes revealed normal progression of the spermatogenic cycle, and the expected cell associations and proportions in the various stages of spermatogenesis were present.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

There was no effect of treatment on pre-coital interval, mating performance and fertility, gestation length or gestation index observed at any dose level.
Conception rate and fertility index were slightly reduced for females that received 100 or 400 mg/kg bw/day when compared to the controls. At 400 mg/kg bw/day one female was sacrificed prematurely on Day 1 of lactation due to total litter loss. 1/10 females in the control group, 1/10 females at 100 and 400 mg/kg bw/day, respectively, failed to litter (although were confirmed as having been pregnant). 1/10 females in the control group, 3/10 females at 100 mg/kg bw/day and 2/10 females at 400 mg/kg bw/day failed to litter and were confirmed as not pregnant. There was, however, no effect of treatment on conception rate or fertility index for females that received 1000 mg/kg bw/day and therefore the slight reductions at 100 or 400 mg/kg bw/day were considered incidental.
8, 6, 6 and 10 litters for females that received 0, 100, 400 or 1000 mg/kg bw/day respectively were available to assess litter size, sex ratio, survival ratio, survival indices and body weights (F1).
For details please refer to Table no. 7 under “Any other information on results incl. tables”.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

There were no treatment related clinical signs in the F1 offspring.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

Post implantation survival index (%), live birth index (%), and viability index (%) Day 4 were all considered to be unaffected by treatment.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There was no effect of parental treatment on birth weights of the offspring.
From Day 4 of age, offspring in all treated groups showed non dose-dependent marginally low body weight gain when compared to controls, such that overall mean offspring body weight gain during Days 1-13 of age was 5-15% lower than control. Mean litter size in the control group was, however, slightly lower than in the treated groups, therefore the control offspring had less competition for sucking from the dam, and therefore the differences in offspring body weight gain in the treated groups were considered attributable to the slightly larger litter sizes and unrelated to treatment.
For details please refer to Table no. 8 under “Any other information on results incl. tables”.

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:

no effects observed

Description (incidence and severity):

The sex ratio was unaffected by threatment with the test substance.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

There was no effect of treatment on ano-genital distances for male and female offspring.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

There were no incidences of nipples observed in male offspring on Day 13 of age.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no treatment related macroscopic abnormalities in the F1 offspring.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Thyroid Hormone Analysis: The mean serum T4 concentrations in samples obtained from F1 male and female offspring on Day 13 of age were unaffected by treatment with the test substance.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

developmental

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Table 1: Body weight (g) and body weight gain (g) group mean values ± SD for males and females before pairing (F0)

Sex

Dose group (mg/kg bw/day)

Days

Change

1

8

15

22

29

36

1-8

8-15

15-22

22-29

22-36

1-15

1-36

Male

0

339 ± 8.0

355 ± 12.4

368 ± 14.9

381 ± 17.7

398 ± 14.8

409 ± 16.6

16 ± 4.9

14 ± 5.0

13 ± 5.5

17 ± 5.8

12 ± 6.1

30 ± 8.1

71 ± 11.4

100

341 ± 8.5

357 ± 11.1

377 ± 13.0

388 ± 11.8

404 ± 15.5

419 ± 19.1

17 ± 4.1

20 ± 6.5*

11 ± 4.4

16 ± 5.6

14 ± 4.8

36 ± 7.8

78 ± 12.0

400

338 ± 9.6

357 ± 9.6

380 ± 10.3

385 ± 12.5

400 ± 14.8

414 ± 21.3

19 ± 6.3

22 ± 5.0**

5 ± 7.3

15 ± 5.5

14 ± 7.6

41 ± 9.4**

76 ± 15.7

1000

339 ± 6.6

352 ± 8.6

367 ± 8.4

376 ± 10.7

390 ± 11.6

401 ± 15.7

13 ± 4.7

15 ± 4.4

9 ± 5.3

14 ± 6.6

11 ± 6.6

28 ± 7.1

62 ± 14.9

Female

0

215 ± 11.9

223 ± 13.1

226 ± 14.1

7 ± 6.2

3 ± 5.1

11 ± 6.7

100

214 ± 7.6

219 ± 13.2

224 ± 14.8

5 ± 8.0

5 ± 4.9

9 ± 10.0

400

214 ± 9.6

219 ± 11.2

225 ± 9.4

4 ± 4.9

7 ± 4.1

11 ± 2.6

1000

211 ± 11.2

219 ± 11.6

227 ± 10.1

8 ± 4.5

8 ± 2.3*

16 ± 4.3*

* and **: statistical significance at p < 0.05 and p < 0.01

 

