Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study according to GLP
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Genamin TA 100

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Italia
- Age at study initiation: (P) 8 wks
- Weight at study initiation: (P) Males: 225 - 250 g; Females: 200 - 225 g
- Fasting period before study: no
- Housing: macrolon cages
- Use of restrainers for preventing ingestion (if dermal): no
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2 °C
- Humidity (%): 55 +/- 5 %
- Air changes (per hr): 15 -20
- Photoperiod (hrs dark / hrs light): 12 hours periodically

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: sesame oil
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 16 h, 7 evenings/week for max. 14 evenings
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: individually
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
Males: 14 days prior to mating, during mating; 28 days maximum
Females: 14 days prior to mating, during mating, during pregnancy, 3 days post-partum (lactation)
Females, positive smear, no offspring: 14 days prior to mating, during mating, 25 days post-mating
Females, negative smear: 14 days prior to mating, during mating, 25 days post-mating
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
mg/kg body weight per day
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
12.5 mg/kg body weight per day
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
50 mg/kg body weight per day
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
150 mg/kg body weight per day
Basis:

No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on dose-range-finder in rats
- Rationale for animal assignment (if not random): random
- Other:

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

OTHER:
Sperm parameters (parental animals):
Sections of testes were stained with PAS-hematoxylin to allow spermatogenesis to be classified into 14 stages, each stage based primariliy upon the changes of the acrosome and head morphology of the younger generation of spermatids.
Litter observations:
number and sex of pups
stillbirths
live births
postnatal mortality
presence of gross anomalies
weight gain (day 0, day 4)
gross examination of dead pups for external and internal abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals on the day after the end of treatment
- Maternal animals: All surviving animals on day 4 of lactation with their pubs

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

HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues were prepared for microscopic examination and weighed, respectively:
- ovaries
- uteri (including horns, cervix, and vagina)
- testes (with special emphasis on stages of spermatogenesis and histopathology of interstitial cell structure)
- epididymides
- accessory sex organs
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed on day 4 of lactation and necropsied

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
salivation, hunched posture at mid- and high dose animals, no changes at low-dose animals
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
decrease in mid- and high dose animals, no effect in low-dose animals
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
decrease in mid- and high dose animals, no effect in low-dose animals
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Test substance intake: lower mean food consumption in mid- and high-dose animals, no effect in low-dose animals

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
decreased mating and fertility indices in high-dose animals

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- 150 mg/kg: salivation, hunched posture, soft stools, piloerection; 6/10 male and 5/10 female animals died
- 50 mg/kg: salivation; 1/10 male and 1/10 female animals died
- 12.5 mg/kg: no changes observed
BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
- 150 mg/kg: decreased body weight gain (males and females)
- 50 mg/kg: decreased body weight gain during pre-mating period
- 12.5 mg/kg: slightly decreased body weight gain (males and females)
TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
- lower food intake in mid and high dose groups (males and females), slightly lower food intake in low dose group during lactation period in females
REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- mating and fertility indices, mean pre-coital time and parturition were unaffected up to 50 mg/kg/day
ORGAN WEIGHTS (PARENTAL ANIMALS)
- lower absolute weight of epididymides and higher value of testis weight relative to body weight at 150 mg/kg/day
- no differences in ovary weights among the experimental groups
GROSS PATHOLOGY (PARENTAL ANIMALS)
- no pathological changes
HISTOPATHOLOGY (PARENTAL ANIMALS)
- histology of testes, epididymides and ovaries of high dose group animals did not show any compound-related changes
- no changes in testicular staging performed in the PAS-hematoxylin stained sections of control and high dose groups
OTHER FINDINGS (PARENTAL ANIMALS)

Effect levels (P0)

Dose descriptor:
NOEL
Effect level:
12.5 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: overall effects

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
no effects up to 50 mg/kg body weight
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
no litters present at highest dose
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
slightly lower pub weight at 50 mg/kg body weight
Sexual maturation:
not examined
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)
- no litters present at 150 mg/kg/day
- no effects on postnatal survival up to day 4 of lactation and no effects on sex ration at 50 mg/kg/day

CLINICAL SIGNS (OFFSPRING)
- no litters present at 150 mg/kg/day
- no significant effects up to 50 mg/kg/day

BODY WEIGHT (OFFSPRING)
- no litters present at 150 mg/kg/day
- slightly lower pup body weight seen in 50 mg/kg/day group
- no effects seen at 12.5 mg/kg/day

GROSS PATHOLOGY (OFFSPRING)
- no abnormalities observed in any pub either at birth or at autopsy on day 4 of lactation neither of the 50 or of the 12.5 mg/kg/day group

Effect levels (F1)

Dose descriptor:
NOEL
Generation:
F1
Effect level:
12.5 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: overall effects

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on findings of general toxicity (death, clinical signs, reduced body weight gain) at daily dosages of 50 mg/kg bw/d a NOEL/systemic toxicity of 12.5 mg/kg bw/d can be derived from the study. Based on the findings of a lower fertility index and a lower conception rate at daily dosages of 50 mg/kg bw/d a NOEL/fertility of 12.5 mg/kg bw/d can be derived from the results of this screening study.

