(Z)-N-octadecyldocos-13-enamide

Endpoint summary

Description of key information

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

NOAEL (OECD 408, oral, rat): >= 1000 mg/kg bw/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

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:

prothrombin time (PT) was described as "unusually high" but no historical control data on PT were provided.

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 oral 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

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

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.

Observations and examinations performed and frequency:

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. On PND 4 and 13, blood was collected from F1 offspring by decapitation.
- Anaesthetic used for blood collection: In adult animals, blood was collected under isoflurane anaesthesia. For the offspring, no anaesthesia was used.
- 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).
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).

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

Sacrifice and pathology:

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.
F1 offspring for thyroid hormone sampling was sacrificed on PND 4 or 13 by decapitation. F1 offspring not selected for thyroid hormone sampling was sacrificed on PND 13 by intraperitoneal injection of sodium pentobarbitone.

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.


For details on terminal procedures regarding reproductive and developmental parameters, please refer to section 7.8.

Statistics:

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

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

Description (incidence and severity):

Body weight adjusted testes weights were statistically significantly high for males that received 100, 400 or 1000 mg/kg bw/day when compared to the controls however, a dose dependent response was not observed. For females that received 1000 mg/kg bw/day, bodyweight adjusted spleen weights were slightly high when compared to the controls and attained statistical significance. Since 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, these organ changes were considered to be incidental and of no toxicological significance.
For details please refer to Table no. 5 under “Any other information on results incl. tables”.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no treatment related macroscopic abnormalities at scheduled termination for males or females.

Neuropathological findings:

not examined

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.

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

Critical 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

 

Conclusions:

Under the conditions of the study, the NOAEL for systemic toxicity was the highest dose tested of >= 1000 mg/kg bw/day in male and female Han Wistar rats.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

GLP guideline study with acceptable restriction. Purity not reported.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

Guideline not stated, purity not reported.

Qualifier:

according to guideline

Guideline:

other: Commission of the European Communities CS/PM/2024, 2 April 1993

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl: CD(SD)BR (VAF plus)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, England
- Age at study initiation: 5 to 6 weeks
- Weight at study initiation: males 141-176 g, females 112-148 g
- Fasting period before study: not applicable, feeding study
- Housing: groups of 5 by sex, in grid-bottomed stainless steel cages (Modular Systems, Kent, England), suspended over paper-lined trays
- Diet (e.g. ad libitum): Powdered diet (SQC Rat and Mouse Maintenance Diet No. 1, Ground Fine, Special Diets Services Limited, Witham, England), ad libitum (except for those periods of deprivation associated with laboratory investigations).
- Water (e.g. ad libitum): Mains water provided in polypropylene bottles, ad libitum (except for those periods of deprivation associated with laboratory investigations).
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-25
- Humidity (%): 28-48
- Air changes (per hr): air conditioned, not further specified
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Diet mixes were formulated weekly.
- Mixing appropriate amounts with (Type of food): Powdered diet (SQC Rat and Mouse Maintenance diet No. 1, Ground Fine, Special Diets Services, Witham, England). A pre-mix was prepared at an appropriate concentration by mixing a weighed amount of test article with a small quantity of diet. This was then mixed with more diet in a rotary mixer and made up to a final quantity with the remaining blank diet in a double-cone blender. To prepare formulated diets appropriate quantities of the pre-mix were mixed with blank diet to make up to the final quantity in a double-cone blender. Formulated diets for males and females were mixed separately.
- Storage temperature of food: not reported

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

To determine the achieved concentration, samples of each diet (including that for the control group), prepared for weeks 1 and 13, were analysed for test article by the Sponsor using a previously validated method. The results are reported separately by the Sponsor.
The stability and homogeneity of test article diets prepared over the concentration ranges used on this study have been assessed by the Sponsor, as well, using the previously validated method.

The achieved dose levels were within 3% of nominal. All individual group values were within 15% of nominal.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

continuously in diet

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

nominal in diet

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

nominal in diet

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

nominal in diet

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Dose levels were selected by the Sponsor after examining preliminary work performed by Quintiles (Quintiles Study Number: CRC/01/C).