Table 2: Haematology parameters at study termination (F0 animals) mean ± SD

Parameter	Males				Females
Dose group (mg/kg bw/day)	0	100	400	1000	0	100	400	1000
WBC (x10^9/L)	4.78 ± 1.318	6.59 ± 0.654*	6.90 ± 1.448*	6.40 ± 1.374*	4.55 ± 0.848	4.58 ± 1.238	4.96 ± 0.950	4.20 ± 1.000
N (x10^9/L)	0.98 ± 0.341	1.30 ± 0.519	1.51 ± 0.554	1.58 ± 0.378	1.62 ± 0.463	1.74 ± 0.654	2.19 ± 0.480	1.84 ± 0.899
M (x10^9/L)	0.08 ± 0.023	0.14 ± 0.064	0.16 ± 0.029*	0.15 ± 0.061*	0.10 ± 0.047	0.11 ± 0.040	0.13 ± 0.055	0.13 ± 0.047
LUC (x10^9/L)	0.04 ± 0.011	0.09 ± 0.033*	0.12 ± 0.098*	0.09 ± 0.033*	0.04 ± 0.015	0.06 ± 0.033	0.07 ± 0.026	0.05 ± 0.018
MCV (fL)	57.4 ± 1.31	56.4 ± 1.00	55.2 ± 1.17	56.8 ± 0.99	60.6 ± 1.99	58.8 ± 1.78	60.3 ± 1.37	57.9 ± 2.11*
PT (sec)	21.6 ±1.19	21.3 ± 0.33	22.5 ± 1.47	20.9 ± 2.05	23.1 ± 4.01	20.9 ± 1.12	20.5 ± 1.22	19.0 ± 1.28**

White blood cell count (WBC), Neutrophils (N), Monocytes (M), Large unstained cells (LUC), mean cell volume (MCV), prothrombin time (PT)

* and **: statistical significance at p < 0.05 and p < 0.01

 

Table 3: Clinical chemistry parameters at study termination (F0 animals) mean ± SD

Parameter	Males				Females
Dose group (mg/kg bw/day)	0	100	400	1000	0	100	400	1000
Gluc (mmol/L)	12.54 ± 1.249	11.15 ± 1.591	10.65 ± 0.942*	9.98 ± 0.691**	7.45 ± 1.000	8.57 ± 2.051	7.42 ± 1.096	7.78 ± 0.775
Na (mmol/L)	145 ± 0.4	146 ± 0.7	145 ± 1.6	147 ± 1.3**	141 ± 1.9	139 ± 1.6	140 ± 2.3	140 ± 1.7
K (mmol/L)	4.11 ± 0.071	4.52 ± 0.340	4.68 ± 0.088*	4.40 ± 0.494*	4.52 ± 0.615	4.55 ± 0.189	4.60 ± 0.323	4.69 ± 0.463
Cl (mmol/L)	105.7 ± 1.16	109.6 ± 0.74**	109.3 ± 1.96**	109.3 ± 0.76**	98.0 ± 0.96	98.9 ± 6.06	100.2 ± 3.95	96.7 ± 2.13
Total Prot (g/L)	63 ± 2.1	64 ± 2.7	63 ± 1.3	63 ± 1.8	60 ± 1.3	57 ± 3.2*	57 ± 2.3*	57 ± 1.5*

 Glucose (Gluc), Sodium (Na), Potassium (K), Chloride (Cl), Total protein (Total Prot)

* and **: statistical significance at p < 0.05 and p < 0.01

 

Table 4: Motor activity measurements during week 5 of treatment (males) or during Days 7-9 of lactation (females), F0 animals, mean ± SD

Sex	Dose group (mg/kg bw/day)	High beam level				Low beam level
		6 min	30 min	60 min	Total	6 min	30 min	60 min	Total
Male	0	97.2 ± 32.7	24.4 ± 23.7	13.6 ± 11.4	291.6 ± 91.5	210.4 ± 80.3	70.6 ± 38.0	79.2 ±.36.1	942.6 ± 323.1
	100	77.0 ± 15.1	18.0 ± 24.1	11.8 ± 13.9	207.4 ± 66.1	201.4 ± 75.9	54.8 ± 59.4	55.6 ± 64.9	743.8 ± 241.7
	400	92.2 ± 22.2	11.6 ± 19.4	10.2 ± 14.1	238.8 ± 121.8	180.6 ± 44.0	30.4 ± 33.0	37.0 ± 41.0	652.0 ± 279.7
	1000	73.6 ± 26.4	0.0 ± 0.0	3.8 ± 5.5	148.6 ± 50.0*	145.8 ± 51.1	4.2 ± 5.7**	25.8 ± 34.4	451.0 ± 147.8**
	HCD^#, mean	82.8	8.8	9.3	232.8	188.1	38.7	33.2	644.2
	HCD^#, range	61.2 - 116.0	0.0 - 24.8	0.6 - 19.0	138.6 - 383.2	125.2 - 240.8	10.8 - 89.2	3.0 - 71.4	506.0 - 917.6
Female	0	87.2 ± 23.2	31.4 ± 25.8	43.4 ± 33.1	336.2 ± 166.1	188.6 ± 41.9	53.6 ± 40.8	71.4 ± 40.6	757.2 ± 181.7
	100	70.8 ± 27.5	27.6 ± 22.8	22.6 ± 22.3	281.0 ± 111.9	133.4 ± 52.6	80.6 ± 39.1	44.0 ± 37.1	736.2 ± 353.8
	400	62.0 ± 19.7	28.0 ± 15.9	35.4 ± 19.4	291.6 ± 156.0	169.8 ± 36.3	54.0 ± 15.9	53.4 ± 21.5	718.2 ± 155.3
	1000	80.2 ± 38.4	28.2 ± 20.1	37.4 ± 17.8	309.6 ± 140.0	153.8 ± 65.6	62.8 ± 54.2	75.6 ± 36.0	705.4 ± 262.5
	HCD^#, mean	69.8	20.9	23.9	258.4	156.8	52.8	53.9	655.2
	HCD^#, range	49.8 - 99.6	11.0 - 40.0	3.6 - 46.2	162.4 - 400.0	127.2 - 191.2	24.8 - 87.8	18.0 - 80.0	486.6 - 846.2

