12-hydroxyoctadecanoic acid, reaction products with 1,3-benzenedimethanamine and hexamethylenediamine

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 13 june 2012 to 8 february 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

432-840-2

EC Name:

-

Cas Number:

220926-97-6

Molecular formula:

No discrete formula is available for this UVCB substance.

IUPAC Name:

12-hydroxy-N-({3-[(12-hydroxyoctadecanamido)methyl]phenyl}methyl)octadecanamide; 12-hydroxy-N-[6-(12-hydroxyoctadecanamido)hexyl]octadecanamide

Test material form:

other: solid

Details on test material:

- Name of test material (as cited in study report): 12-hydroxyoctadecanoic acid, reaction products with 1,3-benzenedimethanamine and hexamethylenediamine (E96095)

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: 10 weeks
- Weight at study initiation: 228 to 286 g at the start of the study (Day 0 of gestation)
- Fasting period before study:no
- Housing:Up to 4 per cage during acclimation then individually housed except during pairing,
- Diet: Ad libitum, standard rodent diet (SDS VRF1 Certified). This diet contained no added antibiotic or other chemotherapeutic or prophylactic agent
- Water: Ad libitum, potable water taken from the public supply.
- Acclimation period: 5 days before they were paired on a 1:1 basis with stock males from the same source.

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 23°C
- Humidity: 40 to 70%
- Air changes (per hr): Each animal room was kept at positive pressure with respect to the outside by its own supply of filtered fresh air, which was passed to atmosphere and not re-circulated
- Photoperiod (hrs dark / hrs light): 12 h continuous light and 12 h continuous dark/24 h.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): homogeneous and stable suspensions were obtained with corn oil as a vehicle
- Concentration in vehicle: 20, 60 and 200 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Type of method: High Performance Liquid Chromatography with UV detection analytical method was used.

Homogeneity and stability of the dosage forms: homogeneity of the test substance in corn oil formulations was assessed with respect to the level of concentration at nominal concentrations of 20 mg/mL and 200 mg/mL. Homogeneity was confirmed following storage at ambient temperature for 2 days and refrigeration for up to 15 days.

Test item concentrations: the test item concentrations in the administered dosage forms analyzed in the first and last weeks of treatment were within +/- 10% of nominal concentrations.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: Only females with a sperm positive vaginal smear or at least two copulation plugs were selected.

Duration of treatment / exposure:

From Day 6 to Day 19 (inclusive) after mating

Frequency of treatment:

Daily

Duration of test:

Sacrifice on Day 20 after mating

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 300 and 1,000 mg/kg bw/day
Basis:
other: nominal doses

No. of animals per sex per dose:

22

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:The doses were selected on the basis of the results of the reproduction/development toxicity screening test (Huntingdon Life Sciences Report Number: FIN0048). In that study, there was no maternal or reproductive/developmental toxicity.

- Rationale for animal assignment (if not random): Animals were allocated to study on Day 0 of gestation, when positive evidence of mating was detected. Females were allocated to group and cage position in sequence of mating, thus ensuring that animals mated on any one day were evenly distributed amongst the groups. The sequence of allocation was controlled to prevent stock males from providing more than one mated female in each treatment group.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: detailed physical examination was performed on each animal on Days 0, 5, 12, 18 and 20 after mating to monitor general health.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each adult was recorded on Days 0, 3, 6-20 after mating.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The weight of food supplied to each adult, that remaining and an estimate of any spilled was recorded for the periods Days 0-2, 3-5, 6-9, 10-13, 14-17 and 18-19 inclusive after mating.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: uterus and ovaries

OTHER: no

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: live and dead fetuses recorded for each uterine horn

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: all per litter

Statistics:

# The following sequence of statistical tests was used for bodyweight, gravid uterine weight, food consumption, corpora lutea, implantations, live young, fetal, placental and litter weight data:
A parametric analysis was performed if Bartlett's test for variance homogeneity was not significant at the 1% level. For pre-treatment data, analysis of variance was used to test for any group differences. Where this was significant (p<0.05) inter group comparisons using t-tests were made. For all other comparisons, the F1 approximate test was applied. If the F1 approximate test was not significant at the 1% level, Williams' test was applied. If the F1 approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test was performed instead.
A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. For pre treatment data, Kruskal-Wallis’ test was used to test for any group differences. Where this was significant (p<0.05) inter group comparisons using Wilcoxon rank sum tests were made. The H1 approximate test was applied. If the H1 approximate test was not significant at the 1% level, Shirley's test was applied. If the H1 approximate test was significant, suggesting that the dose-response was not monotone, Steel's test was performed instead.

