2-octyldodecyl 12-[(1-oxooctadecyl)oxy]octadecanoate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

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 oral exposure:

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

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

Observations and examinations performed and frequency:

MORTALITY AND CLINICAL SIGNS
- Time schedule: at least 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:
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.

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: 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.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
Males: on day 50
Females: on day 5 postpartum
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 5/sex/dose
- Parameters: haemoglobin, erythrocytes, leucocytes, differential blood count (absolute/relative), reticulocytes (count and maturity index), platelets, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, prothrombin time, activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
Males: on day 50
Females: on day 5 postpartum
- Animals fasted: Yes
- How many animals: 5/sex/dose
- Parameters examined: albumin, globulin, albumin/globulin ratio, bile acids, bilirubin, cholesterol (total), creatinine, glucose, urea, total protein, calcium, chlorid, potassium, sodium, alanine aminotransferase (ALAT), alkaline phosphatase (AP), aspartate aminotransferase (ASAT), triglycerides, creatine kinase, gamma-glutamyl-transferase (gGT), inorganic phosphorus,

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
Males: the day before sacrifice and at least two hours after dosing.
Females: on day 4 postpartum.
- Dose groups that were examined: all dose groups (5 animals/group)
- Battery of functions tested:
Functional performance test: Grip strength (fore- and hind limbs) and motor activity
Sensory reactivity assessment: Sensory reactivity to different stimuli (e.g. auditory, visual and proprioceptive)

-OTHER: spermatology stage evaluation

Sacrifice and pathology:

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

HISTOPATHOLOGY: Yes, all organs that were included for fixation (5/sex of control and high dose group)

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.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

males: increase in bile acids at 300 mg/kg bw/day (non adverse)

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

females: lower left ovary weight at 1000 mg/kg bw/day (non adverse)

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
No mortality and 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. In addition, male No. 28 at 300 mg/kg 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.

BODY WEIGHT AND WEIGHT GAIN
No test-item-related differences from the control group were recorded. Some significant differences were observed in group 3 but these were not considered of toxicological relevance.

FOOD CONSUMPTION
Food consumption was similar in all groups.

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

HAEMATOLOGY
No changes of toxicological relevance were recorded.

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

NEUROBEHAVIOUR
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, being it statistically significant at 15 minutes. Slightly lower locomotor activity was recorded in females at 300 and 1000 mg/kg 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).

ORGAN WEIGHTS
At 1000 mg/kg, kidney weights tended to be slightly higher in males. These differences were not statistically significant. Moreover, lower pituitary and adrenal weights were recorded; the differences were statistically significant for pituitary in males. A trend to higher liver weight was recorded in females at 1000 mg/kg, but the differences were not statistically significant.
Concerning reproductive organs, lower left ovary weights were recorded at 1000 mg/kg. This difference was statistically significant in relation to body weight ratio.

GROSS PATHOLOGY
At 1000 mg/kg, large right kidney was observed in one male and reddish thymus in another one. A reddish area on cecum wall was observed in one male and reddish foci in lungs in another one. Likewise, thickened gastric mucosa was observed in one female and small spleen in another one.
At 300 mg/kg, reddish foci were observed in the thymus of one male. No findings were observed in females.
At 100 mg/kg, pale kidneys were observed in one male and reddish foci in lungs in another one. Likewise, left seminal vesicle reduced in size was observed in one male and reddish thymus in three males. Thickened gastric mucosa was observed in one female.
In the control group, one male had left seminal vesicle reduced in size. One male and one female had pale kidneys. In addition, one male had pancreas reduced in size and thymus with reddish foci.
Yellowish-whitish gastric mucosa was recorded in few females from all groups (including control group).

HISTOPATHOLOGY: NON-NEOPLASTIC
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.

Effect levels

Key result

Dose descriptor:

NOAEL

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 toxic effects observed

Target system / organ toxicity

Key result

Critical effects observed:

no