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

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 19 june 2012 to 28 november 2012

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:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

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)

Sex:

male/female

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: 340 to 382 g (males) and 235 to 291 g (females)
- Fasting period before study:no
- Housing: Females: 5 per cage during pre-pairing then individually housed except during pairing,
Males: 5 per cage 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

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

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation (mating period): until mating occurs or 14 days has elapsed
- Proof of pregnancy: vaginal plug or sperm in the morning vaginal lavage 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:

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 remained within an acceptable range of +5.2% to -2% when compared to the nominal values.

Duration of treatment / exposure:

In the males:
− 2 weeks before mating,
− during the mating period (up to 2 weeks),
− until sacrifice in week 6,

In the females:
− 2 weeks before mating,
− during the mating period (up to 2 weeks),
− during pregnancy,
− during lactation until day 6 post-partum inclusive

Frequency of treatment:

daily

Details on study schedule:

- No F1 parents (only one generation mated)
- Age at mating of the mated animals in the study: 13 weeks for males, 13 weeks for females, approximately.

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:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose-levels used in this study (0, 100, 300 and 1000 mg/kg/day) were selected on the basis of the results of a 4-week toxicity study in the rat by oral route followed by a 2-week recovery period ( Study CVP091/993344). The study concluded that 1000 mg/kg bw/day represented the NOAEL for the test substance.The minor changes noted in some of the parameters measured at this dose level, in the absence of any treatment-related pathological changes or effects on food and bodyweight being observed during the treatment period, were considered not to be of toxicological importance. Based on these observations, a dose-level of 1000 mg/kg bw/day was selected for the high dose. The dose levels for the mid and low doses (300 and 100 mg/kg bw/day respectively) were selected to explore any possible dose relationship.

Positive control:

None

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
-detailed physical examination was performed weekly. F0 females were also examined on Days 0, 7, 14 and 20 after mating and on Days 1 and 7 of lactation to monitor general health.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each adult was recorded during acclimatisation, on the day that treatment commenced (Week 0), weekly thereafter and before necropsy. The weight of each F0 female was also recorded on Days 0, 3, 7, 10, 14, 17 and 20 after mating and on Days 1, 4 and 7 of lactation.

FOOD CONSUMPTION :
- 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 cage, that remaining and an estimate of any spilled was recorded on a weekly basis from the first day of treatment until pairing. From these records the mean weekly consumption per animal (g/rat/week) was calculated for each cage.
For each F0 female, the weight of food supplied, that remaining and an estimate of any spilled was also recorded for the periods Days 0-2, 3-6, 7-9, 10-13, 14-16 and 17-19 after mating and Days 1-3 and 4-6 of lactation. From these records the mean daily consumption (g/rat/day) was calculated for each animal.

WATER CONSUMPTION: No

Oestrous cyclicity (parental animals):

For 15 days before pairing, daily vaginal smears were taken from all females. The smears were subsequently examined to establish the duration and regularity of the oestrous cycle. After pairing with the male, smearing was continued using pipette lavage, until evidence of mating was observed.

Sperm parameters (parental animals):

Parameters examined in males of parental generation:
- testis weight (all groups) + microscopic evaluation (control and high-dose groups)
- epididymis weight (all groups) + microscopic evaluation (control and high-dose groups)
- The following tests were performed on sperm:
=>Sperm motility: At least 200 sperm were assessed for each male. The percentages of motile and progressively motile sperm were reported for all groups.
=>Sperm morphology: At least 200 sperm were assessed for each male of control and high-dose groups. The percentages of normal sperm and abnormal sperm were reported.
=>Sperm count: The concentration (Million/g) and total number of sperm were reported for each male of control and high-dose groups.
=>Homogenisation-resistant spermatids count: The concentration (Million/g) and total number of spermatids were reported for each male of control and high-dose groups.
- Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages.

Litter observations:

STANDARDISATION OF LITTERS: No

PARAMETERS EXAMINED:
- number and sex of pups,
- number of live, dead and cannibalized pups,
- presence of gross anomalies, weight gain, clinical signs

GROSS EXAMINATION OF DEAD AND SURVIVING PUPS:
- external and internal abnormalities.

Postmortem examinations (parental animals):

SACRIFICE
The males were sacrificed during week 5 of treatment. The body and selected organs were weighed and a complete macroscopic post-mortem examination was performed. A microscopic examination was performed on the kidneys, liver ,epididymides (only one examined, the other reserved for seminology), prostate, seminal vesicles and testes (only one examined, the other reserved for seminology) from the males in the control- and high dose groups and on all macroscopic lesions.