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily for mortality and morbidity, once daily for clinical signs; if required by clinical condition of an animal, monitoring was adjusted accordingly.
- Cage side observations included: Mortality, morbidity, clinical signs.

BODY WEIGHT: Yes
- Time schedule for examinations: On the day prior to dosing and then weekly thereafter.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each group determined weekly, and mean daily diet consumption per animal calculated as g food/animal/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Two days prior to dosing and on Day 85.
- Dose groups that were examined: All animals before dosing, and control and high dose group on Day 85

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 13
- Anaesthetic used for blood collection: No, blood was taken via tail vein puncture
- Animals fasted: Yes, overnight
- How many animals: All animals
- Parameters examined (in blood taken into EDTA anticoagulant): Erythrocyte count, hemoglobin concentration, leukocyte differential count, mean cell hemoglobin, mean cell hemoglobin concentration, mean cell volume, packed cell volume, platelet count, total leukocyte count.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 13
- Animals fasted: Yes, overnight
- How many animals: All animals
- Parameters examined (in blood taken into lithium heparin anticoagulant): A/G ratio, alanine aminotransferase, albumin, alkaline phosphatase, aspartate aminotransferase, blood urea nitrogen, calcium, chloride, cholesterol, creatinine, glucose, phosphorus, potassium, sodium, total bilirubin, total protein. Coagulation tests (on blood taken into 3.2% aqueous trisodium citrate anticoagulant): activated partial thromboplastin time, fibrinogen, prothrombin time.

URINALYSIS: Yes
- Time schedule for collection of urine: Week 13
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes, overnight
- Parameters examined: Bilirubin, blood pigments, glucose, ketones, leukocytes, microscopic examination of sedimented deposit, nitrites, pH, protein, specific gravity, urobilinogen, volume.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes; all animals were killed by CO2 asphyxiation, weighed and examined externally. The animals were then exsanguinated. The cranial, thoracic and abdominal cavities were opened and examined macroscopically. Any abnormalities were recorded with details of the location, colour, shape and size.

HISTOPATHOLOGY: Yes; with the exception of the eyes, optic nerves and testes, either whole organs or samples of the following tissues were preserved in neutral buffered formaldehyde. The eyes and optic nerves were fixed in Davidson's fluid and the testes were fixed in Bouin's fixative for 24 hours and then transferred to neutral buffered formaldehyde. Organs examined: adrenals, aorta, brain (3 sections), caecum, colon, duodenum, epididymides, eyes (incl. optic nerves), femur (incl. marrow) and joint, heart, ileum, jejunum, kidneys, liver, lungs (incl. mainstem bronchi), mesenteric lymph node, oesophagus, ovaries, pancreas, pituitary, prostate, rectum, salivary gland, sciatic nerve, seminal vesicles, site of mammary gland, skeletal muscle, skin, spinal cord (3 levels), spleen, sternum, stomach, submandibular lymph node, testes, thymus, thyroids, (incl. parathyroids), trachea, urinary bladder, uterus, vagina, all gross lesions. The aforementioned organs were examined from control and high dose animals and from all animals deceased or killed during the study; lungs and all gross lesions were examined from all animals. If target organs were identified in high dose animals they were examined in low and intermediate dose animals, as well.

Other examinations:

ORGAN WEIGHTS
Absolute and relative organ weights from all animals: adrenals, brain, heart, kidneys, liver, ovaries, pituitary, spleen, testes, thymus, thyroids; contralateral organs were weighed together.