* and **: statistical significance at p < 0.05 and p < 0.01

^# Historical control data (HCD), total of 14 studies

 

 

Table 5: Organ weight analysis in parental animals (F0) mean ± SD

Parameter	Males				Females
Dose group (mg/kg bw/day)	0	100	400	1000	0	100	400	1000
Terminal body weight (g)	407.1 ± 15.7	418.8 ± 22.6	410.7 ± 20.4	396.5 ± 19.2	291.9 ± 19.9	303.1 ± 12.3	285.9 ± 13.7	296.2 ± 7.5
Testes weight
Absolute (g)	3.699 ± 0.272	4.131 ± 0.412	4.173 ± 0.275	3.917 ± 0.316	-	-	-	-
Relative to bw (g)	3.703	4.094*	4-164*	3.959*	-	-	-	-
Spleen weight
Absolute (g)	0.722 ± 0.146	0.739 ± 0.053	0.740 ± 0.149	0.747 ± 0.072	0.566 ± 0.049	0.603 ± 0.092	0.569 ± 0.049	0.695 ± 0.098
Relative to bw (g)	0.725	0.730	0.739	0.755	0.572	0.586	0.582	0.691*

* and **: statistical significance at p < 0.05 and p < 0.01

 

Table 6: Microscopic findings in F0 males and females at termination mean ± SD

Parameter	Males				Females
Dose group (mg/kg bw/day)	0	100	400	1000	0	100	400	1000
Liver
Number examined	5	5	5	5	5	5	5	5
Infiltrate, macrophage
Minimal	0	0	0	4	0	0	0	4
Slight	0	0	0	1	0	0	0	1
Total	0	0	0	5	0	0	0	5
Mesenteric lymph nodes
Number examined	5	5	5	5	5	5	5	5
Cellularity, increased, macrophages, foamy
Minimal	0	1	4	0	0	0	5	1
Slight	0	0	1	4	0	0	0	4
Moderate	0	0	0	1	0	0	0	0
Total	0	1	5	5	0	0	5	5
Jejunum
Number examined	5	5	5	5	5	5	5	5
Macrophages, increased, foamy, Lamia propria
Minimal	0	0	3	3	0	0	1	3
Slight	0	0	0	2	0	0	0	1
Total	0	0	3	5	0	0	1	4

 

 

Table 7: Mating performance and fertility – group means (F0)

Sex	Dose group (mg/kg bw/day)	Number mating	Number achieving pregnancy	Conception rate (%)	Fertility index (%)
Male	0	10	9	90	90
	100	10	7	70	70
	400	10	8	80	80
	1000	10	10	100	100
Female	0	10	9	90	90
	100	10	7	70	70
	400	10	8	80	80
	1000	10	10	100	100

 

 

Table 8: Body weight (g) and body weight gain (g) group mean values ± SD for offspring (F1)

Sex	Dose group (mg/kg bw/day)	Day of age					Change
		1	4	7	11	13	1-4	4-7	7-11	11-13	1-13
Male	0	6.6 ± 0.66	9.7 ± 1.34	14.6 ± 1.98	22.2 ± 2.94	26.9 ± 3.40	3.2 ± 0.77	4.9 ± 0.88	7.6 ± 1.24	4.7 ± 0.80	20.3 ± 2.86
	100	6.5 ± 0.85	9.3 ± 1.38	13.7 ± 2.13	20.8 ± 2.97	25.1 ± 3.28	2.8 ± 0.58	4.4 ± 0.96	7.1 ± 0.97	4.3 ± 0.61	18.6 ± 2.55
	400	6.4 ± 0.77	9.0 ± 1.12	13.2 ± 1.40	19.9 ± 2.35	23.7 ± 2.43	2.6 ± 0.45	4.2 ± 0.47	6.7 ± 1.11	3.9 ± 0.22	17.3 ± 1.95
	1000	6.4 ± 0.59	9.3 ± 1.14	13.8 ± 1.60	21.4 ± 2.41	25.6 ± 2.64	2.8 ± 0.64	4.5 ± 0.62	7.6 ± 0.99	4.2 ± 0.46*	19.2 ± 2.28
Female	0	6.1 ± 0.67	9.1 ± 1.18	13.9 ± 1.95	21.6 ± 2.98	26.2 ± 3.46	3.0 ± 0.67	4.8 ± 0.93	7.6 ± 1.28	4.6 ± 0.76	20.0 ± 2.95
	100	6.2 ± 0.81	9.0 ± 1.37	13.4 ± 2.04	20.5 ± 2.95	24.7 ± 3.21	2.8 ± 0.60	4.4 ± 0.89	7.1 ± 1.09	4.1 ± 0.38	18.4 ± 2.48
	400	6.1 ± 0.58	8.6 ± 1.05	12.7 ± 1.35	19.2 ± 2.23	23.1 ± 2.26	2.6 ± 0.54	4.0 ± 0.45	6.5 ± 0.97	3.8 ± 0.26	17.0 ± 1.86
	1000	6.1 ± 0.40	9.0 ± 0.98	13.4 ± 1.47	20.9 ± 2.22	25.1 ± 2.58	2.9 ± 0.63	4.4 ± 0.61	7.5 ± 0.91	4.2 ± 0.52	19.0 ± 2.29

* and **: statistical significance at p < 0.05 and p < 0.01

 