The dams were sacrificed on day 7 of lactation, the body and selected organs were weighed and a complete macroscopic examination was performed.
A microscopic examination was performed on the kidneys, liver, pituitary,uterus, vagina and ovaries in the control and high-dose groups and on all macroscopic lesions. Mammary area was examined only when premature litter death occured.

GROSS NECROPSY
On completion of the treatment period all surviving F0 males and females were killed by carbon dioxide asphyxiation.
- males: after the end of the pairing period (during week 5 of treatment ),
- females: on day 7 of lactation.,
- females which had not delivered: on day 25 after mating (after a body weight recording to check for a possible un-noticed delivery),
- mothers with litter dying entirely: as appropriate.

For females, the numbers of implantation sites in each uterine horn was counted. For females failing to produce a viable litter, the number of uterine implantation sites was re-checked after staining with ammonium sulphide (modification of the Salewski staining technique).

Pups were sacrificed by an intraperitoneal injection of sodium pentobarbital:
- surviving pups: on day7 of age.
- Premature deaths if not autolysed or cannibalised.

HISTOPATHOLOGY
A microscopic examination was performed on:
- Liver, kidneys,epididymides, prostate, seminal vesicles, testes, pituitary, uterus, vagina and ovaries in animals of the control- and high-dose groups sacrificed at the end of the treatment period,
- Mammary area of all females with a premature litter death and sacrificed on completion of the treatment period,
- all macroscopic lesions of all the animals sacrificed on completion of the treatment period,

ORGAN WEIGHTS:
The body weight of each animal sacrificed as scheduled was recorded before sacrifice, and liver, kidney,epididymides (L and R), prostate, testes (L and R), seminal vesicles and ovaries (L and R) were weighed (wet) as soon as possible after dissection.The ratio of organ weight to body weight (recorded immediately before sacrifice) was calculated.

Postmortem examinations (offspring):

SACRIFICE: on day 7 of age

GROSS NECROPSY: on all pups (surviving and found dead)

HISTOPATHOLOGY: No

ORGAN WEIGTHS: No

Statistics:

- For all adult parameters, the analyses were carried out using the individual animal as the basic experimental unit.
- For litter/fetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.

# The following sequence of statistical tests was used for bodyweight, food consumption, organ weights and litter data:
A parametric analysis was performed if Bartlett's test for variance homogeneity was not significant at the 1% level. 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. 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 litter 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.
# For organ weight data, analysis of covariance was performed using terminal bodyweight as covariate, unless non-parametric methods were applied.
# Sex ratio were analysed by generalised mixed linear model with binomial errors.

Reproductive indices:

Percentage Mating = 100 * (Number animals mating/animals paired)
Fertility index = 100 * (Number animals achieving pregnancy / Number of animals mated)
Gestation index = 100 * (Number of live litters born / Number pregnant)

Offspring viability indices:

- Post-implantation survival index= 100 * (Number of pups born / Number of uterine implantation sites)
Post-implantation survival index was expressed as 100% where the number of offspring exceeded the number of implantation sites recorded.
- Live birth index = 100 * (Number of live born pups on Day 1/ Number of delivered pups)
- Viability index = 100 * (Number of surviving pups on day 7 / Number of live born pups on Day 1)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-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

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no deaths and no particular clinical signs.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
There was no effect of treatment on mean bodyweight gain in males and females. and foood consumption of all groups of males receiving the test substance. Food consumption in males treated at 300 or 1000 mg/kg bw/day was marginally high during the two week period before pairing, however the difference was marginal in nature and attributed to normal biological variation and the small study group size. Food consumption in females treated at 100 or 300 mg/kg bw/day before pairing, during gestation and lactation were similar to Control. The marginal difference in food consumption at 1000 mg/kg bw/day throughout gestation, was attributed to lower than expected food consumption in the Control; especially in one female that subsequently had a total litter death.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Assessment of oestrous cycles during the two week pre-pairing period showed that the majority of main phase females had regular 4 or 4/5 day oestrous cycles and it was considered that this parameter was not affected by treatment.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
- There was no effect of treatment on the weight of testes and epididymides. No adverse effects on sperm motility, concentration or morphology were apparent following treatment at 1000 mg/kg/day.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Mating performance and fertility as assessed by percentage mating, conception rate and fertility index, were unaffected by treatment.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Organ weights were not affected by treatment.

