Fatty acids, montan-wax, stearyl esters

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 Dec 2012 - 29 Jan 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 1996

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar 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 endocrine disruptor addressed

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Docosyl docosanoate

EC Number:

241-646-5

EC Name:

Docosyl docosanoate

Cas Number:

17671-27-1

Molecular formula:

C44H88O2

IUPAC Name:

docosyl docosanoate

Test animals

Species:

rat

Strain:

other: Wistar Hannover RccHan

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories Models, S.L., Barcelona, Spain
- Age at study initiation: 11-12 weeks
- Mean weight at study initiation: males: 312 to 364 g; females: 198 to 219 g
- Fasting period before study: no
- Housing: cages with standard, granulated, softwood Lignocel S8/15 bedding
Pretreatment period: 5 per cage
Mating period: 1 male and 1 female per cage
Postmating: single housing
- Diet: pelleted standard Harlan Teklad 2014C rat/mouse maintenance diet; ad libitum
- Water: tap water; ad libitum
(Analyses of diet and water was performed.)
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-24
- Humidity (%): 30-60
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 1% methylcellulose+1% Tween 80 in aqueous solution

Details on exposure:

VEHICLE
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: 18 h
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as day 0 postcoitum
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged individually
- Any other deviations from standard protocol: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of dose formulation was determined in samples taken from the formulation to be administered to Groups 1 to 4 at the start and end of treatment by GC-FID analysis.

Duration of treatment / exposure:

Males: two weeks prior to mating and at least up to and including the day before sacrifice (day 49 of treatment).
Females: two weeks prior to mating and at least up to and including the day before sacrifice (day 4 postpartum).

Frequency of treatment:

Once daily; 7 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

300 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 were selected based on a dose-range finding toxicity study in rats (Harlan Laboratories Study S40412 14-day Oral (gavage) DRF using dose levels of 100, 500 and 1000 mg/kg bw/day.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: 2 hours after dosing
- Detailed clinical observations were performed on all test and control group animals before the first exposure to the test item and once weekly thereafter, at least two hours after dosing. Observations were also performed on females with litters on Day 4 postpartum. These observations were performed outside the home cage, in a standard arena, at least two hours after dosing (where applicable) to ensure that any transient effects of treatment are identified.

BODY WEIGHT: Yes
- Time schedule for examinations:
F0 males: twice weekly during the pre-pairing and pairing period and daily during postpairing period.
F0 females: twice weekly during the pre-pairing and pairing period and daily until day 4-5 postpartum.

FOOD CONSUMPTION: Yes
F0 males: once weekly during the pre-pairing period and two weeks of postpairing period.
F0 females: once weekly during the pre-pairing period and days 0-7, 7-14, 14-21 postcoitum and days 1-4 postpartum.
No food consumption was recorded during the mating period.

WATER CONSUMPTION: Yes
F0 males: one week during the pre-pairing and postpairing period.
F0 females: one week during the pre-pairing, postcoitum period and days 1-4 postpartum.
No water consumption was recorded during the remaining periods.:

Oestrous cyclicity (parental animals):

Smearing of individual females was evaluated during pairing period and was discontinued when sperm are found.

Sperm parameters (parental animals):

Parameters examined in P male parental generations:
testis weight, epididymis weight, qualitative staging of spermatogenesis and histopathology evaluation of interstitial cells of all males from the control
and high-dose groups

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes (males on day 50, females on day 5 postpartum)
- Organ weights: epididymes and testicles, adrenal gland, brain, heart, kidney, liver, ovaries, pituitary, spleen, thymus, thyroid and parathyroids, uterus and oviducts
- Fixation: Adrenals, Aorta (thoracic), Bone with bone marrow- (Sternum), Peyer’s patches, Pharynx, Pituitary, Bone marrow smear (femur) (air-dried smear), Brain (cerebrum, cerebellum, medulla/pons), Epididymides (Bouin's solution), Esophagus, Eyes with optic nerve (Davidson’s fixative), Femur (with articular surface), Heart (with papillary muscle), Intestine, large (cecum, colon and rectum), Intestine, small (duodenum, jejunum and
ileum), Kidneys, Larynx, Liver, Lungs, with main bronchi and bronchioles, Lymph nodes (mandibular and mesenteric), Nose (the entire head will be collected), Mammary gland area, Ovaries, Pancreas, Prostate, coagulating gland and seminal vesicles, Salivary glands (mandibular, sublingual and parotid), Sciatic nerve, Skeletal muscle, Skin (abdominal), Spinal cord (cervical, thoracic and lumbar), Spleen, Stomach (glandular and nonglandular), Testes (Bouin's solution), Thymus, Thyroid (incl. parathyroid gland, if possible), Tongue, Trachea, Urinary bladder, Uterus (cervix, corpus and oviducts), Vagina, All gross lesions

