Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

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:
From 26 JUL 2000 to 02 OCT 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD TG 422)
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

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
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- strain: Wistar Crl:(WI) BR (outbred, SPF)
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: approximately 14 weeks
- Weight at study initiation: group means males: 489-501 g; group means females: 267-271 g
- Fasting period before study: no
- Housing: acclimatisation period: 5 animals per sex per cage, stainless steel suspended cages with wire mesh floors
mating period: 1 female together with 1 male, stainless steel suspended cages with wire mesh floors
after mating: animals were housed individually, Macrolon cages (Type III)
- Diet: standard pelleted laboratory animal diet (Carfil Quality BVBA, Oud-Turnhout, Belgium), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 26 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 3
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: polyethylene glycol 400
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- formulations (w/w) were prepared daily within 4 hours prior to dosing
- storage at ambient temperature

VEHICLE
- vehicle: polyethylene glycol 400, specific gravity: 1.125
- Justification for use and choice of vehicle: based on trial fromulations
- Concentration in vehicle: 0 mg/g; 4.45 mg/g; 17.8 mg/g; 89.9 mg/g
- Amount of vehicle: 10 ml/kg bw
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: up to 14 days
- Proof of pregnancy: copulation plugs in the cage and 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
- Any other deviations from standard protocol: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical study was performed to check accuracy of preparation and to determine stability and homogeneity of test substance preparation.
Test samples were dissolved in chloroform by sonication and analysed spectrophotometrically.
Duration of treatment / exposure:
F0-males:
- for 2 weeks prior to mating, throughout mating and after mating at least until the minimum total dosing period of 28 days had been completed
F0-females:
2 weeks prior to mating, throughout mating, and pregnancy and at least up to, and including the day before sacrifice
Frequency of treatment:
once daily
Details on study schedule:
- Age at mating of the mated animals in the study: 16 weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 50, 200, 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on the results of a dose range finding study with the test item (5 days oral exposure of male and female rats to 50, 200 and 1000 mg/kg bw/day did not cause changes in clinical appearance, body weights, food consumption, macroscopic examination and organ weights)
Positive control:
none

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily in the home cage and twice during the complete study in a standard arena


BODY WEIGHT: Yes
- Time schedule for examinations:
F0-males and F0-females: on the first day of dosing and weekly thereafter
mated femaes were weighed daily from day 0 until day 20 of gestation inclusive
F0-females were weighed daily during lactation
F1-pubs were weighed on day 1 and 4 post partum


FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly, but not during the mating period


WATER CONSUMPTION: Yes
- Time schedule for examinations: subjective appraisal during the study, no quantitative investigation


OTHER:
Observations on haematology, clinical chemistry and neurobehavioural examination are reported in section 7.5.
Sperm parameters (parental animals):
Parameters examined in males: testis weight, epididymis weight, microscopic examination of the integrity of the spermatogenietic cycle
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no


PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
- The duration of gestation
- The number and sex (by assessment of the ano-congenital distance) of pups, of stillbirths,
of live births, and of runts within 24 hours of parturition (day 0 or 1 post partum)
- The number and sex of live pups an day 4 post partum
- The weight of all litters within 24 hours of parturition and an day 4 post partum
- The number of pups with physical or behavioural abnormalities (daily)
- The number of pups with externally visible macroscopic abnormalities (daily)


GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities; stomach was examined for the presence of milk
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: as soon as possible after successful induction of pregnancy had been ensured, at least until the minimum total dosing period of 28 days had been completed
- Maternal animals: On day 4 post partum, or shortly thereafter. The females were never killed sooner than their pups. F0-females showing no evidence of copulation were killed 24-26 days after the last day of the mating period.


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


HISTOPATHOLOGY: Yes
from all animals:
accessory ssex organs
epididymides (fixed in Bouin's fixative)
ovaries
testes (fixed in Bouin's fixative)
all macroscopic lesions

from 5 animals/sex/group:
addrenal glands
bone marrow
brain
cervix
heart
kidneys
liver
lung, infused with formalin
lymph nodes - mandibular, mesenteric
oesophagus
sciatic nerve
small and large intestines (including Peyer's patches)
spinal cord -cervical, midthoracic, lumbar
spleen
stomach
thymus
thyroid including parathyroid
trachea
urinary bladder
uterus
vagina
all gross lesions

Other examinations
ORGAN WEIGHTS
- from all males: epididymides, testes
- from 5 animals/sex/group: adrenal glands, brain, heart, kidneys, liver, spleen, thymus

Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 4 days of age.
- These animals were subjected to postmortem examinations (macroscopic). One abnormal pup was preserved in 96% ethanol for possible further examination.


