Didocosyl sebacate

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:

26 Mar 2013 - 21 May 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to the appropriate OECD test guideline and in compliance with GLP.

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)

Version / remarks:

(1995)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Didocosyl sebacate

EC Number:

255-730-4

EC Name:

Didocosyl sebacate

Cas Number:

42233-75-0

Molecular formula:

Not applicable (UVCB substance)

IUPAC Name:

Decanedioic acid, diesters with Fatty alcohols C20-22 (even numbered)

Test material form:

other: solid

Details on test material:

- Name of test material (as cited in study report): only trade name given
- Physical state: white solid
- Storage condition of test material: room temperature in the dark
- Expiration date of the lot/batch: 30 Nov 2014

Test animals

Species:

rat

Strain:

other: Wistar Han(TM):RccHan(TM):WIST

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK, Ltd., Oxon, UK
- Age at study initiation: 12 weeks
- Weight at study initiation: 300 - 344 g (males), 192 – 223 g (females)
- Housing: Initially, all animals were housed in groups of four in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing phase, the animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male/one female basis. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- Diet: Rodent 2018C Teklad Global Certified Diet (Harlan Laboratories UK, Oxon, UK), ad libitum
- Water: (tap/filtered) water, ad libitum
- Acclimation period: five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test item formulations were prepared daily during the study period due to the chemical characteristics of the test item and its limited solubility in organic and aqueous media.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Due to the chemical characteristics of the test item and its limited solubility in organic and aqueous media, arachis oil was chosen as the vehicle.
- Concentration in vehicle: 7.5, 75 and 250 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: Animals were paired on a 1 male/1 female basis within each dose group, for a period of up to fourteen days.
- Proof of pregnancy: vaginal plug/sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical validation of the test item was previously determined using an earlier batch of ST1797KS in Harlan Study No. 41203177. In this earlier study, verification work carried out by Harlan Laboratories Ltd., Shardlow, UK Analytical Services established that due to the chemical characteristics of the test item a chromatographic method of analysis could not be developed due to the test items limited solubility in typical HPLC/GC solvents (for this reason there was no alternative but to use the gravimetric approach which involves weighing a set amount of test item into arachis oil (vehicle) to make the required formulation concentration and then analyzing by taking an aliquot onto a glass sintered crucible and then rinsing with a solvent i.e. acetone to remove the oil. The insoluble test item is then retained on the crucible and weighed to confirm the concentration present. The homogeneity determinations were performed using an earlier batch of ST1797KS under Harlan Laboratories Ltd. Study number 41203177. The test item formulations were sampled and analyzed within two days of preparation. The accuracy determinations were performed under Harlan Laboratories Ltd. Study number 41203177. The analytical method has been considered to be sufficiently accurate for the purpose of this study. The test sample results have not been corrected for recovery. The analytical method has been satisfactorily validated in terms of accuracy for the purposes of the study. The results indicate that the prepared formulations were within ± 9 % of the nominal concentration.

Duration of treatment / exposure:

(P) Males: for up to 43 days (beginning during 14 days of pre-mating period)
(P) Females: maximum period of 8 weeks (14 days pre-mating until post-natal day 5)

Frequency of treatment:

daily, 7 days/week

Doses / concentrations

Remarks:

Doses / Concentrations:
30, 300, 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

12 P males, 12 P females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were chosen based on the results of previous work (Harlan Laboratories Ltd., Project No: 41203177). The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to men.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before dosing, soon after dosing, and one hour and five hours after dosing during the working week. Animals were observed immediately before dosing, soon after dosing and one hour after dosing at weekends and public holidays (except for females during parturition where applicable).

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until mating was evident. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.

Oestrous cyclicity (parental animals):

No data

Sperm parameters (parental animals):

Parameters examined in P male parental generations:
- testis weight, epididymis weight, sperm morphology, spermatogenesis

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, live births, stillbirths, postnatal mortality, surface righting, weight gain, clinical signs, presence of gross anomalies

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were killed on Day 43.
- Maternal animals: All surviving animals were killed on Day 5 post partum.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations.

HISTOPATHOLOGY / ORGAN WEIGHTS
- The tissues of coagulating gland, epididymides, ovaries, mammary gland, pituitary, prostate, seminal vesicles, testes (including spermatogenesis), uterus cervix, vagina were prepared for microscopic examination.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring was sacrificed at 5 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination).

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations.

Statistics:

The homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup variance were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covarities. Any transformed data were analyzed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for nonparametric data. If no dose response was found but the data shows non-homogeneity of means, the data were analyzed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric). Data not analyzed by the Provantis data capture system were assessed separately using the SPSS statistical package. Initially, the homogeneity of the data was assessed using Levene’s test. Where Levene’s test was shown to be non-significant (p=0.05), parametric analysis of the data was applied, incorporating analysis of variance (ANOVA). If this data was shown to be significant, this analysis was followed by pair-wise comparisons using Dunnett’s test. Where Levene’s test was significant, non-parametric analysis of the data was analyzed incorporating the Kruskal-Wallis test which if significant, was followed by the Mann-Whitney U test. Dose response relationship was also be investigated by linear regression. Where the data was unsuitable for these analyses, then pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric). Due to the preponderance of non-normally distributed data, reproductive parameters (implantation losses, offspring sex ratio and offspring surface righting) were analyzed using nonparametric analyses. Probability values (p) are presented as follows: p<0.001 ***, p<0.01 **, p<0.05 *, p>0.05 (not significant).