Statistics:

For details on statistical methods, please refer to "Any other information on materials and methods incl. tables

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Hairloss was noted in the treated animals, which is a common finding in animals of this strain and age and lacked any dose-relationship.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One mid dose female was killed in extremis on day 50, clinical signs included peribuccal swelling, teeth abnormalities and black periorbital staining. Histopathology revealed peribuccal ulceration. In view of the isolated nature of this finding it was considered to be unrelated to treatment.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Bodyweight and bodyweight gains were considered to be unaffected by treatment. Body weight gains were slightly reduced in all treated male groups, however these values were within 8% of controls and therefore considered not to be an effect of treatment. Female groups showed bodyweight gains within 3% of controls. The slight bodyweight loss noted in week 13 of the study was considered to be attributable to blood sampling procedures.
For details please refer to the document "OECD 408_tables" under "Attached background material".

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was unaffected by treatment and was similar in all groups.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related findings at ophthalmoscopic examination.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

A non-dose-related increase in monocytes was apparent in all treated male groups. Group mean values for the low and the mid dose groups were within the expected background ranges. However, 5/10 males of the high dose group exceeded the normal range. Due to the small magnitude of this change and in the absence of any related pathological changes, this was considered to be without toxicological significance.
All treated females showed reduced activated partial thromboplastin times compared to controls; however, this was considered to be due to two animals in the control group having high values and not to any effect of the test substance.
For details please refer to the document "OECD 408_tables" under "Attached background material".

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

There were no changes considered to be of toxicological significance. The statistically significant values noted in several parameters (creatinine, albumin) were minor, and all group mean values were within the normal ranges found in this laboratory.
For details please refer to the document "OECD 408_tables" under "Attached background material".

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no changes attributable to treatment. The trace and small amounts of epithelial cells and casts noted were apparent throughout the dose groups, and in the absence of any supporting pathology this was considered to be unrelated to the test substance.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

A statistically significant reduction in absolute liver weights was observed in all treated male groups, and a statistically significant reduction in bodyweight-related liver weights was observed in males of the mid and high dose group. In the absence of corresponding histopathological findings this was considered not to be of toxicological significance.
A statistically significant reduction in absolute brain weight was apparent in all treated males. However, there was no effect on bodyweight-related brain weights, and therefore this finding was considered to be unrelated to treatment.
Body weight-related heart weights were statistically higher than in controls in all treated female groups. As this change was minor and lacked a dose-related manner, it was considered not to be of any toxicological significance.
For details please refer to the document "OECD 408_tables" under "Attached background material".

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related macroscopic findings.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related microscopic findings. The small number of histopathological findings recorded were within the normal range of background alterations which may be seen in untreated rats of this age and strain.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

There were no neoplastic findings reported.

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 adverse effects observed up to the highest dose tested.

Key result

Critical effects observed:

not specified

Conclusions:

The repeated daily administration of the test substance in the diet to the Crl:CD(SD)BR(VAF plus) strain for 13 weeks at dose levels of 250, 500 and 1000 mg/kg bw/day did not reveal any evidence of toxicity. Therefore, the NOAEL was determined to be 1000 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

The available information comprises adequate, reliable (Klimisch score 1-2) and consistent studies, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, of Regulation (EC) No 1907/2006.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Oral

Subacute

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, 2021). 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.

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. Changes in haematology parameters were not considered adverse and were limited to a non-dose-dependent increase in total white blood cell concentrations in all groups of treated males when compared to the controls, attributable to increases in neutrophil, monocyte and large unstained cell concentrations, and a slight decrease in mean cell volume in females given 1000 mg/kg bw/day. When compared to controls, analysis of the blood plasma at scheduled termination revealed a dose dependent reduction in glucose concentration and an increase in potassium concentration in males given 400 or 1000 mg/kg bw/day, increased chloride concentrations in all groups of treated males, increased sodium concentrations in males given 1000 mg/kg bw/day, and low total protein concentrations in all groups of treated females. These findings were not considered adverse. 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 and a slight increase in spleen weights in females given 1000 mg/kg bw/day. Test item-related but non-adverse histopathological findings were observed in the liver, mesenteric lymph node and jejunum for F0 males and females. 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. 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. Based on the absence of adverse effects, it was concluded that≥1000 mg/kg bw/day was the No Observed-Adverse-Effect (NOAEL) for systemic toxicity.

