Reaction mass of N,N’-ethane-1,2-diylbis(12-hydroxyoctadecan-1-amide), Octadecanamide, 12-hydroxy-N-[2-[(1-oxooctadecyl)amino]ethyl]- and Octadecanoic acid, 12-hydroxy-, 1-hexyl-12-[[2-[(12-hydroxy-1-oxooctadecyl)amino]ethyl]amino]-12-oxododecyl ester

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on generations indicated in Effect levels (migrated information)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From 20 july 2012 to 04 january 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Read across study hence maximum reliability rating of 2 assigned according to ECHA guidance.

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 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

Necropsies for the males were scheduled for Week 6 of treatment instead of Week 5. The OECD 421 guideline requires a necropsy of the males after a dosing period of at least 4 weeks. Therefore this deviation did not affect the the integrity of the study.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Octadecanoic acid, 12-hydroxy-, reaction products with ethylenediamine

IUPAC Name:

Octadecanoic acid, 12-hydroxy-, reaction products with ethylenediamine

Constituent 2

Reference substance name:

100545-48-0

Cas Number:

100545-48-0

IUPAC Name:

100545-48-0

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material: Octadecanoic acid, 12-hydroxy-, reaction products with ethylenediamine

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: 344 to 386 g (males) and 241 to 278 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:

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 10 mg/mL and 200 mg/mL. Homogeneity was confirmed during distribution between the bottles, during magnetic stirring for 2 hours, and on re-suspension following storage at ambient temperature for 1 day and refrigeration for up to 15 days. At each time-point, the mean analysed concentration for the three samples remained within 7% of the initial time zero value and the coefficient of variation was less than 4%.

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 +10% to -15% 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 14-day repeated- dose preliminary study in the CD rat. In that study, the substance was generally well tolerated at dose levels up to 1000 mg/kg/day with no noteworthy effect of treatment on clinical signs, bodyweight gain, food consumption, macroscopic findings or organ weights.

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 (all groups)
- epididymis weight (all groups) + microscopic evaluation (control and high-dose groups)
- microscopic evaluation of stages of the spermatogenic cycle and testicular interstitial cells (control and high-dose groups)
- detailed qualitative examination was made of the testes, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell- or stage-specificity of testicular findings was noted.

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 6 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 and epididymis from the males in the control- and high dose groups, testes from all 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 and ovaries in the control and high-dose groups and on all macroscopic lesions.

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 6 of treatment ),
- females: on day 7of 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 and ovaries in animals of the control- and high-dose groups sacrificed at the end of the treatment period and for female that were sacrificed prematurely,
- all macroscopic lesions of all the animals of the low- and intermediate-dose groups sacrificed on completion of the treatment
period,
- Testes of males from control, low-, mid- and high-dose groups,

Special emphasis was paid to the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

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 other 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:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

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 and foood consumption of all groups of males receiving the test substance. Overall bodyweight, bodyweight change and food consumption of females receiving the test substance at 100, 300 or 1000 mg/kg/day were lower than that of the Controls during the first week of treatment although a dose response was not apparent. There was no conclusive effect of treatment during the second week of treatment, and weight gain and food consumption throughout Days 1-7 of lactation was similar in all groups.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Oestrous cycle length was unaffected by treatment.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
- There was no effect of treatment on the weight of testes and epididymides.

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)
Statistically significant increased prostate weight for males receiving the test substance at 1000 mg/kg/day was noted. As the prostate was not assessed microscopically, the toxicological significance of these slight increases was uncertain, however in the absence of any effects on fertility or reproductive performance, in isolation these changes were considered not to be adverse.

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

HISTOPATHOLOGY (PARENTAL ANIMALS)

- Slight to minimal bilateral tubular atrophy, with partial or complete depletion of germ cells from a few scattered tubules, was respectively seen at
300 mg/kg/day in 1 animal and at 1000 mg/kg/day in 2 animals. In the epididymides minimal amounts of degenerated spermatogenic cells in the ducts were also observed in the two males treated with 1000 mg/kg/day and were considered secondary to the changes seen in the testes.
Although the distribution pattern was suggestive of a treatment effect, these changes were small in incidence or severity and no particular cell- or spermatogenic stage specificity was observed. As reported in literature, tubular atrophy in the testes, even more extensive, can sometimes be seen as background finding in young adult rats (Creasy, 2011). Although an effect of the treatment cannot completely be discounted, the minor changes in the testes were more likely to be considered as background changes.
- A mammary adenocarcinoma was detected in 1 female at 300 mg/kg/day. This tumour was considered incidental based on the occurrence in a single animals and the lack on any proliferative changes in the mammary gland in the other treated animals. Although mammary adenocarcinomas are uncommon in young rats, they can occasionally occur spontaneously even at an early age, as reported in literature.
There was no evidence of test article related changes in the other examined tissues.

OTHER FINDINGS (PARENTAL ANIMALS)
Two females in the Control group and one female dosed at 300 mg/kg/day failed to litter, and were found to have no sign of any implantations pre- or post-staining on Day 25 after mating.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive performance

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

reproductive performance

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 8, 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 at 100 or 300 mg/kg bw/day on mean number of implantations, live litter size on Day 1 and offspring survival up to Day 7 of age. At 1000 mg/kg/day, the mean number of implantations showed no adverse effect of parental treatment but the post-implantation survival index (86.1% ) was slightly lower than in Controls (94.4%) and the other study groups. This resulted in marginally low total and live litter sizes. However, none of the differences attained statistical significance, and the post-implantation survival index was within the recent background control range (lowest value 84.9%): therefore no effect of treatment was inferred.

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 and subsequent bodyweight gain up to Day 7 of age was similar to control values for the litters of females receiving the test substance at 100, 300 or 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)

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental toxicity

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: offspring growth and survival

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

It was concluded that the No-Observed-Adverse-Effect-Level (NOAEL) was 1000 mg/kg/day (the top dose) for reproductive performance and offspring growth and survival in the CD rat following oral gavage administration in a standard screening test.

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 five 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. The clinical condition, litter size and survival, sex ratio and bodyweight of all offspring were assessed and macroscopic pathology investigations were undertaken.  

There were no deaths, and the general appearance and behaviour of the animals were not affected by treatment. There were no adverse effects of treatment on adult bodyweights, bodyweight gains or food consumption and no effects on oestrous cycles, pre-coital interval, mating performance, fertility, conception rate or fertility index; gestation length and gestation index.

At 1000 mg/kg/day, the post-implantation survival index was slightly decreased compared to controls (86.1% versus 94.4% in controls). However, this slightly low value remained in the historical range of the laboratory (84.9% to 100%). As a consequence, the mean live litter size on Day 1 was slightly lower than in controls (14.3 versus 15.1 in controls).

There were no effects of treatment on offspring survival and sex ratio and offspring bodyweight gain to Day 7 of age. There were no macropathological findings in the adults or offspring. Analysis of the weight of the selected organs at scheduled termination revealed statistically significant increased prostate weight for males receiving 1000 mg/kg/day. The weight of all other selected organs was unaffected by treatment with Bisamide 80005005 and there were no particular microscopic findings.

It was concluded that the NOAEL for the test substance was 1000 mg/kg/day (the limit dose) for reproductive performance and offspring growth and survival in the CD rat following oral gavage administration in a standard screening test.