Postmortem examinations (offspring):

On day 4 postpartum, all live pups were sacrificed by an intraperitoneal injection of sodium pentobarbital and examined macroscopically.
Any F1 offspring that died during the study was examined externally and necropsied, except those excessively cannibalized. Samples of the organs and tissues with macroscopic alterations were taken and preserved in neutral phosphate buffered 4% formaldehyde solution for possible microscopic examination.

Statistics:

The following statistical methods were used to analyse food consumption during postpairing period, body weight, clinical laboratory data, organ weights, postnatal development and reproduction data, as well as macroscopic findings:
The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables can be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data cannot be assumed to follow a normal distribution.
Fisher's exact-test was applied to the macroscopic findings.
Armitage/Cochran Trend Test [5] for non-neoplastic lesions, if appropriate.
Bonferroni-test was applied to some reproduction parameters.

Reproductive indices:

- Percentage Mating [%] = Number of females mated/Number of females paired x 100
- Fertility Index female [%] = Females achieving pregnancy/Number of females paired x 100
- Gestation Index [%] = Number of litters with live pups/Number of pregnant rats x 100
- Concenption rate [%] = Females achieving pregnancy/Number of females mated x 100
- Pre-implantation loss [%] = Number of corpora lutea - Number of implantation sites/Number of corpora lutea x 100
- Post-implantation loss [%] = Number of implantation sites - Total number of offspring born/Number of implantation sites x 100

Offspring viability indices:

- Post-natal loss [%] = Number of offspring/Number of offspring alive on day 4 x 100
- Viability Index [%] = Number of offspring alive on day 4/Number of offspring alive on day 1 x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No relevant clinical signs were observed in males and females. One male and female from the control group and three males and one female at 100 mg/kg showed occasionally up of the product (presumably immediate reaction to the administration of test substance). In addition, male No. 28 at 300 mg/kg bw/day had missing upper incisors in week 4 of treatment. No clinical signs were observed in females at 300 or 1000 mg/kg, while in males no clinical signs were oberved at 1000 mg/kg bw/day.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No test-item-related differences from the control group were recorded. Some significant differences were observed at 1000 mg/kg bw/day but these were not considered of toxicological relevance.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

effects observed, non-treatment-related

Description (incidence and severity):

Slight increase in water consumption was recorded in females at 300 and 1000 mg/kg bw/day during pregnancy period. These differences were not statistically significant. In males no differences from the control group were recorded.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

No changes of toxicological relevance were recorded in females. Bilirubin, cholesterol and triglyceride values tended to be higher at 300 and 1000 mg/kg bw/day in females. An increase in bile acid values was observed at 300 mg/kg bw/day in males (non adverse). The differences recorded were statistically significant with respect to the control group.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

No differences in fore- or hind limb grip strength were recorded in males and females. Slightly lower locomotor activity was recorded in males at 100 mg/kg bw/day, being it statistically significant at 15 minutes. Slightly lower locomotor activity was recorded in females at 300 and 1000 mg/kg bw/day for 15-30 min, but the differences were not statistically significant. No differences were recorded in the remaining groups. All animals responded positively to the different reflexes such as blink, pinna, righting and iridic reflexes and pain, auditory startle and handling response (push-off).

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

All microscopic findings recorded were considered to be within the range of normal background lesions that may be seen in rats of this strain and age and under the experimental conditions used in this study.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

All findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age. There were only single cases with isolated tubules that showed minor changes. They consisted of single sperm resorption in stage VII. In epididymides, there was only one animal in group 1 with unilateral monounclear cell foci of a minimal serverity.