In addition, there is a further study available on the subacute oral toxicity of (Z)-N-octadecyldocos-13-enamide (CAS 10094-45-8) in female Holtzman rats via oral gavage, which was not performed according to a specific test guideline (Leberco Laboratories, 1963). During 5 consecutive days, 10 animals were administered daily the test substance in olive oil in 2 dose intervals of 5 mL total volume, amounting to a daily total dose of approx. 5000 mg/kg bw/day. During the treatment and a subsequent 20-day post-exposure recovery period, no mortalities and clinical signs of toxicity were noted. The animals showed a normal body weight gain during the entire study period. At sacrifice, no gross pathological abnormalities were noted in any of the treated animals. Based on the results of this study, the NOEL of the test substance in female rats was considered to be approx. 5000 mg/kg bw/day.

 

Subchronic

The subchronic oral toxicity of (Z)-N-octadecyldocos-13-enamide (CAS 10094-45-8) was investigated in male and female Crl: CD(SD)BR (VAF plus) rats in a GLP-conform study according to the requirements of the Commission of the European Communities CS/PM/2024 (2 April 1993) and similar to OECD 408 (Quintiles Toxicology, 1998). Based on a preliminary dose-range finding study, the test substance was administered daily ad libitum for a period of 13 weeks to groups of 10 animals per sex at dietary dose levels of 250, 500 and 1000 mg/kg bw/day, respectively. A similar constituted control group received the plain diet. On Day 50 of the study, one female from the 500 mg/kg bw/day dose group was killed in extremis due to clinical signs including peribuccal swelling, teeth abnormalities and black periorbital staining. Histopathology of this animal revealed peribuccal ulceration, which in view of the isolated nature of this finding was considered to be unrelated to treatment. In treated animals, hair loss was noted among all dose groups, which, in the absence of any dose-response relationship, was not considered to be a treatment-related, since it was reported to be a common finding in animals of this strain and age. Body weights as well as food and water consumption in treated animals were not significantly altered compared to those of controls. At the terminal ophthalmoscopic examination, no adverse findings on eyes attributable to treatment were noted. Clinical chemistry analysis revealed sporadic differences in some parameters which were not dose-related and within the normal range of control values, and thus not toxicologically relevant. At haematological analysis, a non-dose-related increase in monocytes was apparent in males of all treatment groups. However, group mean values for animals treated with 250 and 500 mg/kg bw/day were within the expected background ranges of the controls. Only in 5/10 males of the high dose group the normal range in the number of monocytes was exceeded, but this change was considered to be without toxicological significance due to the small magnitude and in the absence of any corresponding pathological changes. In urine, trace and small amounts of epithelial cells and casts were apparent throughout the dose groups, but in the absence of any supporting pathology this was considered to be unrelated to the test substance. Determination of organ weights revealed a statistically significant reduction in absolute liver weights in males of all treatment groups, whereas a statistically significant decrease in relative liver weights was only noted in males of the 500 and 1000 mg/kg bw/day dose group. In the absence of any corresponding histopathological findings, the changes in liver weight were considered to be non-adverse, and thus of no toxicological significance. All other statistically significant effects on organ weights (brain and heart) were not considered to be treatment-related as they occurred in the absence of any histopathological finding, and were thus considered to be of no toxicological relevance. Gross necropsy and histopathological (neoplastic and non-neoplastic) examination, including male and female reproduction organs, did not reveal any pathological findings related to treatment with the test substance. Based on the overall effects of the study, a NOAEL of ≥ 1000 mg/kg bw/day for male and female rats was derived.

In addition, a subchronic oral study performed with the source substance (Z)-N-octadec-9-enylhexadecan-1-amide (CAS16260-09-6) was included in the technical dossier as bridging study to show a similar lack of effects in source and target substance supporting the read-across for toxicological endpoints (Acute oral and dermal toxicity) in accordance with the Read-Across Assessment Framework (RAAF) (ECHA, 2017).

A detailed justification for the grouping of chemicals and read-across is provided in the technical dossier (see IUCLID section 13.2).

Justification for classification or non-classification

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