(Z)-N-octadecyldocos-13-enamide

Toxicity to reproduction

Administrative data

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

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

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

Test material

Test material information

Constituent 1

Reference substance name:

(Z)-N-octadecyldocos-13-enamide

EC Number:

233-226-5

EC Name:

(Z)-N-octadecyldocos-13-enamide

Cas Number:

10094-45-8

Molecular formula:

C40H79NO

IUPAC Name:

(Z)-N-octadecyldocos-13-enamide

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

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.

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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”.

Effect levels (P0)

1

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

2

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

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

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.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

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

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

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

 

Applicant's summary and conclusion

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.