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating Performance:
Median precoital time and mean precoital time (approx. 3 days) were similar in animals treated with the test item at 100 and 1000 mg/kg bw with respect to the control group. However, mean and median precoital time was longer at 300 mg/kg bw (5.7 and 4 days, respectively). In addition, female no. 68 from this group did not mate with the first male (no. 28) after a pairing period of 14 days and was mated a second time with male no. 23. This female showed an anestrus cycle of 11 days. Moreover, some animals from all groups took more than 2-4 days to mate. Females nos. 43 (control group), 53 (100 mg/kg), 61 and 70 (300 mg/kg) and 73 (1000 mg/kg) took 12, 12, 13, 14 and 13 days, respectively, to mate and showed an anestrus cycle.
Fertility:
No treatment-related differences were recorded in the percentage of mating or the gestation index in females at the three doses with respect to the control group. Although fertility index and conception rate was lower in test-item-treated groups (100% vs. 90%), it was considered to be within the range of normal background findings which may be seen in rats. Females no. 53 at 100 mg/kg bw, no. 70 at 300 mg/kg bw and no. 77 at 1000 mg/kg bw, which mated with males nos. 13, 30 and 37, respectively, were not pregnant despite the presence of sperm in the vaginal smear and vaginal plug in the last two females. Duration of the mating period of those couples was 12, 14 and 4 days, respectively. Females no. 69 at 300 mg/kg bw and no. 74 at 1000 mg/kg bw, which mated with males nos. 29 and 34, did not show signs of pregnancy and were mated again to ensure eight pregnant females per group. These animals were identified as 81 and 82 to record the data and their second mating led to pregnancy. To check the fertility of the male whose first pairing did not result in mating, male no. 28 from the 300-mg/kg bw group was mated a second time with one female from a reserve group and mating led to pregnancy.
Reproduction data:
No treatment-related differences from the control group were recorded in the mean of implantation sites per litter or corpora lutea. No treatment-related differences from the control group were recorded in the percentage of pre- or postimplantation losses. Female no. 45 from control group showed 100% implantation site loss. No differences in sex ratio were recorded.
Breeding data:
The length of pregnancy was similar in all groups with a mean of 21-22 days.Female no. 78 at 1000 mg/kg bw had not enough milk and did not nurse correctly its pups; consequently, the pups died or were devoured between days 2 and 3 of lactation. Despite the fact that the pups had milk in the stomach when alive, they did not have any at necropsy and showed hypothermia and pallor. Female no. 78 showed slightly enlarged mammary glands. Even the percentage of pups showing milk in the stomach was similar in all groups. One pup in the litter of females no. 43 from control group, no. 63 at 300 mg/kg bw, no. 78 and two pups from no. 73 at 1000 mg/kg bw showed no milk in stomach on day 1 postpartum and died.

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 were observed up to and including the highest tested dose level

2

Key result

Dose descriptor:

NOAEL

Remarks:

reproduction

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 were observed up to and including the highest tested dose level

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):

No treatment-related alterations were recorded in the morphological examination of the pups.
One pup from control group had a hematoma on toes and one pup from 300 and 1000 mg/kg bw group had a wound on scapula or limbs.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

No treatment-related differences from the control group were recorded in the mean of dead pups at the first litter check and living pups. During the first 4 days postpartum, the percentage and mean of postnatal losses was slightly higher at 1000 mg/kg bw; however the number of litters affected was similar to that of the control group (2 vs 1 litters) due to the fact that female no. 78 (1000 mg/kg bw) lost all its litter. The resulting viability index was lower than in the control group (84.7% versus 99.1%, respectively). No differences were recorded at 100 and 300 mg/kg bw.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No differences were recorded between groups and sexes. At 1000 mg/kg bw, one runt pup *no.7 from litter no.54) was observed.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No noteworthy findings were recorded in the control, 100, 300 or 1000 mg/kg bw pups.

Histopathological findings:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

A significantly lower percentage of fetuses with positive response in the surface-righting reflex (righting reflex) at 1000 mg/kg bw was recorded compared to the control group. There were no treatment-related differences compared to the control animals at 100 or 300 mg/kg bw.
Righting reflex was only assessed on Day 1 postpartum. The clinical relevance of this parameter remained unclear. The study report did not contain exact age matching of pups (time point in hours after birth at which the reflex was tested), nor considered biologically possible range of gestation length. No follow up behavioural testing at later time points during development of pups was done. Testing of righting reflex is not required following OECD guideline 422.

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 were observed up to and including the highest tested dose level

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no