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no findings at macroscopic examination that could be attributed to treatment with the test substance.

HISTOPATHOLOGY (PARENTAL ANIMALS)
There were no findings which were considered to be related to the administration of the test substance. In the seminiferous tubules, no cell or stage specific abnormalities were noted.

OTHER FINDINGS (PARENTAL ANIMALS)
One Control female was killed for welfare reasons following the death of her litter.The animal had an extended parturition and the last surviving pups died before Day 1 post-partum recordings were made.

Effect levels (P0)

1

Dose descriptor:

NOEL

Remarks:

Reproductive performance

Effect level:

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

Based on:

test mat.

Sex:

female

2

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

The assessment of litter responses detailed below is made based on the 9, 10, 9 and 10 litters in each of groups 0, 100, 300 and 1000 mg/kg bw/day, respectively.

VIABILITY (OFFSPRING)
There was no effect of parental treatment with the test substance on mean number of implantations, live litter size on Day 1 and offspring survival up to Day 7 of age.

CLINICAL SIGNS (OFFSPRING)
The general appearance and behaviour of the offspring were not affected by maternal treatment.

BODY WEIGHT (OFFSPRING)
Offspring bodyweight on Day 1 of age was not affected by parental treatment. Subsequent bodyweight gain up to Day 7 of age appeared to be marginally higher in offspring with maternal treatment at 1000 mg/kg/day.


GROSS PATHOLOGY (OFFSPRING)
Macroscopic examination of offspring killed at scheduled termination on Day 7 of age revealed no abnormalities.

OTHER FINDINGS (OFFSPRING):
There was no effect of parental treatment with the test substance on sex ratio.

Effect levels (F1)

Dose descriptor:

NOEL

Remarks:

Developmental toxicity

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

It was conclude that oral administration of the test substance to Sprague-Dawley rats for at least four weeks at doses of 100, 300 and 1000 mg/kg/day was well tolerated with no toxicologically significant systemic effects and there was no effect of treatment on reproductive performance, including mating performance, fertility and offspring survival and development up to Day 7 of age.

Executive summary:

In a screening study for reproductive / developmental effects performed according to OECD 421 , the objective was to evaluate the potential toxic effects of the test substance following daily oral administration (gavage) to male and female rats from before mating, through mating and gestation until Day 6 post-partum.

Three groups of 10 male and 10 female rats received the test substance by gavage at doses of 100, 300 or 1000 mg/kg/day. Males were treated for 15 days before pairing up to the day before necropsy after a minimum treatment period of four consecutive weeks.  Females were treated daily for a minimum of 15 days before pairing, throughout mating and gestation until Day 6 of lactation. A similarly constituted Control group received the vehicle, corn oil, at the same volume-dose. 

During the study, clinical condition, bodyweight, food consumption, organ weights, macroscopic and microscopic pathology investigations were undertaken in all adults.  Oestrous cycles and gestation length were assessed and parturition observations were performed for females.  Sperm analysis and sperm morphology were performed for males.The clinical condition, litter size and survival, sex ratio and bodyweight of all offspring were assessed and macroscopic pathology investigations were undertaken.  

Overall treatment with the test substance was well-tolerated at all doses, with no adverse effect on general condition or signs following administration related to treatment. Bodyweight gain was unaffected by treatment. There were some marginal intergroup variations in food consumption which were attributed to normal biological variation and the small study group size, as the differences were marginal in nature and there was no effect on bodyweight gain. In addition, one animal in the Control group was humanely killed following the death of her litter and data showed that this animal had low food consumption, when compared to other animals in the Control group and an extended parturition.

There was no effect of treatment on oestrous cycles, pre-coital interval, mating performance, fertility, conception rate or fertility index; gestation length, gestation index; litter size, offspring survival and sex ratio or offspring bodyweight gain or clinical signs to Day 7 of age. There were no macropathological findings in the adults or offspring and no difference in adult organ weights or sperm analysis/ morphology. Histopathological examination revealed no change in any observed tissues or change in seminiferous tubules of the testes with respect to spermatogenic cycle and the integrity of the various cell types present within the different stages.

It was concluded that the NOEL for the test substance was 1000 mg/kg bw/day (the limit dose) for reproductive performance and offspring growth and survival. For the general toxicity, the NOAEL was considered to be 1000 mg/kg bw/day.