Fatty acids, C18-unsatd., trimers, compds. with oleylamine

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented and reported study fully adequate for assessment. The study was conducted according to an internationally accepted technical guideline and in compliance with GLP in a recognized contract research organization.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test) of 1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Sprague Dawley rats, strain: Crl:CD(SD) with appropriate range of bodyweight at study start.
- Source: Charles River (UK) Ltd.
- Age at treatment start: Ca. 70 days.
- Weight at treatment start: Males: minimum 341 g, maximum 390 g,
Females: minimum 212 g, maximum 277 g.
- Housing Inside a barriered rodent facility:
all animals pre-pairing + toxicity subgroups: In groups up to 5 by sex in solid floor polycarbonate cages.
during pairing (1 male+1 female/cage): In RB3 modified polypropylene cages with stainless steel grid-floor over absorbent paper-lined trays.
males after pairing: In groups up to 5 in solid floor polycarbonate cages.
females during gestation and lactation: Females housed individually (+litter) in solid floor polycarbonate cages.
- Bedding material (in solid floor cages): Wood based bedding, sterilised by autoclaving before use.
- Cage enrichment: Aspen chew block + plastic shelter (except during pairing or post gestation day 20).
- Diet (ad libitum): Standard rodent diet (SDS VRF1 Certified) without antibiotic, chemotherapeutic or prophylactic agent.
- Fasting (diet withheld): Main phase males and Toxicity phase females overnight before blood sampling for clinical pathology.
- Water (ad libitum): Potable drinking water from the public supply.
- Acclimation period: 7 days before treatment start, under laboratory conditions.

Routine analysis of the batch of diet used and water, chew blocks and bedding material did not provide evidence of contamination that might have prejudiced the study.

IN-LIFE DATES:
- Duration of test, males & toxicity phase females: Five weeks
Duration of test, main phase females (i.e. reproductive subgroup): From 14 days prior to pairing to day 7 of lactation.
Duration of test, offspring: From birth to day 7 of lactation.

ENVIRONMENTAL CONDITIONS

Air conditioned room kept at positve pressure without re-circulation of the filtered fresh air supplied to the room.
Controlled environment, environmental conditions were set at:
- Temperature (°C): 21 ± 2°C
- Relative Humidity (%): 40 to 70%
- Photoperiod (artificial lighting): 12 hrs day / 12 hrs night
- Rate of air exchange: At least 15 changes/h
Deviations from the target ranges for temperature and relative humidity were not evident.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

Treatment of parental animals by oral gavage administration. Test substance was not directly administered to F1 animals.

- Concentration in vehicle: The concentration of the test material in vehicle varied between dose groups thus allowing constant dosage volume in terms of mL/kg bw/day.
- Amount (dose volume by gavage): 5 mL/kg bw/day.
Actual dose volumes were calculated at about weekly or shorter intervals accounting for the latest body weight.

- For concentrations of test material in vehicle at different dose levels, see Table 1 in "Any other information on materials and methods incl. tables"

- Justification for choice of vehicle:
The suitability of propylene glycol as a vehicle was established visually and by chemical analysis during the 7-day range-finding study:
Endpoint study record "7.5.1 Repeated dose toxicity: oral - 7d_range-finding_gavage_HLS_GAH0029".
In addition, in the present main study, concentrations of dose formulations were chemically analysed.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Chemical analysis of test material formulations by high performance liquid chromatography coupled with a mass spectrometer (HPLC-MS/MS).
- Concentrations (verified at first, second and last treatment week) of the test material formulations were lower than expected from nominal concentrations (see Table 1).
- Mean concentrations of WS400151 in prepared formulations attained by chemical analysis were 70.5% to 87.2% of the corresponding nominal concentration. Evaluation of a number of aspects of the analytical and formulation procedures did not lead to identification of the cause for the lower concentrations achieved by chemical analysis. In order to take a conservative approach, it was concluded that the concentrations determined by chemical analysis reflected the concentrations of test substance formulation administered to the animals.

Details on mating procedure:

- Male/female ratio per cage: 1/1
- Length of cohabitation: At the most 14 days, until proof of pregnancy was confirmed. 
- Proof of successful mating: Formation of at least one copulation plug and a sperm positive vaginal smear.
The day this was found was referred to as day 0 of gestation.
(During cohabitation, females were checked every morning for pregnancy).

Duration of treatment / exposure:

- Treatment period, males & toxicity phase females: Daily, for five consecutive weeks, in males commencing 14 days prior to mating
- Treatment period, main phase females (i.e. reproductive subgroup): 44 to 47 days (from 14 days prior to mating to day 6 of lactation)
- Offspring were not dosed

Frequency of treatment:

Daily, 7 days/week (during parturition, dosing omitted as appropriate)

Duration of test:

- Duration of test, all F0 males & toxicity subgroup females: Five weeks
Duration of test, reproductive subgroup females (F0): From 14 days prior to mating to day 7 of lactation.
Duration of test, offspring: From birth to day 7 of lactation.

