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Toxicity to reproduction

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Administrative data

screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report

Materials and methods

Test guideline
according to guideline
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. QA statement)
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:

Test animals

Details on test animals or test system and environmental conditions:
- Wistar rats, strain: Hsd:HanWIST with appropriate range of bodyweight at study start.
- Source: Advinus Therapeutics Ltd. in-house random bred (conventionally bred)
- Age at treatment start: 11-12 weeks old, both sexes
- Weight at treatment start: 241-294 g for males, 185-210 g for females
- Housing in polysulfone cages with stainless steel top grill
during pre-pairing dosing period: In groups of 2 by sex
during pairing: 1 male+1 female/cage
males after pairing: In groups of 2 by sex
females during gestation and lactation: Females housed individually (+litter).
- Bedding material (in polysulfone cages): steam sterilised clean corn cob
- Diet (ad libitum): Teklad certified Global 14% protein Rodent maintenance diet - pellets, Harlan, 5800 AN Venray, The Netherlands
- Water (ad libitum): Deep bore-well water passed through activated charcoal filter and UV irradiated, regular quality control
- Acclimation period: 5 days before treatment start, after examination for health and suitability.

The animal room was maintained at (target ranges for temperature and relative humidity):
- Temperature (°C): 19 - 24°C
- Relative Humidity (%): 57 - 67 %
- Photoperiod (artificial lighting): 12 hrs day / 12 hrs night
- Ventilation: 12-15 times/h

Administration / exposure

Route of administration:
oral: gavage
polyethylene glycol
PEG 300
Details on exposure:
- 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. Concentrations amounted to 15, 30, and 100 mg/ml vehicle, accordingly.
- Amount (dose volume by gavage): 10 mL/kg bw/day.
Actual dose volumes were calculated at about weekly or shorter intervals accounting for the latest bodyweight.

- Frequency of preparation of dose formulations: Once a week

Treatment of parental animals by oral gavage administration. Test substance was not directly administered to F1 animals, as these pups were sacrificed on Day 4 post partum, i.e. Day 4 of lactation.
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 pregnancy: Formation of vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy.
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
For analysis of the test substance in the vehicle GC / FID was used with a DB-35 column (30 m long, 0.53 mm i.d., 0.5 μm film thickness). The analytical method was validated. The mean contents of the test substance in dose formulations were found to be well within the acceptance limit (within +/- 15%) of dose theoretical concentration; the relative standard deviation was equal to or less than 10%.
Duration of treatment / exposure:
- Treatment period, parental males: 42 days (14 days before mating and 28 days including up to 14 days for mating)
- Treatment period, parental females (dams): approx. 40-45 days (from 14 days prior to mating to Lactation Day 4)
- Pups were not treated directly (possibly via milk): until Lactation Day 4.
Frequency of treatment:
Doses / concentrationsopen allclose all
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Control animals:
yes, concurrent vehicle
Positive control:
Not included in the study.


Parental animals: Observations and examinations:
Clinical observations performed and frequency:
- Clinical signs: Daily (covering external appearance, motor activity & morbidity in each animal)
- Body weight, Males: Weekly from Treatment Day 0 to 42 (i.e. from the first treatment day until the day before necropsy).
Body weight, Females: Weekly for pre-pairing & pairing period, Gestation Days 0, 7, 14, 20 & Lactation Days 0 (i.e. the day post partum) & 4
- Food consumption, Males: Weekly for pre-pairing period
Food consumption, Females: Weekly for pre-pairing period (Days 0-7 & 7-14) and for Gestation (Days 0-7, 7-14, 14-20) & Lactation (Days 0-4, i.e. the day post partum until the day of necropsy).
Oestrous cyclicity (parental animals):
not observed
Sperm parameters (parental animals):
Parameters examined in male parental animals: testis weight, epididymis weight, histopathology with special emphasis on stages of spermatogenesis in male gonads and of interstitial testicular cell structure
Litter observations:
- Delivery or post-implantation survival index (Total no. of pups born / Total no. of implantation sites) x 100
- Total no. of pups (alive and dead), sex
- Body weight of live pups (on Lactation Days 1 and 4)
- No. of pups alive, dead or cannibalised on Lactation Days 1, 2, 3, 4
- Live birth index (No. of live pups on Lactation Day 1 / Total no. of pups born) x 100
- Viability index (No. of live pups on Lactation Day 4 / No. of live pups on Lactation Day 1) x 100
- Sex ratio  (Nos. of male pups and Nos. of female pups)
- Clinical signs, on each day after birth

Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes, see below
WEIGHING OF ORGANS: Yes, see below
HISTOPATHOLOGY: Yes, see below

Terminal sacrifice
- Males: Killed on the day after the 42-day treatment period.
- Terminal sacrifice, Females (dams): Killed on Lactation Day 4
- Terminal sacrifice, unmated Female: 1 mid dose female (300 mg/kg/day) which had failed to mate was killed on the day after 26 daily doses

Gross pathology: Necropsy with tissue collection.
The number of implantation sites and corpora lutea was recorded for all dams

Organs Weights: The following organs were weighed at necropsy and their ratios to terminal bodyweight determined:
testes, epididymides.

