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Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-06-03 to 2016-01-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted 27 July 1995
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
strain: Crl:CD (SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Italia s.r.l, Via Indipendenza. 11, 23885 – Calco (Lecco), Italy
- Age at arrival: 8 weeks
- Weight at arrival: Males:260.0-300.4 g; Females: 168.2-210.4 g
- Housing: double or single (depending on the part of the experiment)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 2°C
- Humidity (%): 55% ± 15%,
- Air changes (per hr): 15 - 20 air changes per hour filtered on HEPA,
- Photoperiod (hrs dark / hrs light): 12 / 12 hours

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
PEG 300
Details on exposure:
VEHICLE
- Justification for use and choice of vehicle (if other than water): Solubility in vehicle PEG 300
- Concentration in vehicle: 0, 10, 30, and 100 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg
- Lot/batch no. of PEG300: K45904702 (Merck KGaA)
Details on mating procedure:
- Male/female mating ratio: 1/1
- Length of cohabitation: 7 days/week with a maximum of 14 days
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of the formulated test item was checked twice during the dosing period (i.e., on formulations prepared on one day of the pre mating and post mating period).
The used HPLC analytical method was validated. The homogeneous distribution and stability of the test item in the vehicle PEG300 was demonstrated.
Duration of treatment / exposure:
F0 Males were treated for 2 weeks before the start of the mating period and throughout the same.Males were further dosed after the mating period until the minimum total dosing period of 28 days was completed.
F0 Females were treated for 2 weeks before the start of the mating period and throughout the same. Treatment continued throughout pregnancy and until day 3 of lactation.
A concurrent control group received PEG300 with the same dosing regimens.
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 (Total number of animals: 80 (10 males +10 females per group; 4 groups)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on a 28 day study (rat, oral, gavage) with the test item
Positive control:
No

Examinations

Parental animals: Observations and examinations:
The following observations and examinations were performed:
- Mating
- Clinical signs (twice daily)
- Body weight (before dosing (-1 day), on the first day of dosing and then weekly)
- Food consumption (weekly) and water consumption (twice a week)
- Mortality and abortion
- Reproduction parameters (Mating index, fertility index, pregnancy index, pregnancy period, mean mating time, pre-implantation losses, post-implantation losses)
- Gross necropsy and histopathology (Ovaries, Epididymis, Prostate, Seminal vesicle, Testis, all tissues showing abnormalities)
Oestrous cyclicity (parental animals):
Not examined
Sperm parameters (parental animals):
A detailed qualitative examination of one testis and the equilateral epididymis was done in 10 male rats of the control (vehicle) group, as well as in 10 males of the high dose group, 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 disorganization of the germinal epithelium.
Litter observations:
On Day 0 of lactation, the pups of each litter were identified with a mark made by appropriately coloring different sites of the body; this mark was renewed when necessary.
Live pups were counted and sexed and litters weighed within 24 hours of parturition (Day 0 of lactation) and on Day 4 post partum.