Conclusions:

Based on the results of this study, the NOAEL for systemic toxicity was >= 1000 mg/kg bw/day in male and female Han Wistar rats. The NOAELs for reproduction and for development were both determined to be >= 1000 mg/kg bw/day for male and female rats.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

The available information comprises an adequate, reliable (Klimisch score 1) and consistent study, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, of Regulation (EC) No 1907/2006.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Toxicity to reproduction – fertility (screening)

A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test was conducted in Han Wistar rats with administration of (Z)-N-octadecyldocos-13-enamide (CAS 10094-45-8) by oral gavage for at least five weeks (Covance Laboratories Limited, 2021a). The study was performed according to OECD 422 and in compliance with GLP regulations. Three groups of ten male and ten female rats received the test substance at doses of 100, 400 or 1000 mg/kg bw/day by oral gavage administration. Males were treated daily for two weeks before pairing, up to necropsy after a minimum of five consecutive weeks of treatment. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 12 of lactation. A similarly constituted control group received the vehicle, propylene glycol, at the same volume dose as treated groups. Females were allowed to litter, rear their offspring and were sacrificed on Day 13 of lactation. The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. During the study, clinical conditions, detailed physical examinations and arena observations, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, haematology (peripheral blood), blood chemistry, thyroid hormone analysis, oestrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations were undertaken. The clinical condition, litter size and survival, sex ratio, body weight, ano-genital distance, thyroid hormone analysis and macropathology for all offspring were also assessed. Nipple counts were performed on male offspring on Day 13 of age.

There were no mortalities related to treatment. Sensory reactivity observations and grip strength in males and females were unaffected by treatment and motor activity was also considered unaffected by treatment. Group mean body weight gain and food intake values for males and females that received 100, 400 or 1000 mg/kg bw/day were unaffected by treatment throughout the duration of study. For females that received 100, 400 or 1000 mg/kg bw/day, there was no effect of treatment on pre-coital interval, mating performance and fertility, gestation length, gestation index, conception rate, fertility index or pre-terminal oestrous cycles. At scheduled termination, there were no test item-related macroscopic abnormalities observed, and differences in organ weight parameters were limited to a non dose-dependent increase in testes weights in all groups of treated males. There was no effect of treatment on circulating levels of thyroxine (T4) in adult males treated at 100, 400 or 1000 mg/kg bw/day, or in the F1 male and female offspring on Day 13 of age. Litter size, sex ratio, survival and anogenital distance were unaffected by treatment with the test substance. The male offspring did not develop nipples and there were no parental treatment-related macroscopic findings in the offspring. There was no effect of parental treatment on birth weights or subsequent body weight gain of the offspring to Day 13 of age. Administration of the test substance at doses up to and including 1000 mg/kg bw/day was well tolerated and no adverse effects were apparent. It was therefore concluded that≥1000 mg/kg bw/day was the No Observed-Adverse-Effect (NOAEL) for systemic toxicity and reproductive and developmental toxicity.

 

Toxicity to reproduction - fertility (extended one-generation toxicity study)

According to Regulation (EC) No 1907/2006, Annex IX, 8.7.3, column 1, an extended one-generation reproductive toxicity study is not indicated as the available subchronic oral (90-day) toxicity and OECD 422 screening studies in male and female rats conducted with the test substance do not indicate any adverse effects on reproductive performance, organs or tissues as assessed by gross and histopathological examination. Therefore, on behalf of animal welfare, the conduct of additional animal studies on reproduction toxicity with the test substance by any route of exposure is considered scientifically unjustified.

Effects on developmental toxicity

Description of key information

Oral (OECD 414), rat: NOAEL embryo-foetal development >= 1000 mg/kg bw/day

Oral (OECD 414), rat: NOAEL maternal toxicity >= 1000 mg/kg bw/day

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 May - 07 Aug 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adpted in 2018

Deviations:

yes

Remarks:

No special attention paid to external foetal sex (gross examination) compared with internal (gonadal) sex; no indication of incomplete testicular descent /cryptochordism noted in males

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 1998

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

2016

Qualifier:

according to guideline

Guideline:

other: JMAFF, 12 Nohsan No. 8147

Version / remarks:

adopted 2000

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

RccHan™;WIST

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS Limited
- Age at study initiation (females): 77 to 83 days old
- Weight at study initiation (females): 180 to 218 g
- Fasting period before study: no
- Housing: Prior to mating, the animals were housed in groups of up to 4/sex in polycarbonate cages with a stainless steel mesh lid and with softwood based bark-free fiber bedding. During mating, males and females were housed in a 1 : 1 ratio in grid bottomed cages with absorbent paper. After mating, females were housed individually. Throughout the study, the cages were enriched with an aspen chew block and a plastic shelter (except during pairing).
- Diet: SDS VRF1 certified pelleted diet, ad libitum
- Water: potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum
- Acclimation period: Five days before commencement of pairing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-70
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 01 June 2020 To: 25 June 2020

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of test item was ground in a mortar using a pestle and mixed with some vehicle to form a paste. Further amounts of vehicle were gradually added and mixed to produce a smooth, pourable suspension. The suspension was quantitatively transferred and diluted to volume and finally mixed using a high-shear homogenizer.
A series of suspensions at the required concentrations were prepared by dilution of individual weighings of the test item.
Test item dosing formulations were prepared weekly and stored at ambient temperature (15 to 25°C).

