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Toxicological information

Toxicity to reproduction

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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:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

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

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
- Source: Charles River (Europe) Laboratories Inc, Toxicoop Ltd. Hungary, 1103 Budapest, Cserkesz u. 90
- Age at study initiation: 10-11 weeks old at starting, 12-13 weeks old at mating
- Weight at study initiation: 351-414 g (males), 204-261 g (females)
- Housing: type II and III polypropylene/polycarbonate
- Diet: ssniff SM R/M-Z+H "Autoclavable complete feed for rats and mice - breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany, ad libitum
- Water: tap water from municipal supply, ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19.9-24.0 °C
- Humidity: 30-60 %
- Air changes: 15-20 changes/hour
- Photoperiod: 12 hours dark / 12 hours light

IN-LIFE DATES: From: 29 October 2009 (animal arrival) To: 25 December 2009 (last necropsy)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.1 % Tween 80 in PEG 400 (w/v)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle at the appropriate frequency to allow the use of formulations within 3 days while stored at 5 ± 3 °C.

VEHICLE
- Justification for use and choice of vehicle: the test item is not soluble in water. According to the analytical method, a mixture of 0.1 % Tween 80 in PEG 400 (w/v) was considered a suitable vehicle to be used for the preparation of dose formulations for oral administration.
- Concentration in vehicle: 0, 18.75, 75 and 250 mg/mL
- Amount of vehicle: 4 mL/kg bw
- Lot/batch nos.: 1435799 (PEG 400), 1435771 (Tween 80)
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: max. 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged individually. Bedding material suitable for nesting was provided.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Top, middle and bottom samples were taken for analysis of concentration and homogeneity, in duplicate from each of the test item dosing formulations (low, mid and high dose) on 3 occasions, approximately during the first, mid and last week of treatment as practical. Similarly, one sample was taken in duplicate from the control solution for concentration measurements.
Duration of treatment / exposure:
Main males were dosed for at least 28 days (14 days pre-mating and 14 days mating plus an optional extended post-mating period), then they were euthanized and subjected to necropsy examination or alternatively were retained and continued to be dosed for the possible conduction of a second mating if considered appropriate.
Main females were dosed for 14 days pre-mating, for up to 14 days mating, through gestation period and up to and including the day before necropsy (at least 4 days post-partum dosing). Females showing no evidence of copulation were sacrificed 24-26 days after the last day of the mating period.
Recovery animals (not used for the assessment of reproduction/developmental toxicity) were kept at least for further 14 days after the first scheduled euthanasia of dams, without treatment to detect delayed occurrence, persistence of or recovery from toxic effects.
All F1 offspring was terminated on postpartal day 4 or shortly thereafter.
Frequency of treatment:
Once daily
Details on study schedule:
- Age at mating of the mated animals in the study: 12 weeks old
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 75, 300 and 1000 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
Main study: 12 animals
Recovery: 5 animals (control and high dose only; not used for the assessment of reproduction/developmental toxicity)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: doses were selected based on a preceding dose range finding study (LAB Ltd., study code 09/174-220PE).
Positive control:
None

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS:
- Time schedule: once daily, after treatment

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: once weekly, after treatment

BODY WEIGHT:
- Time schedule for examinations:
Main study: parent animals were weighed on the first day of dosing (Day 0), at least weekly thereafter and at termination. Parent females were weighed on gestation Days 0, 7, 14 and 20 and on postpartal Days 0 and 4. Body weight of females was additionally weighed on gestation Days 10 and 17 in order to give accurate treatment volumes, but these data were not evaluated statistically.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: at least weekly

OTHER: number of pairings, fertile pairings, infertile males, pregnant females, sperm positive but non-pregnant females, non-mated females, corpora lutea/dam, implantations/dam and dams with live pups on Day 0 and 4, duration of pregnancy, pre-implantation mortality, intrauterine mortality and total intra- and extrauterine mortality.

Various ophthalmology, clinical pathology, urinalysis and neurobehaviour parameters were assessed in parental animals according to OECD guideline 422; please refer to "7.5.1 Repeated dose toxicity: oral".
Oestrous cyclicity (parental animals):
Examination of the oestrous cycle in parental females was performed during the mating period, until a sperm positive vaginal smear or a vaginal plug was identified.
Sperm parameters (parental animals):
Parameters examined in male parental generation: testis weight, epididymis weight, weight of seminal vesicles with coagulating glands as well as histological examination of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: mean pup body weight on postnatal Days 0 and 4, mean pup body weight gain (per litter) between postnatal Days 0 and 4, number of live births per litter and number of viable pups per litter on postnatal Days 0 and 4

GROSS EXAMINATION OF DEAD PUPS:
yes, at least for external abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: all surviving animals, after at least 28 days of dosing
- Maternal animals: all surviving animals, after at least 4 days of postpartum dosing

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
Samples for histopathology were collected from 5 parental animals/sex/group. The following organs and tissues (or representative samples) were preserved: adrenals, aorta, brain, epididymides, eyes with optic nerve, oesophagus, femur with marrow, heart, kidneys, large intestine, lachrymal gland with Harderian glands, liver, lungs with bronchi, lymph nodes, mammary gland, ovaries with oviduct, pancreas, pituitary, prostate, salivary gland, sciatic nerve, seminal vesicles with coagulating glands, skeletal muscle, skin and subcutis, small intestine, spinal cord, spleen, sternum with marrow, testes, thymus, thyroid with parathyroids, tongue, trachea, urinary bladder, uterus and vagina.
Histological examinations were performed in control and high dose animals only. Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries covered the follicular, luteal and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.