Reproductive indices:

- Pre-coital interval = Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating
- Mating index [%] = Number of animals mated/Number of animals paired x 100
- Pregnancy index [%] = Number of pregnant females/Number of animals mated x 100
- Gestation length = Calculated as the number of days of gestation including the day for observation of mating and the start of parturition
- Parturition index [%] = Number of females delivering live offspring/Number of pregnant females x 100

Offspring viability indices:

- Live Birth Index [%] = Number of pups born alive on day 1/Total number born (live + dead) x 100
- Viability Index [%] = Number of pups alive on day 4/Number of pups live on day 1 x 100
- Pre-implantation loss [%] = Corpora lutea - implantations/Corpora lutea x 100
- Post-implantation loss [%] = Implantations - number of pups born alive/Implantations x 100
- Sex ratio: [% males] = Number of male offspring/Total number of offspring x 100

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:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No treatment-related clinical findings were observed throughout the study. Incidental findings were characterized by sporadic episodes of pilo-erection observed in two 30 mg/kg bw/day males and in a similar number of males treated at 1000 mg/kg bw/day between Weeks 4 and 5. In addition, noisy respiration was observed in one control female (Day 9) and in one female at 30 mg/kg bw/day (Day 42). It is reasonable given the isolated nature and absence of a dose related trend that both findings be considered a result of biological variability and not to be a result of treatment. One 300 mg/kg bw/day female developed a small swelling on the left side of the thorax from Day 19 persisting through to termination. This may have resulted from a mal-dose although there was no evidence of the lesion at necropsy. One 1000 mg/kg bw/day male was observed between Days 28 and 34 to have developed a small scab in otherwise unaffected skin, this minor lesion had regressed by the start of the fifth week of treatment. Given the transient and isolated nature of this finding it was considered to be incidental and not related to treatment. No unscheduled mortality occurred during the study period.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
There was no convincing evidence or dose dependent trend to indicate an adverse body weight response to treatment in any of the male test animals when compared to controls. No adverse effects on body weight change were detected for female test animals during the pre-mating, gestation or lactation phases of the study when compared to controls.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
No treatment-related adverse effect on dietary intake or for food efficiency (the ratio of body weight gain to dietary intake) was identified for either sex of test animals in comparison with controls throughout the treatment-period. The sporadic instances of variances that did occur were considered attributable to biological variability. Daily visual inspection of water bottles did not reveal any significant intergroup differences between control and treated animals.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No treatment-related effects were detected in mating performance. The majority of paired animals mated within the first four days of pairing. Statistical analysis of the pre-coital interval data did not reveal any significant intergroup differences. No treatment-related effects on fertility were detected for treated animals when compared to controls. Three control females, one female at 30 and one female at 300 mg/kg bw/day did not achieve pregnancy following evidence of mating. In the absence of any histopathological correlates in the reproductive organs to elucidate the cause of the non-pregnancy in either the paired female or male partner which did not produce a pregnancy, these intergroup differences were considered to be incidental and of no toxicological importance. No treatment-related effects were detected in the length of gestation for treated females when compared to controls. All animals showed gestation lengths between 22 to 23½ days. Statistical analysis of the data did not reveal any significant intergroup differences.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No treatment related effects were detected in the reproductive organs weighed in males from any treatment group.

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no treatment-related macroscopic abnormalities detected in animals killed at study termination. Incidental findings were confined to one control male observed to have small and flaccid testes and small epididymides. Such observations represent common sporadic findings amongst rats of the strain and age used in the study and as such was considered to have arisen fortuitously.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Under the conditions of this study, the test item ST1797KS produced no histological evidence of toxicological properties in the organs and tissues examined in this study. No treatment-related effects on the testicular histomorphology were identified including spermatogenesis and interstitial cell structure.

Effect levels (P0)

Dose descriptor:

NOEL

Remarks:

systemic

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No mortality and no clinical signs of toxicity occurred during the study period

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)

VIABILITY (OFFSPRING)
In total nine females from control, eleven females from 30 and 300 mg/kg bw/day dose groups and twelve females from 1000 mg/kg bw/day dose group gave birth to a live litter and successfully reared young to Day 5 of age. No significant differences were detected for corpora lutea counts for treated animals when compared to controls. Litter sizes and viability for treated groups were also comparable to controls. There were no convincing treatment-related intergroup differences in sex ratio (percentage male offspring) for litters from treated groups compared to controls.

CLINICAL SIGNS (OFFSPRING)
No clinical observable signs of toxicity were detected for offspring from all treatment groups.

BODY WEIGHT (OFFSPRING)
Offspring body weight gain and litter weights at birth and subsequently on Day 1 and Day 4 post partum were comparable to controls. There were no significant differences in litter weights or mean offspring body weights between control and treated animals. Statistical analysis of the data did not reveal any significant intergroup differences.

GROSS PATHOLOGY (OFFSPRING)
No treatment-related macroscopic abnormalities were detected for interim death or terminal kill offspring. The incidental findings observed were those occasionally observed in reproductive studies of this type and were considered to be unrelated to toxicity of the test item.

Effect levels (F1)

Dose descriptor:

NOAEL

Remarks:

reproduction

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The oral administration of ST1797KS to rats for a period of up to eight weeks (including two weeks pre-mating, gestation and early lactation period for females) at dose levels of up to 1000 mg/kg bw/day resulted in no treatment related effects. There were no convincing treatment-related responses observed throughout the study nor treatment-related effects detected in the reproductive parameters observed namely for mating performance or fertility. Furthermore, there was no evidence of any developmental effects observed in offspring from treated litters and no effect on the reproductive organs was evident following post mortem assessments or during histopathological assessments. On this basis, the ‘No Observed Effect Level’ (NOEL) for systemic toxicity and reproduction for either sex was considered to be greater than 1000 mg/kg bw/day.