VEHICLE
- Justification for use and choice of vehicle (if other than water):
Due to the low water solubility of the test item, propylene glycol was selected as the vehicle as it had proved suitable for dissolving the test substance and because there was an acceptable analytical method available for analysis of the test formulations.
- Concentration in vehicle: 16.66, 66.66 and 166.66 mg/mL
- Amount of vehicle: 6 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each of the first and last preparation formulations were analysed for achieved concentration of the test item after extraction in and dilution in tetrahydrofuran (THF) followed by gas chromatographic analysis with flame ionisation detection (FID). Sample concentrations were determined with reference to external standards prepared in the concentration range of 20 µg/mL to 200 µg/mL. The mean concentrations were within 10% of the nominal concentration, confirming the accuracy of formulation, with the exception of Week 1 Group 3 samples (400 mg/kg bw(day) which were +10.1% from nominal. This minor excursion from the accepted range of +/-10% was considered not to have affected the study. The difference from mean remained within 4%, confirming precise analysis. Procedural recoveries remained within the range established during the validation, confirming the continued accuracy of the analytical procedure. The procedural recoveries prepared with last week samples were between 98.6% and 101.6%. The procedural recoveries prepared with first week samples were not reported due to an incorrect vehicle reagent number transcribed in the raw data. This deviation from the study protocol is not considered to have an impact on the outcome of the study as results were not corrected for recoveries.
In addition, the homogeneity and stability of formulations during storage were confirmed as part of another study. It was demonstrated that formulations in the concentration range of 2 and 166.66 mg/mL were stable at ambient temperature (15 to 25°C) for 15 days or refrigerated (2 to 8°C) for 15 days.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1 with identified stock males
- Length of cohabitation: up to 7 days
- Proof of pregnancy: Ejected copulation plugs in cage tray or sperm in vaginal smear referred to as Day 0 of gestation.

Duration of treatment / exposure:

From Gestation Day 6 to Gestation Day 19 (after mating)

Frequency of treatment:

daily, 7 days/week

Duration of test:

From Gestation Day 0 (proof of pregnancy) to Gestation Day 20 (necropsy)

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

400 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

20 females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels selected for investigation in this main embryo-foetal development study (0, 100, 400 and 1000 mg/kg bw/day) were selected in conjunction with the sponsor and were based on the results of a preliminary embryo-foetal development study and a 14-day repeat dose bridging study with the test substance.
In the preliminary embryo-foetal development study, daily oral gavage doses of 100, 400 and 833 mg/kg bw/day (the highest feasible dose level in the arachis oil vehicle), were administered from Day 6 to 19 of pregnancy. There were no premature deaths, no clinical signs and no effects on food intake. Slightly low body weight gain was observed at 833 mg/kg bw/day on Days 19 and 20 of gestation however, at scheduled termination there were no macroscopic abnormalities detected, no toxicologically significant findings in litter or foetal weights and no findings at the external examination of the foetuses.
The 14-day repeat dose bridging study was employed to determine if there was a change in the toxicity profile of the test substance when formulated using propylene glycol as opposed to arachis oil, which was used in previous toxicology studies. There were no premature deaths and no signs observed in relation to the administration of dose. For males that received 1000 mg/kg bw/day, there was a suggestion of slightly reduced body weight gain from Days 11 to 15 when compared to males that received 400 mg/kg bw/day. There was no effect of treatment on food intake and there were no macroscopic abnormalities detected at scheduled termination on Day 15.
Therefore, 1000 mg/kg bw/day was selected as the high dose level for the current study with 100 and 400 mg/kg bw/day selected as the low and intermediate dose levels, respectively, to assess any dose-response relationship.

- Rationale for animal assignment: Females showing at least two copulation plugs as positive proof of mating were allocated to group and cage position in the sequence of mating. Females mating on any one day were evenly distributed amongst the groups.
- Fasting period before blood sampling for (rat) dam thyroid hormones: no
- Time of day for (rat) dam blood sampling: To minimize any potential confounding effect of the time of day of blood sampling, the time of blood sampling was controlled to allow satisfactory inter-group comparisons.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily
- Cage side observations included: signs associated with dosing, clinical signs of ill-health and mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A detailed physical examination was performed on each animal on Days 0, 5, 12, 18 and 20 after mating to monitor general health.

BODY WEIGHT: Yes
- Time schedule for examinations: on Days 0, 3 and 6-20 after mating

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
For the periods Days 0-3, 3-6, 6-10, 10-14, 14-18 and 18-20 after mating inclusive

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.
- Organs examined:
All abnormalities and the thyroid gland were fixed in 10% neutral buffered formalin. The thyroid gland was weighed, fixed and subjected to a gross and histopathological examination

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes, including cervix and ovaries
For each ovary/uterine horn:
- Number of corpora lutea: Yes
- Number of implantations sites: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Number of foetuses (live and dead).

For apparently non pregnant animals:
The number of uterine implantation sites were checked after staining with ammonium sulphide [modification of the Salewski staining technique].

Blood sampling:

- Plasma: No
- Serum: No

- Other: Thyroid Hormone Analysis
Blood samples were collected at termination from the sublingual vein under isoflurane anaesthesia. Parameters investigated were:
Triiodothyronine (T3), Thyroxine (T4) and Thyroid stimulating hormone (TSH).