At birth and during the lactation period, all the young were individually observed for:
- Live and stillbirths
- Runts (pups that are significantly smaller than control pups)
- Mortality ensuing after live birth, ascertained daily. Whenever possible, any pup found dead was examined externally and internally in an attempt to determine the cause of death;
- Sex (on Day 0 of lactation by an external examination and measurement of the anogenital distance and by internal examination, at sacrifice, on Day 4 of lactation);
- External abnormalities at birth;
- Pup weight (on Days 0 and 4 of lactation) Females were killed on Day 4 of lactation together with their pups.
- Reproductive parameters (Birth index, Viability index)
Postmortem examinations (parental animals):
- Gross pathology
Any abnormalities or pathological changes, with special attention to the reproductive system organs. Number of implantation sites and corpora lutea was recorded in pregnant females.
- Organ weight:
testes and epididymides of all male adult animals
- Histopathology
Reproductive organs, female: Ovary (2)
Reproductive organs, male: Epididymis (2), Prostate, Seminal vesicle, Testis (2)
All tissues showing abnormality
Postmortem examinations (offspring):
Gross pathology
Statistics:
For the statistical analysis of the frequencies of dams or litters control and treated groups were compared using the Cochran-Armitage Trend Test followed by pairwise comparison with the control group using the Fisher’s Exact Test with Bonferroni-Holm correction.
For all other data a decision tree was applied consisting of a data preprocessing for Normality test (Shapiro-Wilks) and homogeneity of variances test (Bartlett) and a main data analysis with parametric (Williams test, ANOVA, Dunnett test) and non-parametric analysis (Shirley-Williams test, Kruskal-Wallis and Steel test).
All variables were checked for Normality and for Homogeneity of Variances. Normal and homogeneous variables followed a parametric approach. Variables were checked under no transformation and then under a log-transformation.
Analysis of cumulative survival data or conditions that develop over time were done using Log Rank Tests with Kaplan-Meier Test.
Reproductive indices:
- Mating index: percent ratio between the animals with positive smear + females found to be pregnant but without positive smear and the animals mated.
- Fertility index: percent ratio of females having evident signs of pregnancy with respect to the females that had positive vaginal smear and females found to be pregnant but without positive smear.
- Pregnancy index: the percent ratio of females with live births with respect to the pregnant females.
- Pregnancy period: the duration of pregnancy was determined for all those dams that reached pregnancy term as being the time that elapsed between the positive vaginal smear and the start of parturition.
- Preimplantation losses
- Postimplantation losses
Mean mating time: was calculated on the dams proved pregnant and was expressed for each group as the mean time lapse (in days) between the beginning of the mating period and the ascertainment that copulation occurred (positive smear).
Offspring viability indices:
- The mean F1 body weight was obtained by averaging the mean weight of each litter at the various times.
- The F1 probability of survival was analyzed per group. Animals that died owing to accidental causes or were sacrificed at the end of lactation were statistically censored.
- The mean value per litter of live pups (number of males and females and total) was calculated at different times (Days 0, 4 of lactation).
- The following indices of each litter were calculated:
Birth index:
No. of live newborns at birth
----------------------------------- x 100
No. of implantations

Viability index on Day 4:
No. live pups on Day 4 after birth
----------------------------------------- x 100
No. of live births