Based on the results of this study, 12.5 mg/kg/day is the NOEL for maternal toxicity and the NOEL for the offspring.
Executive summary:

Groups of 10 rats (Crl:CD (SD) BR) per sex were treated with dosages of 0, 12.5, 50, and 150 mg/kg bw/day of Genamin TA 100 by gavage (administration volume 10 ml/kg bw/day) using sesame oil as a vehicle. Males were treated daily from 14 days prior to mating until the end of the mating period for a maximum of 28 days. Females were treated daily for 14 days before start of the mating period, throughout the same, during pregnancy and until day 3 of lactation. The animals were mated one male with one female.

At daily doses of 150 mg/kg bw 6/10 males and 5/10 females died between day 9 and day 25 of treatment (during the premating and mating period). At daily doses of 50 mg/kg bw 1/10 males and 1/10 females died on day 13 respectively on day 24 of treatment. No animals died at 12.5 mg/kg bw/day, and in the control group 1 female died by accident.

Clinical observations at 150 mg/kg bw/day revealed salivation after treatment, hunched posture and in some cases soft stools and piloerection. The only clinical sign present at 50 mg/kg bw/day was salivation. No changes were seen at 12.5 mg/kg bw/day.

Body weight loss of about 22 g at the 150 mg/kg bw dose group and statistically significant (p > 0.01) lower body weight gain at the 50 mg/kg bw dose group during the premating period together with a lower mean food consumption was observed in the males. Also in the females body weight loss of about 17 g at the 150 mg/kg bw dose group and statistically significant lower body weight gain at the 50 mg/kg bw dose group together with a lower mean food consumption during the premating period was observed.

At sacrifice of the parental animals organ weights of ovaries, testes and epididymides were determined from all experimental groups. Histopathology was carried out on testes, epididymides and ovaries of the controls and of the animals of the 150 mg/kg bw dose group. A statistically significant(p> 0.01) lower absolute and relative weight of epididymides and a statistically significant(p> 0.01) higher value of relative testis weight was observed at 150 mg/kg bw/day. No significant differences were noted in ovary weights among the various experimental groups. Histopathology of testes and epididymides of high dose group animals did not show any compound-related changes. In particular, no changes were seen in the testicular staging performed in the PAS-hematoxylin stained sections of final sacrificed animals of control and of the high dose groups. In the ovaries, moderate increased frequency of atrophic corpora lutea was seen in the animals of the high dose group which died or were sacrificed, compared to the controls, this finding was considered a secondary effect to the decrease in body weight growth induced by treatment in this group.

At the dosages of 150 mg/kg bw/day only 3 females out of 7 mated females had positive vaginal smears and of these only one was pregnant, but with only implantation sites and no live pups. At this dosage level also the mean pre-coital interval was longer (13.4 days) than that of the control group (2.3 days). With regard to the mating index in the control, the 12.5 and the 50 mg/kg bw dose level groups 9/9 (100%), 9/10 (90%) and 9/9 (100%) mated females were sperm positive. With regard to the fertility index 9/9 (100%), 8/10 (80%) and 7/9 (78%) of the mated females became pregnant in the control, the 12.5 and the 50 mg/kg bw dose level groups. Mean pre-coital time and parturition were unaffected in the low and mid dose treated groups.

With regard to the conception rate in the control, the 12.5 and the 50 mg/kg bw dose level groups 9/9 (100%), 8/9 (89%) and 7/9 (78%) of the sperm positive females became pregnant and delivered live pups. Numbers of corpora lutea had not been determined during this study. Thus, any pre-implantation loss was not evaluated. Staining for the presence of implantation sites was only performed with the uteri of apparently non-pregnant females. The mean number of visible implantation sites per dam in the control, the 12.5 and the 50 mg/kg bw dose level groups were 17.6, 13.9 and 15.0. There were no stillborns or litters with only implantations in the control, low, and mid dose groups. The mean number of total pups born per litter was 16.9, 12.3 and 14.3 in the control, the 12.5 and the 50 mg/kg bw/day dose groups. Thus, the mean litter index for post-implantation loss was calculated to 3.4, 9.9 and 4.5% in the control, the low and the mid dose groups.

No effects were noted on the pup sex ratio. No abnormalities were observed in any pup either at birth or at autopsy on day 4 of lactation neither in the 12.5 nor in the 50 mg/kg bw/day treated groups. Lower mean values of live born per litter and of pups per litter alive at day 4 were found for both the low and the intermediate dose groups in comparison to those of the control group, however, the decreases were not dose dependent and the differences were statistically significant for the 12.5 mg/kg bw dose group only. Thus, these findings were considered unlikely to be related to the compound. A slightly lower pup body weight was observed in the 50 mg/kg bw/day treated group in comparison with the control group at birth and at day 4 of lactation but not for the pups of the 12.5 mg/kg bw/day treated group. Based on general toxicity findings a NOEL(systemic toxicity) of 12.5 mg/kg/day can be derived from stis study. Additionally, based on findings of a lower fertility index and a slightly lower conception rate at the mid dose level a NOEL(fertility) of 12 .5 mg/kg body weight per day is deducible.Further, during an oral 28-day study (OECD TG 407) with the same test substance Genamin TA 100 in rats (see section 7.5.1) also reproductive organ weights and reproductive organ histopathology of testes and epididymides, as well as organ weights of ovaries and uteri had been investigated. No substance-related findings had been observed during this study on these organs at oral doses up to and including 150 mg/kg bw/day.