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter] (fixed in Bouin's fluid)
- Skeletal examinations: Yes: [half per litter] (fixed in Industrial Methylated Spirit (IMS) and stained with Alizarin Red)
- Head examinations: No
- Anogenital distance of all live rodent pups: Yes

Detailed foetal examinations:
Findings observed were classified, according to severity and incidence, as:
Major abnormalities: normally rare, definitely detrimental to normal subsequent development, possibly lethal, e.g. ventricular septal defect.
Minor abnormalities: minor differences from normal that are detected relatively frequently considered to have little detrimental effect and may be a transient stage in development e.g. bipartite centrum, dilated ureter.
Variants: alternative structures or stages of development occurring regularly in the control population, e.g. number of ribs, incomplete ossification of 5th and 6th sternebrae.
Observations on repeated structures like ribs, vertebrae and sternebrae are reported as the first and last affected element, in the form ‘5th 13th bilateral ribs’, which should be interpreted as ‘5th to 13th bilateral ribs’.

Statistics:

Please refer to the document "Statistical Analyis_OECD 414" under "Attached background material".

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 foetuses / Number of implantations) x 100

Historical control data:

Historical Control data supplied for selected observations where this information is considered to assist interpretation of study data. In this instance minor skeletal historical control data is included

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

On Day 16 and 17 of gestation, piloerection was recorded in one female that received 400 mg/kg bw/day however an association of this finding with treatment was considered unlikely in the absence of similar signs in other females at this or higher dose levels. There were no clinical signs recorded throughout the duration of study that were attributable to treatment with the test material.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no premature decedents.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Minor variations in body weight gain were observed in some instances attaining statistical significance, however these marginal variations were considered incidental and of no biological or toxicological significance. Thus, there was no effect of treatment on group mean body weight gain from Day 6 to 20 of gestation.
For details please refer to Table no. 1 under “Any other information on results incl. tables”.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There were minor variations observed in food consumption attaining in one instance statistical significance, however these marginal variations were considered incidental and of no biological or toxicological significance.
Thus, there was no effect of treatment on group mean food intake for females that received the test substance at doses up to and including 1000 mg/kg bw/day.
For details please refer to Table no. 2 under “Any other information on results incl. tables”.

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):

There was no effect of treatment on thyroid gland weights at any dose level investigated.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

All macroscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls), and/or were as expected for Han Wistar rats of this age; therefore, they were considered not treatment-related.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At terminal sacrifice, an increase in the incidence of thyroid follicular cell hypertrophy was seen in females administered 1000 mg/kg bw/day.
However, the finding was minimal and not affecting the functionality of the follicular cells and consequently considered non-adverse.
For details please refer to Table no. 3 under “Any other information on results incl. tables”.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Thyroid Hormone Analysis:
Administration of the test material at 400 or 1000 mg/kg bw/day was associated with a dose dependent statistically significant increase in serum TSH concentrations when compared to controls. However, the extent of the increase was not considered adverse, and in the absence of a concomitant decrease in serum T3/T4 concentrations or any associated effects on maternal reproductive performance or the survival and development of the foetuses, these differences in serum TSH concentration were considered to be not toxicologically relevant.
There were no changes in serum T3 or T4 concentrations when compared to the controls in the 100, 400 or 1000 mg/kg bw/day groups.
For details please refer to Table no. 4 under “Any other information on results incl. tables”.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There was no effect of treatment on pre- and post-implantation loss.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Description (incidence and severity):

There was no effect of treatment on resorptions (early or late).

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Two control females (No. 19 and 81) were found to be not pregnant at scheduled termination, all other females assigned to study were confirmed as being pregnant. The following assessment of litter data is therefore based on a total of 18, 20, 20 and 20 females with live young at termination on Day 20 of gestation in the Control group and at 100, 400 and 1000 mg/kg bw/day respectively.

Other effects:

no effects observed

Description (incidence and severity):

Placental weight:
There was no effect of maternal treatment on mean placental weight at any dose level investigated.

Key result

Dose descriptor:

NOAEL

Remarks:

maternal toxicity

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: no adverse effects observed

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There was no effect of maternal treatment on mean foetal weights at any dose level investigated. It was noted that mean foetal weights at 1000 mg/kg bw/day were marginally higher than control, with differences in female foetal weights attaining statistical significance, however this marginal increase was considered incidental and of no biological or toxicological significance.
For details please refer to Table no. 5 under “Any other information on results incl. tables”.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

There was no effect of maternal treatment on the number of live young.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There was no effect of maternal treatment on the sex ratio.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

There was no effect of maternal treatment on litter size and weights.

Anogenital distance of all rodent fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

The adjusted mean anogential distance of male foetuses at 1000 mg/kg bw/day was marginally shorter than control, with the difference attaining statistical significance, however in the absence of a similar difference in female foetuses, this marginal decrease was considered incidental and of no biological or toxicological significance.
Thus, there was no effect of maternal treatment on foetal anogenital distance at all dose levels investigated.
For details please refer to Table no. 6 under “Any other information on results incl. tables”.

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

Detailed external foetal examination did not reveal any abnormalities which were considered to be related to maternal treatment with the test substance.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg bw/day there was an increased foetal incidence of 14th short supernumerary ribs and ossified cervical vertebral centra, both of which were within the Historical Control Data range.
As short supernumerary 14th ribs are variants and ossified cervical centra is a marker of foetal maturity, both findings were considered not to be adverse.
For details please refer to Table no. 7 and Table no. 9 under “Any other information on results incl. tables”.