At the time of termination, body weight and weight of the following organs of all parental animals were determined:
uterus (with and without cervix), vagina, testes, epididymides (total and cauda), prostate, seminal vesicles with coagulating glands, brain, ovaries and pituitary gland
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring was sacrificed on postnatal Day 4.
- These animals were subjected to postmortem macroscopic examination.

GROSS NECROPSY
- Pups euthanised at postnatal Day 4 were carefully examined at least externally for gross abnormalities. Any found dead pups were subjected to necropsy with internal and external macroscopic examinations.
Statistics:
The statistical evaluation was performed with the statistical program package SPSS PC+4.0. The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test the Kruskal-Wallis analysis of variance was used and the inter-group comparisons was performed using Mann-Whitney U-test. Chi square test was performed where applicable.
Reproductive indices:
Male and female mating indices, male and female fertility indices and gestation index
Offspring viability indices:
Survival index on postnatal Days 0 and 4, sex ratio on postnatal Days 0 and 4

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:
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
There was no mortality or relevant clinical findings.

BODY WEIGHT AND FOOD CONSUMPTION
Body weight and food consumption were not affected by the treatment with the test item.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
There were no test item related differences in the oestrous cycle evaluated during the mating period.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
There was no test item-related increase in infertile males.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No effects were noted on mating, fertility and gestation indices.

ORGAN WEIGHTS
In main male animals, statistically higher mean liver weights were noted at 300 and 1000 mg/kg bw/d, with an apparent dose response, and higher kidney weights were noted at 1000 mg/kg bw/d, as absolute values (p<0.01) and when adjusted for body and brain weight relative values which showed statistical significance at 75 mg/kg bw/d (p<0.05). Similar effects were observed in females (p<0.05 for absolute kidneys weight at 1000 mg/kg bw/d; p<0.01 for absolute liver weight at 300 and 1000 mg/kg bw/d) and the brain and body weight relative liver weights were also statistically higher than control at 300 and 1000 mg/kg bw/day. No statistically significant variations were noted in the absolute or relative liver or kidneys weights after a 14-day recovery period in the female animals; in the recovery male animals, the relative mean liver weight referring to body weight was slightly higher than control at 1000 mg/kg bw/d.
Although these organ weight changes were considered potentially related to test item administration, in the absence of any concomitant clinical, clinical pathological, macroscopical, histopathological or physiological reproductive adverse effects, these generally transient liver and kidney weight changes were regarded as adaptive rather than reflecting a toxic response.

GROSS PATHOLOGY
There were no test item-related gross lesions.

HISTOPATHOLOGY
There was no microscopical evidence of test item-related findings.

OTHER FINDINGS
No test item-related changes were noted in clinical laboratory, urinalysis, ophthalmoglogical, neurobehavioural and developmental parameters.

Effect levels (P0)

Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no reproductive effects were noted

Results: F1 generation

General toxicity (F1)

Clinical signs:
not specified
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
There were no statistically significant variations noted in the pre/postimplantation loss and total intrauterine mortality values in the treated animals compared to control animals.

BODY WEIGHT (OFFSPRING)
Body weights at birth and at post natal Day 4 was not affected by the treatment with the test item.

SEXUAL MATURATION (OFFSPRING)
There were no changes in the sex ratio.

GROSS PATHOLOGY (OFFSPRING)
No macroscopic changes were seen in F1 offspring generation euthanized and examined at scheduled termination.

Effect levels (F1)

Dose descriptor:
NOEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no F1 offspring effects were noted

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Dose Formulation Analysis:

Dose formulations were homogenous. The measured concentrations varied from 99 to 103 % of nominal concentrations and these results were considered suitable for the study purposes. Assessment of test item stability in the vehicle, 0.1% Tween 80 in PEG 400 (w/v), (LAB study code 09/174-316AN) indicated an up to 24-hour stability at room temperature, and 72 -hour stability at 5 ± 3 °C, at concentrations from approximately 1 to 250 mg/mL in the vehicle with a recovery within the acceptable range of 100 ± 10 %.

Applicant's summary and conclusion

Executive summary:

In a subacute reproduction / developmental toxicity screening study (Kubaszky, 2010a) Montanol 800 (≥98 %) in 0.1 % Tween 80 in PEG 400 (w/v) was administered to 12 Wistar rats/sex/dose level by oral gavage (4 mL/kg bw) at dose levels of 0 (vehicle only), 75, 300 or 1000 mg/kg bw/d. Unmated recovery animals (5 rats/sex at control and high dose) were kept for further 14 days, after the first scheduled necropsy of dams, without treatment to detect delayed occurrence, or persistence of, or recovery from toxic effects. In the reproductive/developmental toxicity screening part of the study, possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition and on the development of the F1 offspring from conception to day 4 post partum were assessed. There were no test item-related effects in mortality, clinical signs, body weight, food consumption, ophthalmology, haematology, clinical chemistry, urinalysis, neurobehaviour, gross pathology, histopathology, reproductive or developmental endpoints. Transient liver and kidney weight changes noted at 300 and 1000 mg/kg bw/d were considered to represent adaptive responses. Accordingly, the NOEL for effects on reproduction in males and females was 1000 mg/kg bw/d.

This screening study is acceptable and satisfies the requirement for test guideline OECD 422.