Group mean values were calculated from individual data in two ways:
Mean A: calculated on all the surviving females having evident signs of pregnancy, includingthose that presented 100% post-implantation losses.
Mean B: calculated only on those females with viable fetuses at term.
The external, visceral and skeletal malformations and variations found were described for each litter.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical observations or behavioural changes were noted in any experimental animal during the study.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female of group 2 (150 mg/kg bw/day) was found dead on day 7 of the study. The reason is considered to be not treatment-related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males: At 500 mg/kg bw/day a minimal lower mean body weight (statistically significant on day 14 of the study) together with significantly lower mean body weight gain was observed in males during the first two weeks of treatment (day 1-7 and 7-14) with recovery thereafter. Males at 50 and 150 mg/kg did not show any compound-related effects on body weight at any time during the study in comparison to controls.
Females: No toxicologically relevant changes in body weight during the course of the study.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 500 mg/kg bw/day significantly lower mean food consumption in males at day 1-7 and 7 - 14 periods in comparison to control. No effects were observed at low and mid dose.
No relevant effects on food consumption was observed in females.
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Males: Statistically significantly lower mean water consumption was observed at 150 mg/kg bw/day on Days 11-14 and at 500 mg/kg on Days 4-7, 7-11 and 11-14.
Females: During the 2-week pre-mating period no differences in water consumption were observed between treated and control females. The statistically significantly lower mean value at 500 mg/kg in the Day 7-11 period in comparison to controls is not considered treatment-related since confined only to this interval.
During pregnancy, slightly lower mean water consumption was observed at 150 mg/kg, after one week of treatment, and at 500 mg/kg, in comparison to the control group; statistically significantly lower values were reached on days 14-17 in Group 3 and on days 7-11 and 14-17 in Group 4.
During the lactation period lower mean water consumption was still observed in the 500 mg/kg/day treated group in comparison to controls, although without any statistical significance.
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:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
The oral application of the test material led to no treatment-related findings in testes, epididymides, prostate, seminal vesicle, and ovary.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
The randomly scanned seminiferous tubules of 10/10 males of the control and 9/10 males of the high dose group showed the appropriate cell layers in their approximate normal numbers, this means that stages I-VIII contained a layer of spermatogonia, a layer of pachytene spermatocytes and several layers of round spermatids interspersed with elongated spermatids. Stages IX-XIV contained a layer of spermatogonia and prepachytene spermatocytes, several layers of late pachytene or dividing (stage XIV) spermatocytes and several layers of elongating spermatids. In stage IX-XII only one population of elongated spermatids at the lumen was observed. Residual bodies in stage VII and VIII appeared normal. There was no evidence of degenerating spermatocytes or spermatids. The estimated stage frequency was proportional to stage duration. One high dose group male (Animal-no. 70) showed a marked tubular atrophy with loss of spermatocytes and spermatids. This single case is considered spontaneous and incidental and not related to treatment.
Reproductive performance:
no effects observed
Description (incidence and severity):
No differences were seen in the mating index (ratio between mated and paired animals) or fertility index (ratio between pregnant and mated animals) among the various experimental groups. The mean pre-coital time was similar in all groups.
One female only (No. 80 of Group 4) never showed a positive smear, but this could be related to its male (No. 70), which at histology showed marked tubular atrophy with loss of spermatocytes and spermatids. This single case was considered spontaneous and incidental and not related to treatment.
Pregnancy length and pregnancy index (ratio between females with live births and pregnant females) among the different experimental groups showed no differences.
The mean number of corpora lutea in Groups 2, 3 and 4 were slightly higher in comparison to the control group, though without dose-relationship.
A slightly lower mean value of implantations, nevertheless significant, in the highest dose treated group was observed in comparison to controls, while no changes were observed in the low and mid dose groups. However, this value is in the lower range of historical control values of the laboratory and even exceeding the historical control ranges of Charles River and considered not relevant.
Higher mean pre-implantation losses were observed in all treated groups in comparison to controls, however, not significant and without dose-relationship. This effect is due to single animals and, as the number of corpora lutea and implantation sites are within historical control data of the laboratory, considered not relevant.
No differences were observed in the mean value per litter of live pups at birth except for a slight reduction in the mean number of live pups at the highest dose (13.38 vs. 14.90 of the control group) which is mainly due to one animal in Group 4 (No. 78 which lost 5 of 12 pups). No stillborns were found in any group. Furthermore post implantation losses and birth index values showed no relevant differences in treated groups in comparison to controls.

Details on results (P0)

No compound-related deaths and no clinical signs were observed during the study.
At 500 mg/kg lower mean daily food and water consumption was observed in males during the first two weeks of treatment, together with lower body weight gain in these animals.
No relevant changes were seen in body weight, or food and water consumption of females.
At necropsy no relevant macroscopic changes were seen in any animal and no differences were noted in the mean weight of testes and epididymides of treated males in comparison to controls.
No treatment-related changes were recorded during histological examination performed on the ovaries, testes and epididymis.
No effects were observed on the reproductive performance of animals, i.e. no differences were noted comparing the mating and fertility indices (ratio between mated and paired animals and ratio between pregnant and mated animals, respectively) of the treated groups with those of the control group. In addition the mean pre-coital time was similar in all experimental groups.
No differences were seen in the pregnancy length and in the pregnancy index (ratio between females with live births and pregnant females) between treated and control groups.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no adverse effects observed
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
Remarks on result:
other: slight effects, not adverse