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

Detailed visceral foetal examinations revealed an increased incidence of effects on umbilical artery (left) in treatment groups compared with controls. As no clear dose-response-relatioship was observed with regard to this finding (incidence: 16, 20, 18 and 21 at 0, 100, 400 and 1000 mg/kg bw/day), this finding was considered to be of no biological or toxicological relevance.
For details please refer to Table no. 8 under “Any other information on results incl. tables”.

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Remarks:

embryo-foetal survival and development

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Table 1: Body weight (g) and body weight gain (g) group mean values ± SD during gestation

Sex

Dose group (mg/kg bw/day)

Days

Change

0

6

10

16

19

20

0-3

3-6

7-8

9-10

15-16

19-20

6-20

Female

0

199 ± 8.1

218 ± 8.9

231 ± 10.4

265 ± 11.4

294 ± 14.3

306 ± 15.9

10 ± 4.0

9 ± 3.7

4 ± 2.8

3 ± 3.3

9 ± 3.7

12 ± 3.7

88 ± 10.8

100

196 ± 7.5

214 ± 7.4

226 ± 7.7

255 ± 8.3

282 ± 9.3

294 ± 9.7

10 ± 3.1

9 ± 3.3

3 ± 2.8

5 ± 2.3*

8 ± 2.4

12 ± 3.2

80 ± 6.3

400

199 ± 7.2

217 ± 9.0

228 ± 9.8

258 ± 14.3

287 ± 18.0

299 ± 21.1

9 ± 2.8

9 ± 3.1

3 ± 2.0

5 ± 3.1*

8 ± 2.6

12 ± 4.6

83 ± 14.7

1000

200 ± 7.4

219 ± 10.0

230 ± 11.9

262 ± 14.8

291 ± 17.5

303 ± 19.8

10 ± 3.3

9 ± 3.6

3 ± 3.0*

4 ± 2.3*

8 ± 3.8

11 ± 4.5

83 ± 12.4

* and **: statistical significance at p < 0.05 and p < 0.01

 

Table 2: Food consumption (g/animal/day) group mean values ± SD during gestation

Sex	Dose group (mg/kg bw/day)	Change
		0-3	3-6	6-10	10-14	14-18	18-20
Female	0	16 ± 2.1	18 ± 1.7	15 ± 1.7	18 ± 1.7	19 ± 1.5	19 ± 1.8
	100	15 ± 1.7	17 ± 1.5	15 ± 1.5	17 ± 2.1	18 ± 1.5*	18 ± 1.9
	400	15 ± 2.7	18 ± 2.0	15 ± 1.8	17 ± 2.2	19 ± 2.0	18 ± 2.7
	1000	16 ± 2.1	18 ± 1.4	16 ± 2.2	18 ± 2.4	20 ± 1.9	19 ± 3.2

* and **: statistical significance at p < 0.05 and p < 0.01

 

Table 3: Microscopic findings in females on Day 20 of gestation

Parameter	Females
Dose group (mg/kg bw/day)	0	100	400	1000
Thyroids
Number examined	20	20	20	20
Follicular cell hypertrophy
Minimal	1	1	2	8
Total	1	1	2	8

 

 

Table 4: Serum TSH, T3 and T4 concentration data, mean ± SD

Parameter	Females
Dose group (mg/kg bw/day)	0	100	400	1000
TSH (pg/mL)	1150 ± 531	1130 ± 798	1700 ± 806*	2000 ± 867**
T3 (pg/mL)	449 ± 105	435 ± 104	416 ± 109	445 ± 86.1
T4 (pg/mL)	21800 ± 4680	21400 ± 3630	21300 ± 6450	20100 ± 3960

* and **: statistical significance at p < 0.05 and p < 0.01

Williams test (two-tailed) ANOVA model

 

Table 5: Litter and foetal weight (g) on Day 20 of gestation, group mean values ± SD

Sex	Dose group (mg/kg bw/day)	Total litter weight	Male foetal weight	Female foetal weight	Overall foetal weight
Female	0	40.98 ± 6.833	3.54 ± 0.186	3.31.± 0.212	3.42 ± 0.199
	100	35.43 ± 5.465	3.46 ± 0.338	3.26 ± 0.297	3.35 ± 0.287
	400	38.33 ± 9.299	3.58 ± 0.163	3.36 ± 0.180	3.47 ± 0.142
	1000	40.11 ± 7.979	3.62 ± 0.266	3.47 ± 0.232*	3.55 ± 0.239

* and **: statistical significance at p < 0.05 and p < 0.01

 

Table 6: Anogenital distance - Absolute and adjusted values (mm) on Day 20 of gestation

Sex	Dose group (mg/kg bw/day)	Absolute (mean ± SD)	Adjusted (mean)
Male	0	3.9 ± 0.55	3.9
	100	3.7 ± 0.54	3.7
	400	3.6 ± 0.55	3.6
	1000	3.5 ± 0.53	3.5*
Female	0	2.6 ± 0.36	2.6
	100	2.4 ± 0.33	2.5
	400	2.4 ± 0.36	2.4
	1000	2.4 ± 0.35	2.4

* and **: statistical significance at p < 0.05 and p < 0.01

 

Table 7: Foetal examinations – minor skeletal abnormalities and variant findings (group incidences)