Doses / concentrations

Remarks:

Doses / Concentrations:
0 (vehicle control), 7.1, 21.9, 75 mg/kg bw/day
Basis:
other: dose levels derived from chemical determination of administered dose formulations

No. of animals per sex per dose:

Toxicity phase animals: */ 5 females
Main phase animals (i.e. reproductive subgroups): 10 males / 10 females
*Explanatory note by the notifier:
Examinations assigned to the toxicity phase females to meet the requirements of a 28-day repeat dose oral toxicity study were also assigned to 5 (for some examinations to 10) main phase males per dose group. Therefore, these 5 main phase males per dose group are called also "toxicity subgroup" in the present robust study summary for clarification. After pairing with main phase females, all males were killed at the same time (Week 6).

Control animals:

yes, concurrent vehicle

Details on study design:

This study was conducted to examine both repeated dose toxicity and  reproductive/developmental toxicity as an OECD screening combined study
(OECD 422 test guideline).  Therefore, animals initially entering the study were divided into toxicity subgroup animals (toxicity phase) and reproductive subgroup animals (main phase), whereby 5 of the 10 F0 males (used for pairing) per dose group formed the toxicity male subgroups.

Dose selection was based on the results of a 7-day preliminary oral toxicity study in young adult rats (females nulliparous and non-pregnant) in which a NOAEL could not be established at dose levels of 100, 300 or 1000 mg/kg/day.

Examinations

Maternal examinations:

Clinical observations performed and frequency:
- Clinical signs : At least twice a day (before and after administration)
- Detailed physical examination
and arena observations: Before treatment start and at least once a treatment week.
- Body weight: Weekly for pre-mating and mating period; on gestation days 0, 6, 13, 20; on lactation days 1 & 7.
- Body weight: Weekly for pre-pairing period; on gestation days 0, 6, 13, 20; on lactation days 1 & 7.
- Food consumption: Weekly for pre-pairing period, during gestation for days 0-6, 6-13, 13-20, during lactation for days 1-4 & 4-7.
- Frequency of vaginal estrus: Daily by examination of vaginal smears taken from the beginning of the treatment period to the day of confirmed copulation.
(During the pairing period, females were checked every morning for pregnancy).

Additional parameters examined:
- No. of animals mating (evidence of successful copulation, i.e. sperm positive vaginal smear and at least one copulation plug)
- Pre-coital interval (pairing days until detection of mating)
- No. of animals achieving pregnancy
- No. of living pregnant females
- From Day 20 post copulation 3 times a day checks for evidence of parturition, any difficulties and numbers of live and dead offspring.
- Gross pathology
- Organ weights
adrenals, brain, heart, kidneys, liver, lungs & bronchi, ovaries, pituitary, spleen, thymus, thyroid with parathyroids, uterus with cervix & oviducts.
- Histopathology (gross lesions).

Explanatory note
This study was conducted to examine both repeated dose toxicity and  reproductive/developmental toxicity as an OECD screening combined study
(OECD 422 test guideline).  Therefore, animals initially entering the study were divided into toxicity subgroup animals (toxicity phase) and reproductive subgroup animals (main phase), whereby 5 of the 10 F0 males (used for pairing) per dose group formed the toxicity male subgroups. Examinations confined to toxicity subgroup animals and/or to parental males (F0) are detailed in the separate endpoint study records,
"7.5.1 Repeated dose toxicity: oral - Repeat dose tox combined_gavage_rat_HLS_GAH0030" and
"7.8.1 Toxicity to reproduction - Tox to reproduction screening combined_gavage_rat_HLS_GAH0030"

Ovaries and uterine content:

The ovaries and uterine content were macroscopically examined after termination on day 7 of lactation, i.e. day 7 post partum.
Numbers of uterine implantation sites were recorded and post implantation survival determined.
In addition, gestation length (time elapsing between detection of mating and commencement of parturition) was recorded.