Histopathology: The following organs were microscopically observed for the control and 1000 mg/kg bw/day groups:
testes, epididymides, prostate, seminal vesicles and coagulating glands, ovaries.
Postmortem examinations (offspring):
On Lactation Day 4 external macroscopic examination of all survivors for gross abnormalities.
The statistical analysis of the experimental data was carried out using the validated package in Excel and also using licensed copies of SYSTAT Statistical package ver.12.0. All quantitative variables like body weight, food consumption, organ weights and organ weight ratios were tested for normality (Shapiro-Wilk test) and homogeneity of variances (Levene’s test) within the group before performing a one-factor ANOVA modeling by treatment groups. Non-optimal (non-normal or heteroschedastic) data were transformed, before using ANOVA. Means between treatment and control groups were compared using Dunnett’s test if the overall ‘F’ test was found to be significant.
Pre-implantation loss (%), post implantation loss (%), no. of corpora lutea, implantations, pre-coital interval and gestation length (days) were analyzed after suitable transformation (√ x + ½) of the data. One-way analysis of variance (ANOVA) was carried out for the transformed data. Dunnett’s pairwise comparison of the treated means with the control mean was done for the significant group differences.
Z test was performed for testing the differences in proportions for mating and fertility indices.
All analyses and comparisons were evaluated at the 5% (P≤0.05) level.
Reproductive indices:
- Pre-coital interval (pairing days until detection of mating)
- No. of animals mating (evidence of successful copulation, i.e. at least one copulation plug or a sperm positive vaginal smear)
- No. of animals achieving pregnancy
- Percentage mating or copulation index (No. of animals mating/No. of animals paired) x 100
- Conception rate or fertility index (No. of animals achieving pregnancy/No. of animals mated) x 100
- Gestation length (time elapsing from detection of mating until the day prior to confirmation of parturition)
- No. of pregnant animals
- No. of pregnant animals with parturition
- Gestation Index (No. of animals achieving pregnancy with parturition/No. of animals achieving pregnancy) x 100
- No. of corpora lutea
- No. of implantation sites
- Implantation index (No. of implantation sites/No. of corpora lutea) x 100
Offspring viability indices:
- Mean litter size per group (number of implantations - number of live fetuses / number of implantations) x 100
- Day 4 survival index (No. of live pups on Lactation Day 4 / No. of live pups born) x 100.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
semi-solid feces in 2/10 males at 1000 mg/kg/day from Day 5 to 9.
mortality observed, non-treatment-related
Description (incidence):
one dam from the 1000 mg/kg/d group was found dead on treatment day 37 (GD21); attributed to spontaneous dystocia thus not related to treatment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Significantly lower mean body weight was observed in males at 1000 mg/kg Bwt/day on Day 43; mean body weight gain was lower in males between Days 1-8, 36-43 and 1-43. Reduced body weight gain in females at 1000 mg/kg/day between Day 1-8 and 11-15. See attached Tables in "Attached background material".
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
only reproduction organs were examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
represented by testes & epididymis weights and absence of histopathological findings.
Reproductive performance:
no effects observed
Description (incidence and severity):
See attached Tables in "Attached background material".

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
reproductive toxicity
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: see 'Remark'
Key result
Dose descriptor:
parental toxicity
Effect level:
> 300 - < 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
other: with regard to parental toxicity the NOAEL was derived at the dose level of 300 mg/kg/d based on reduction in body weight gain primarily in males of the high dose group

Target system / organ toxicity (P0)

Key result
Critical effects observed:
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
other: body weight gain
other: body weight gain
Treatment related:

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
See attached Table on survival data of pups in "Attached background material".
Body weight and weight changes:
no effects observed
Description (incidence and severity):
See attached Table on body weight of pups in "Attached background material".
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

Day 4 survival index was significantly lower at 300 and 1000 mg/kg Bwt/day when compared to the control group. This finding was mainly due to the loss of an entire litter from a single dam (Animal No. Ro2741) at 300 mg/kg Bwt/day and also due to litter losses in two dams (Animals No. Ro2751 and Ro2754) at 1000 mg/kg Bwt/day during the lactation period. A similar incidence of total litter loss was spontaneously observed in a previous OECD 421 study performed in-house. The observed low Day 4 survival index in both 300 and 1000 mg/kg Bwt/day groups was therefore considered to bear no relationship to treatment with test item.

Effect levels (F1)

Key result
Dose descriptor:
developmental toxicity
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: NOAEL = highest dose tested.

Target system / organ toxicity (F1)

Key result
Critical effects observed:

Overall reproductive toxicity

Key result
Reproductive effects observed:

Applicant's summary and conclusion

In this screening study there were no treatment related effects on reproduction or developmental toxicology parameters. The no-observed-adverse- effect-levels (NOAEL) for parental toxicity regarding reprotoxic endpoints and for foetal toxicity are 1000 mg/kg bw/day. The NOAEL for general parental toxicity was derived at 300 mg/kg bw/day based on reduction of weight gain at the highest dose.