# For corpora lutea, implantations, live young, fetal, placental and litter weight data,if 75% of the data (across all groups) were the same value, for example c, Fisher’s Exact tests were performed. Treatment groups were compared using pairwise comparisons of each dose group against the control both for i) values c, as applicable.

# Pre/post implantation loss and sex ratio were analysed by generalised mixed linear model with binomial errors.

Indices:

Pre-implantation loss was considered to reflect losses due to non-fertilisation of ova. It was calculated from the formula:
Pre-implantation loss (%) = [(Number of corpora lutea – Number of implantations)/ Number of corpora lutea] x 100

Where the number of implantations exceeded the number of corpora lutea observed, pre implantation loss was assumed to be zero.

Post-implantation loss was considered to exclude the first two to three days post-implantation as deaths occurring at this stage are considered to leave no remains visible at Day 20 of gestation. It was calculated from the formula:
Post-implantation loss (%) = [(Number of implantations – Number of live fetuses)/Number of implantations] x 100

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
- There were no clinical signs and no effect of treatment on bodyweight gain.
- The food consumption of females receiving 1000 mg/kg/day was statistically high, when compared with Control. However this intergroup difference was small and these females had eaten marginally more than Control before commencement of treatment, therefore no toxicological significance is attached to this finding.
- Macroscopic examination of females on Day 20 of gestation did not reveal any findings that were considered to be related to treatment.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
- All females were pregnant. Therefore, the assessment was based on the 22 females with live young in the Control group and each treatment group at termination, on Day 20 of gestation:
Litter data as assessed by mean corpora lutea, implantations, early, late and total resorption counts, live young, sex ratio, and pre- and post-implantation loss, for females receiving 100, 300 or 1000 mg/kg/day was similar to Control and considered unaffected by treatment.

- Mean male and female fetal weights were marginally higher than Control following maternal treatment at 1000 mg/kg/day and was associated with marginally higher overall mean fetal weight. However, as the range of individual fetal weights was similar to Control, the difference was considered fortuitous and not biologically relevant or toxicologically important. Litter size, litter or placental weights were considered unaffected by treatment.

- The incidence of major and minor abnormalities and skeletal variants showed no relationship to treatment.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The repeated daily oral administration of the test substance to pregnant Sprague-Dawley rats from Day 6 to 19 of gestation at doses of 100, 300 or 1000 mg/kg bw/day was well tolerated. It was therefore concluded that 1000 mg/kg bw/day was the no-observed-adverse-effect-level (NOAEL) for both maternal toxicity and embryo-fetal development.

Executive summary:

In a prenatal development toxicity study performed according to OECD 414 , the objective was to evaluate the potential toxic effects of the test substance on the pregnant female rat and on embryonic and fetal development, following oral administration (gavage) from Day 6 to Day 19 post-coitum, inclusive. 

Three groups of 22 female rats received the test substance at doses of 100, 300 or 1000 mg/kg bw /day from Day 6 to 19 after mating. A similarly constituted Control group received the vehicle, corn oil, at the same dose volume throughout the same period. Animals were killed on Day 20 of gestation for reproductive assessment and fetal examination.

Clinical observations, bodyweight and food consumption were monitored. Adult females were examined macroscopically at necropsy on Day 20 of gestation and all fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral or skeletal examination.

Maternal clinical condition, bodyweight, food consumption, macropathology, pregnancy outcome, embryo-fetal survival, growth and development were unaffected by maternal treatment at doses up to and including 1000 mg/kg/day.

It was therefore concluded that 1000 mg/kg bw/day was the no-observed-adverse-effect-level (NOAEL) for both maternal toxicity and embryo-fetal development.