GROSS NECROPSY
- Gross necropsy and external examinations if practically possible

HISTOPATHOLOGY / ORGAN WEIGTHS: No
Statistics:
- Dunnett-test
- Steel-test
- exact Fisher-test



Reproductive indices:
- percentage mating: (number of females mated)/(number of females paired) * 100
- fertility index: (number of animals pregnant or siring a litter)/(number of females paired) * 100
- conception index: (number of animals pregnant)/(number of females mated) *100
- gestation index: (number bearing live pups)/(number pregnant) * 100
Offspring viability indices:
- percentage live males at first litter check: (number of live male pups)/(number of live pups) * 100
- percentage live females at first litter check: (number of live female pups)/(number of live pups) * 100
- viability index: (number of live pups on day 4 P.P.)/(number of live pups born alive) * 100

Results and discussion

Results: P0 (first parental animals)

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
Gross pathological findings:
no effects observed
Histopathological findings: non-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
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No treatment related clinical signs were observed (see section 7.5.1).

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Body weights and body weight gain of treated animals remained in the same range as controls (see section 7.5.1).


TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
There were no differences in food consumption (absolute and relative) between treated and control animals (see section 7.5.1).


REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
not examined

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Microscopic examination of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Four females (one female in each group) were non-pregnant.

The average number of implantation sites in pregnant females receiving 50, 200 or 1000 mg/kg/day was 13.7, 16.3 and 15.2, respectively, compared with a mean figure of 14.7 for the control group. These values are within the range expected in control rats of this age and strain, and give no indication of a treatment-related disturbance.

The majority of pairs mated during the first 4 days of the mating period. Mean pre-coital times were similar for treated and control groups.

For each dose group, 100% mating was confirmed. The fertility and conception indices were similar for treated and control groups. The gestation Index was 100% for all treatment groups.


ORGAN WEIGHTS (PARENTAL ANIMALS)
Organ weights and organ:body weight ratlos of treated animals were considered to be similar to those of control animals.


GROSS PATHOLOGY (PARENTAL ANIMALS)
Macroscopic observations of the remaining animals at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.


HISTOPATHOLOGY (PARENTAL ANIMALS)
There were no microscopic findings recorded which could be attributed to treatment with the test substance.

OTHER FINDINGS:

BREEDING DATA:
The mean duration of gestation of treated females was similar to that of the control females and ranged between 21.4 and 22.0 days.


Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: see 'Remark'

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)
The number of living and dead pups at first litter check revealed similar values for treated and control groups. Postnatal loss between days 0-4 post partum showed a statistical significant increase in the 50 mg/kg dose group. Consequently, the viability Index of this group was statistically significantly decreased. This was considered to be caused mainly by the loss of one litter, therefore no toxicological significance was attached to this finding.

CLINICAL SIGNS (OFFSPRING)
No unexpected clinical signs were recorded among pups of any dose group. The incidences of the small number of findings were within expected limits and showed no relationship to dose. They included no milk in stomach, hypothermia, smell appearance, open skull and back, curled tail, prothrusion of the tongue, autolytic pup, yellow skin, haemorrhage on stomach, and broken tail.

BODY WEIGHT (OFFSPRING)
No statistically significant group differences were recorded for body weights of pups at days 1 and 4 post partum.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: no changes in litter observations that were considered to be an effect of treatmemt

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
There were no changes in clinical appearance, functional observations, body weights, food consumption, clinical laboratory investigations, reproduction, litter observation, macroscopic examination, organ weights and microscopic examination that were considered to be an effect of treatment.
From the results presented in this report a definitive No Observed Adverse Effect Level (NOAEL) for parental and reproduction/developmental toxicity for the test item of 1000 mg/kg/day was established.
Executive summary:

 

A combined repeated dose toxicity study with reproduction/developmental toxicity screening test with the test item administered by oral gavage in Wistar rats (10/sex/group) was performed according to OECD TG 422. The dose levels for this study were 0, 50, 200 and 1000 mg/kg bw/day. Oral dosing of male and fernale Wistar rats with the test item at dose levels of 50, 200 or 1000 mg/kg b.w./day for at least 28 days, revealed no treatment-related findings on parental animals, on fertility, on embryo-foetal development, or on pup development. From the results presented in this report a definitive No Observed Adverse Effect Level (NOAEL) for parental and reproduction/developmental toxicity for the test item of 1000 mg/kg/day was established.