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Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
(1995)
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan- Nederland, Kreuzelweg 53, 5960 AD Horst, The Nederlands
- Age at study initiation: about 11-12 wks
- Weight at study initiation: Males: 309-355 g (mean: 332 g); Females: 210-243 g (mean: 225 g)
- Housing: Except for mating individually in Makrolon cages Type IIIh
- Diet and water: ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 5
- Air changes (per hr): > 10
- Photoperiod (hrs dark / hrs light): 12
During light phase low-noise radio music program was broadcasted to the animal room.
Route of administration:
oral: gavage
Vehicle:
other: 1% aqueous methylcellulose
Details on exposure:
Administration Volume: 10 mL/kg body weight
Details on mating procedure:
During the cohabitation period the first F0 male was co-housed with the first female F0 animal within the group and so on overnight (afternoon up to next morning) at a planned maximum of 14 times during the two-week cohabitation period. As a rule inseminated females were not further co-housed. Insemination was established by investigating vaginal smears prepared in the morning after co-housing or by occurrence of a vaginal plug. In case that F0 females were sperm-positive on the first mating day, but were found not to be pregnant (body weight gain was missing), these females were remated with the same male over 6 days following the regular mating period (14 days). During this re-mating period no vaginal smears were taken.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The contents (control and test item formulations) and homogeneity (low and high dose) of the test substance were verified twice, at start and near termination of the study. The method established could be described as followed: Caused by the hydrolysis of the zinc containing substance in water, the content of the test substance in the formulations was determined via a zinc measurement. The test substance concentration in the formulation was calculated using a factor considering the content of zinc in the test item. The content check revealed that all dose formulations were prepared correctly with the actual content being within +/-20 % of the nominal values.
Duration of treatment / exposure:
Males: 35 days, females: up to 60 days
Frequency of treatment:
Once daily
Details on study schedule:
Treatment of the F0 animals with the test substance started 2 weeks prior to the cohabitation period. Males were treated for a total period of at least 4 weeks before undergoing necropsy (actually 36 days). Females were treated up to day 4-6 p.p., so that pre-mating, mating, gestation and early lactation were covered by the treatment.
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose selection was based on results of a subacute (28 days) repeated dose toxicity study which was conducted with the test item administered once daily in nominal doses of 0 (vehicle control), 15, 150 and 1000 mg/kg b.w. to 5 male and 5 female animals per dose group (see chapter repeated dose toxicity: oral). The no-observed-adverse-effect-level (NOAEL) of the study was set at 1000 mg/kg b.w., and the no-observed-effect level (NOEL) at 150 mg/kg b.w. (next lower dosage). In summary, all doses including the high dose of 1000 mg/kg were well tolerated. Therefore, 0, 100, 300 and 1000 mg/kg were used as dosages in the present study. 1000 mg/kg is the limit dose recommended in the guideline. 100 mg/kg was expected to be a NOAEL.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes, all animals
- Time schedule: observations for morbidity and mortality twice a day (once on weekends and public holidays), general clinical observation (in the home cage) were made daily (especially findings occurring during littering e.g. prolonged parturition) some time (about 30 to 60 minutes) after the administration and recorded.

DETAILED CLINICAL OBSERVATIONS: Yes, all F0-animals
This investigation included the evaluation of the general state of health, behaviour, condition of the fur, and the orifices as well as excretory products.
- Time schedule: Males - prior to the treatment and then weekly up to necropsy; females - prior to the treatment and then weekly during premating and mating period and, additionally, during gestation and lactation period daily.

BODY WEIGHT: Yes, all F0-animals
- Time schedule for examinations: Individual body weights of all parental animals were determined just prior before the first treatment of animals and then daily thereafter. Furthermore, body weights were recorded immediately before scheduled necropsies for calculation of relative organ weights.

FOOD CONSUMPTION:
The food intake of F0 males was measured weekly during the premating period on a seven day basis. In F0 females food intake was measured in the same way during the premating period. During gestation period determination of food intake was done on post-coital days 0-7, 7-14 and 14-20.
During lactation period determination of food intake was done on day 0-4 p.p.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
Litter observations:
The numbers of live and dead pups as well as the sex of the pups (including those of dead pups, if possible) were determined shortly after birth (on postpartum day 0) and on day 4 p.p. At these time points individual body weights and clinical signs were recorded as well. Note was taken of any apparent malformations.
Postmortem examinations (parental animals):
Selected organs (liver, testes, epididymides, ovaries with oviducts) of the F0 parental animals were weighed and organs were subjected to macroscopic and histopathological investigations. In F0 females the number of implantation sites was counted after staining the uterus with 10% ammoniumsulfide. At necropsy also the number of corpora lutea in the right and left ovary was determined.
Postmortem examinations (offspring):
Unscheduled Necropsies: Pups that were found dead at birth, that died during lactation, as well as those killed (with carbon dioxide) in moribund condition were macroscopically inspected after opening the body cavities, with particular attention on the organs of reproduction except for cases of autolysis or cannibalism. This included also visible skeletal abnormalities as far as possible. A lung flotation in water was performed during the necropsy of pups found dead on the day of the first litter inspection to determine whether pups had breathed at birth or not.
Scheduled Necropsies: When pups were 4-6 days old they were killed under carbon dioxide anesthesia and were examined for macroscopical alterations as described above.
Statistics:
Analysis of Variance (ANOVA) and in case of significant results Dunnett test as post hoc test for: Body weights and body weight gains, Food consumption, Number of implantation sites per female, Number of viable pups, Prenatal loss per litter, Organ weights at necropsy, Time to insemination, Live birth and viability rate, Sex ratio, Duration of gestation, Pup weights.
2*N CHi2 test; in case of significant differences Fisher's exact test with Bonferroni correction for: Number of viable pups per group based on the number of implantations, Insemination gestation and fertility index.
CHi2 test and Fisher’s Exact test for: Stillborn as well as died, missing and/or cannibalized pups.
Reproductive indices:
The following reproductive indices were determined: Insemination index (%), Fertility index (%), Gestation index (%)
Offspring viability indices:
The following offspring viability indices were determined: Live birth index (%), Viability index (%)
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
All animals survived until scheduled necropsy. No toxicologically relevant clinical findings were observed at daily cage-side or weekly detailed clinical observations up to 1000 mg/kg.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
No toxicologically relevant changes in body weights or body weight gain were found in male and female rats at any phase of the study. Food intake was comparable in control and test item treated groups throughout the study.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No dose-dependent toxicological relevant effects could be observed concerning the insemination, fertility and gestation indices as well as the mean duration of gestation and number of females with live born pups up to 1000 mg/kg bw. No toxicologically relevant changes in mating performance were evident up to 1000 mg/kg. No toxicologically relevant effect on the number of implantation sites or prenatal losses could be seen up to 1000 mg/kg. No toxicologically relevant, dose-dependent changes occurred concerning the total numbers of pups born, stillborn pups, the live birth index, percentage of males born, the litter size at birth and the viability index up to 1000 mg/kg.