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Slightly lower mean number of live pups were observed at 500 mg/kg bw/day. Viability index on day 4 (mean values per litter) slightly lower at 500 mg/kg bw /day mainly due to one female with a value of 58.33% vs. 100% of control group.
Body weight and weight changes:
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Anogenital distance (AGD):
effects observed, non-treatment-related
Description (incidence and severity):
A statistically significantly longer anogenital distance was observed in female pups at 150 and 500 mg/kg. In males a slightly longer anogenital distance in comparison to controls was observed at 500 mg/kg, though without reaching statistical significance. The above finding can be explained by the slightly higher weight of the pups at birth.
Day 0 Gr.1 (0 mg/kg) Gr.2 (50 mg/kg) Gr.3 (150 mg/kg) Gr.4 (500 mg/kg)
AGD males (mm) 3.20±0.399 3.31±0.340 3.18±0.257 3.48±0.323
AGD females (mm) 1.63±0.175 1.66±0.207 1.80±0.1.34* 1.95±0.114**
Mean Male Pup BW 6.96±0.575 6.77±0.661 6.51±0.434 7.24±0.730
Mean Female Pup Bw 6.40±0.520 6.38±0.597 6.34±0.830 6.84±0.787
** P<0.001
Furthermore the latter finding was not confirmed by the external sex evaluation, since the correspondence between internal and external sex was 100% in all experimental groups at autopsy Day 4.
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
During autopsy no changes were noted in pups, except for a few pups showing a whitish content in the bladder:
Group 2: Animal No. 36 pup 2, animal No. 39 pup 1
Group 3: Animal No. 56 pup 12
Group 4: Animal No. 74 pup 5
This finding was not considered as having any toxicological relevance since it was sporadically observed in a few pups in all groups.
Histopathological findings:
not examined
Other effects:
no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

No differences were observed in the mean value per litter of live pups at birth except for a slight reduction in the mean number of live pups at the highest dose (13.38 vs. 14.90 of the control group). No stillborns were found in any group. Furthermore post implantation losses and birth index values showed no relevant differences in treated groups in comparison to controls.
No post-natal losses were observed in the control group, while in groups 2, 3 and 4 the following
post-natal losses were observed:
- Group 2: 1 cannibalized pup of one female 32.
- Group 3: 1 cannibalized pup of female No. 52, 1 cannibalized and 1 pup found dead of
female No. 53; 1 cannibalized pup of female No. 60
- Group 4: 1 cannibalized pup of female No. 72; 1 found dead and 4 cannibalized pups of
female No. 78.
Slightly lower mean viability index on Day 4 was observed at 500 mg/kg/day, in comparison to controls, mainly due to Female No. 78 treated at 500 mg/kg, which lost 5 pups.
No differences were seen in the sex ratio among the different experimental groups.
No abnormality at birth was seen in any pup.
The mean weight of male and female pups at birth was slightly higher in Group 4 without any statistical significance.
A statistically significantly longer anogenital distance was observed in female pups at 150 and 500 mg/kg. In males a slightly longer anogenital distance in comparison to controls was observed at 500 mg/kg, though without reaching any statistical significance. The above finding can be explained by the slightly higher weight at birth, observed in single pups in dosed groups. Furthermore the latter finding was not confirmed by the external sex evaluation, since the correspondence between internal and external sex was 100% in all experimental groups at autopsy Day 4.
No differences were observed in pup body weight on Day 4 of lactation between treated groups and controls.
During autopsy no changes were noted in adult animals or pups, except for a few pups showing a whitish content in the bladder. This finding was not considered as having any toxicological relevance since it was sporadically observed in a few pups in all groups.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
In conclusion, the test item given orally to male rats for twenty-eight consecutive days and to females during the pre-mating, mating and gestation periods and up to day 3 of lactation at the doses of 0, 50, 150 and 500 mg/kg/day induced no treatment-related adverse effects to reproduction up to the highest dose tested.
On the basis of the above findings, 150 mg/kg was considered the NOELsystemic tox and 500 mg/kg bw/day the NOAELreproductive tox.
Executive summary:

Study Design

In this study the evaluation of possible effects on reproduction and/or development of the test item was assessed in Crl:CD (SD) rats.