Parameter	Foetuses				Litters
Number examined	110	105	111	114	18	20	20	20
Dose group (mg/kg bw/day)	0	100	400	1000	0	100	400	1000
Minor skeletal abnormalities
Number of 14^th ribs:
Short supernumerary	18	23	28	32	10	12	13	14
Full supernumerary	0	2	0	4	0	2	0	3
Total	18	23	28	33	10	12	13	14
Increased ossification
Cervical vertebral centra (all ossified)	1	2	7	13	1	1	5	4

 

Table 8: Foetal examinations – minor visceral abnormalities and necropsy findings (group incidences)

Parameter	Foetuses				Litters
Number examined	106	107	110	114	18	20	20	20
Dose group (mg/kg bw/day)	0	100	400	1000	0	100	400	1000
Visceral abnormalities
Umbilical artery (left)	16	20	18	21	11	14	12	14

 

Table 9: Fetal examinations – minor skeletal historical control data

 

		8437167								HCD Range
		Fetuses				Litters				Fetuses	Litters
Group		1	2	3	4	1	2	3	4
Number Examined		110	105	111	114	18	20	20	20	1016	187
Minor skeletal abnormalities
Cranial	sutural bone(s)	0	1	1	0	0	1	1	0	0-1	0-1
Ribs	branched with costal cartilage	0	1	0	0	0	1	0	0	0-1	0-1
Sternebrae	hemisternebra	0	0	1	0	0	0	1	0	0-1	0-1
Rib and vertebral configuration
	short supernumerary	18	23	28	32	10	12	13	14	15-36	5-17
Number of 14th ribs	full supernumerary	0	2	0	4	0	2	0	3	0-2	0-2
	total	18	23	28	33	10	12	13	14	15-37	5-17
Thoracolumbar vertebrae	20	0	1	1	1	0	1	1	1	0-3	0-2
Delayed/Incomplete ossification/unossified
Vertebrae	cervical	0	0	0	2	0	0	0	2	0-1	0-1
Appendicular	metatarsals	0	1	0	0	0	1	0	0	0-1	0-1
Increased ossification
Cervical vertebral centra	all ossified	1	2	7	13	1	1	5	4	4-29	3-10

 

 

Conclusions:

Based on the results of this study, the No-Observed-Adverse-Effect-Level (NOAEL) for maternal toxicity and embryo-foetal survival and development was concluded to be >= 1000 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

The available information comprises an adequate, reliable (Klimisch score 1) and consistent study, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, of Regulation (EC) No 1907/2006.

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

Toxicity to reproduction – Effects on developmental toxicity

A GLP-compliant pre-natal developmental toxicity study with (Z)-N-octadecyldocos-13-enamide (CAS 10094-45-8) in Han Wistar rats according to OECD 414 was performed to assess the influence of the substance on embryo-foetal survival and development when administered during the organogenesis and foetal growth phases of pregnancy (Days 6 to 19 after mating) (Covance Laboratories Limited, 2021b). Three groups of 20 females received the test substance at doses of 100, 400 or 1000 mg/kg bw/day by oral gavage administration. A similarly constituted control group received the vehicle, propylene glycol at the same volume dose as the treated groups. Animals were sacrificed on Day 20 after mating for reproductive assessment and foetal examination. Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 20 after mating, blood samples were taken for thyroid hormone analysis and the gravid uterus weight and thyroid weights were recorded. Microscopic pathology investigations of the thyroid glands were also undertaken. Anogenital distance was measured for foetuses and all foetuses were weighed and examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination.

There were no premature deaths throughout the duration of the study and there were no clinical signs or signs associated with dose administration attributable to treatment with the test substance. Serum TSH concentrations were proportionally increased with higher dose levels at 400 or 1000 mg/kg bw/day in pregnant females but was unaffected at 100 mg/kg bw/day, when compared to controls. These differences in serum TSH concentrations were considered incidental and non-adverse. There was no effect of treatment on serum T3 or T4 concentrations at 100, 400 or 1000 mg/kg bw/day. There was no effect of treatment on group mean absolute or adjusted maternal body weight gain, gravid uterine weight or food intake, and there were no macroscopic findings in the dams on Day 20 of gestation considered to be related to treatment at all dose levels investigated with the test substance. At scheduled termination, it was found that there were no treatment-related changes in thyroid gland weights. Microscopic examination revealed an increased incidence of thyroid follicular cell hypertrophy for females that received 1000 mg/kg bw/day. This observation was minimal however, and did not affect the functionality of the follicular cells. Two control females were found not to be pregnant at macroscopic examination, all other females were pregnant with live young. Litter data, as assessed by the mean numbers of implantations, resorptions (early or late), live young, sex ratio or the levels of pre or post implantation losses, was unaffected by treatment at all dose levels investigated. There was no effect of maternal treatment on mean placental, total litter weights or foetal weights, or on ano-genital distance of the foetuses at any dose level investigated. Detailed foetal examination did not reveal any abnormalities clearly related to maternal treatment with the test substance. Based on the results of this study, the No-Observed-Adverse-Effect-Level (NOAEL) for maternal toxicity and embryo-foetal survival and development was concluded to be≥1000 mg/kg bw/day.

Justification for classification or non-classification

The available data on toxicity to reproduction of (Z)-N-octadecyldocos-13-enamide (CAS 10094-45-8) do not meet the criteria for classification according to Regulation (EC) No 1272/2008 and are therefore conclusive but not sufficient for classification.