Fetal examinations:

Litters were examined post partum as follows (day of birth = day 0):
- Number:  Daily until day 7 post partum.
- Sex: In total litter: 1st day; in live litter 1st and 7th day
- Live births/mortality: Daily until day 7 post partum.
- Clinical signs: Daily until day 7 post partum
- Body weight of live pups: 1st, 4th, 7th day and weight change from days 1-4 and 1-7.
- Necropsy:  7th day (Full macroscopic examination of all pups including assessment of the presence of milk in the stomach, where possible.
(Missing or grossly autolysed or cannibalised pups could not be examined)

Statistics:

As detailed in Endpoint study record "7.5.1 Repeated dose toxicity: oral - Repeat dose tox combined_gavage_rat_HLS_GAH0030"

Indices:

- Percentage mating (No. of animals mating/No. of animals paired) x 100
- Conception rate (No. of animals achieving pregnancy/No. of animals mated) x 100
- Fertility index (No. of animals achieving pregnancy/ No. or animals paired) x 100
- Gestation index (No. of live litters born on day 0/No. of living pregnant females) x 100
- Post-implantation survival index  (Total no. of pups born/Total no. of uterine implantation sites) x 100
- Live birth index (No. of live pups on day 1 after littering/Total no. of pups born) x 100
- Sex ratio expressed as percentage males and calculated for total offspring on Day 1 and for live offspring on Days 1 & 7
(No. of male pups in litter/No. of offspring in litter) x 100
- Viability index (No. of live pups on day 4 after littering /No. of live pups on day 1 after littering) x 100
- Lactation index (No. of live pups on day 7 after littering /No. of live pups on day 1 after littering) x 100

Historical control data:

Not included in the study report.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: Based mainly on histopathology in nulliparous and non-pregnant females after 5 weeks of treatment

Details on maternal toxic effects:
CLINICAL SIGNS, NEUROBEHAVIOUR AND MORTALITY
Three high dose females (2 of the toxicity subgroup and 1 of the reproductive subgroup) were found dead following intubation errors. One low and one high dose females without viable litter were killed on Day 25 after evidence of mating. Mortality or clinical signs attributable to treatment with the test material or effects on sensory reactivity, grip strength or motor activity were not evident.

BODY WEIGHT AND WEIGHT GAIN
At 75 mg/kg/day, overall bodyweight gain was low in females during the first two treatment weeks and during gestation.

FOOD CONSUMPTION
At 75 mg/kg/day, food consumption was slightly low during the first two treatment weeks and lower than concurrent controls overall during gestation and lactation. At lower dose levels (7.1 and 21.9 mg/kg/day) foodconsumption was unaffected by treatment with the test material.

REPRODUCTIVE ENDPOINTS
Oestrous cycles, pre-coital interval mating performance, fertility, gestation length, gestation index and litter size were not affected by treatment.

GROSS PATHOLOGY
After five treatment weeks at 75 mg/kg/day, the jejunum of all surviving nulliparous non-pregnant toxicity phase females was thickened, the mesenteric lymph nodes were enlarged in 2 of these females, and the duodenum was thickened 2 females. At lower dose levels after 5 treatment weeks and at all dose levels in dams killed on Lactation Day 7, toxicologically relevant macroscopic lesions were not evident. In addition, mean numbers of uterine implantations were considered unaffected by treatment with the test substance.

ORGAN WEIGHTS
Following lactation, adjusted mean kidney weight was low with relationship to treatment. This was considered to be fortuitous and toxicologically not significant, as there was no such change in toxicity phase animals after 5 treatment weeks.

HISTOPATHOLOGY
In general, tissues from maternal animals were not histopathologically examined. In nulliparous, non-pregnant females histopathology findings indicative of adverse effects on reproductive organs were not evident. After five treatment weeks, the presence of foamy macrophages in the mucosa of the jejunum and duodenum at 75 mg/kg/day, and foamy histiocytosis in the sinuses of the mesenteric lymph nodes at 21.9 and 75 mg/kg/day were attributed to treatment with the test material and were considered potentially adverse, but were not considered to represent reproductive toxicity endpoints.

Further details of histopathology findings in adult animals (main phase males and nulliparous, non-pregnant toxicity subgroup females) of the present study are given in separate endpoint study record "7.5.1 Repeated dose toxicity: oral - Repeat dose tox combined_gavage_rat_HLS_GAH0030".

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

7.1 other: mg/kg bw/day (dose level derived from chemical analysis of administered dose formulations)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOEL

Effect level:

75 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

In the present study, the no-adverse-effect-level (NOAEL) for maternal toxicity was 7.1 mg/kg/day. This NOAEL was derived mainly from histopathology findings in nulliparous, nonpregnant females after 5 weeks of treatment, because, in general, histopathology was not conducted on dams. The no-effect-level (NOEL) for developmental toxicity has been 75 mg/kg/day, i.e. the highest dose level tested. Dose levels substantially higher than this were not selected for the present study, because of general toxicity. Effects indicative of reproductive or developmental toxicity of WS400151 were not evident. Therefore, classification and labelling regarding reproductive/developmental toxicity according to EU classification rules [DIRECTIVE 67/548/EEC and REGULATION (EC) 1272/2008] was not inferred.