The test item was administered to 10 animals/sex/group, daily by oral route, at 50, 150 and 500 mg/kg bw/day using PEG 300 as the vehicle.

Treatment of F0 males started two weeks before mating and lasted for 28 days.

F0 Females were treated continuously from two weeks before mating up to Day 3 of lactation.

A concurrent control group of males and females received the vehicle PEG 300 with the same dosing regimens.

All animals were observed for survival and clinical signs; any abnormality was recorded for parent generation and offspring.

Body weight, and food and water consumption were recorded at scheduled times during the premating, pregnancy and lactation periods.

On the day of parturition (Day 0 of lactation) pups were counted and weighed; then they were sexed by an external examination and the ano-genital distance measured.

The presence of live and dead pups, runts and stillbirths was checked in all litters. Any pup found dead was examined in an attempt to determine the cause of death.

F0 males were sacrificed at the end of the treatment period, on Day 29 of the study.

F0 females were sacrificed on Day 4 of lactation together with their litters. Females that never showed evidence of copulation were killed 27 days after the end of the mating period while females with sign of copulation which did not litter were sacrificed 26 days after the day of positive smear.

The uteri of apparently non-pregnant females were stained with Salewski solution for thepresence of implantation sites.

During autopsy all animals were macroscopically examined for any abnormality or pathological changes.

Special attention was paid to the reproductive system organs of adult animals; furthermore testes and epididymides of all adult males were weighed.

The number of implantation sites and corpora lutea was recorded in females.

The anogenital distance of pups was measured and internal sex evaluation was performed on them at autopsy.

Detailed histological examination was performed on the ovaries, testes and epididymis (with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cellstructure) of the animals of the highest dose group and control group.

Results

No compound-related deaths and no clinical signs were observed during the study.

At 500 mg/kg bw/day lower mean daily food and water consumption was observed in males during the first two weeks of treatment, together with lower body weight gain in these animals.

No relevant changes were seen in body weight, or food and water consumption of females.

At necropsy no relevant macroscopic changes were seen in any animal and no differences were noted in the mean weight of testes and epididymides of treated males in comparison to controls.

No treatment-related changes were recorded during histological examination performed on the ovaries, testes and epididymis.

No effects were observed on the reproductive performance of animals, i.e. no differences were noted comparing the mating and fertility indices (ratio between mated and paired animals and ratio between pregnant and mated animals, respectively) of the treated groups with those of the control group. In addition the mean pre-coital time was similar in all experimental groups.

No differences were seen in the pregnancy length and in the pregnancy index (ratio between females with live births and pregnant females) between treated and control groups.

Slightly lower mean number of implantations, but nontheless significant, and consequently higher mean pre-implantation losses (namely corpora lutea minus implantations, not significant) and slightly lower mean number of live pups (not significant) were observed at 500 mg/kg bw/day in comparison to the control values. However, all these values were within historical control data and considered not adverse.

The viability index on day 4 showed slightly lower mean values per litter in the 500 mg/kg bw/day treated group in comparison to controls, mainly due to one female with a value of 58.33% vs. 100% of the control group and not considered treatment-related.

The anogenital distance of female pups of the mid and high dose group was slightly longer than that control pups which can be explained by incread body weights of these pups. Moreover, at internal examination on Day 4 the correspondence between internal and external sex was 100% for all the animals.

Necropsy of pups on Day 4 did not evidence any compound-related changes.

Conclusions

In conclusion, the test item given orally to male rats for 28 consecutive days and to females during the pre-mating, mating and gestation periods and up to day 3 of lactation at the doses of 0, 50, 150 and 500 mg/kg bw/day induced lower mean body weight gain, together with lower food and water consumption in the highest dose treated males.

The test material induced no treatment-related adverse effects to reproduction up to the highest dose tested.

On the basis of the above findings, 150 mg/kg was considered the NOELsystemic tox and 500 mg/kg bw/day the NOAELreproductive tox.