ORGAN WEIGHTS (PARENTAL ANIMALS)
None of the absolute and relative organ weights was statistically significantly or toxicologically relevantly changed.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No relevant gross pathological changes were observed.

HISTOPATHOLOGY (PARENTAL ANIMALS)
In the organs/tissues evaluated histopathologically in males and females (liver, the thyroid, testes and epididymides, and ovaries/ oviducts), no findings were detected which were assessed as treatment related.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No treatment related effect on general tolerance or reproductive parameters in parental rats up to the highest dose tested (1000 mg/kg)
CLINICAL SIGNS (OFFSPRING)
No clinical signs with a dose-dependent distribution were observed in F1 pups during the five days lactation period at levels up to 1000 mg/kg.

BODY WEIGHT (OFFSPRING)
No toxicologically relevant effects on pup weights at birth or on day 4 was observed up to 1000 mg/kg.

GROSS PATHOLOGY (OFFSPRING)
No macroscopical alterations with a remarkable incidence were noted at pup necropsies up to 1000 mg/kg. Therefore the findings observed are found to be chance finding.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No treatment related findings in F1 pubs up to the highest dose tested (1000 mg/kg)
Reproductive effects observed:
not specified
Executive summary:

A screening for developmental and reproduction toxicity was conducted according to OECD TG 421. 12 male and 12 female rats in each dose group were administered daily via gavage the substance formulated in 1% methylcellulose at doses of 0 (vehicle control), 100, 300 and 1000 mg/kg bw, starting 2 weeks prior to mating until scheduled necropsy. Males were treated for a total period of at least 4 weeks (actually 36 days). Females were treated up to day 4-6 p.p, so that pre-mating, mating, gestation and early lactation were covered by the treatment.

Investigations were performed on general tolerance of the test compound by the parental animals as well as on effects on reproduction including early postnatal development of F1 pups. The animals were regularly observed and weighed, food intake and reproduction parameters were determined. Selected organs were weighed and organs were subjected to macroscopic and histopathological investigations.

All animals survived until scheduled necropsy. No toxicological relevant findings were observed at daily cage-side or weekly detailed clinical observations. Body weight and body weight gain and food intake were comparable between compound-treated animals and controls. Necropsy, organ weight analysis and histopathology did not reveal any relevant effect.

No toxicological relevant effects were observed concerning the insemination, fertility and gestation indices as well as the mean duration of gestation and number of females with live born pups, mating performance, number of implantation sites or prenatal losses, total numbers of pups born, stillborn pups, the live birth index, percentage of males born, the litter size at birth and the viability index.

No toxicological relevant findings were observed in F1 pups during the five days lactation period, on pup weights at birth or on day 4 or any macroscopical alterations with a remarkable incidence.

Overall, no indication for systemic availability could be concluded from the study. The NOAEL was set 1000 mg/kg for general toxicity of F0 animals, for reproduction toxicity and for developmental toxicity.

 

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Additional information

A screening for developmental and reproduction toxicity was conducted according to OECD TG 421. 12 male and 12 female rats in each dose group were administered daily via gavage the substance formulated in 1% methylcellulose at doses of 0 (vehicle control), 100, 300 and 1000 mg/kg bw, starting 2 weeks prior to mating until scheduled necropsy. Males were treated for a total period of at least 4 weeks (actually 36 days). Females were treated up to day 4-6 p.p, so that pre-mating, mating, gestation and early lactation were covered by the treatment.

Investigations were performed on general tolerance of the test compound by the parental animals as well as on effects on reproduction including early postnatal development of F1 pups. The animals were regularly observed and weighed, food intake and reproduction parameters were determined. Selected organs were weighed and organs were subjected to macroscopic and histopathological investigations.

All animals survived until scheduled necropsy. No toxicological relevant findings were observed at daily cage-side or weekly detailed clinical observations. Body weight and body weight gain and food intake were comparable between compound-treated animals and controls. Necropsy, organ weight analysis and histopathology did not reveal any relevant effect.

No toxicological relevant effects were observed concerning the insemination, fertility and gestation indices as well as the mean duration of gestation and number of females with live born pups, mating performance, number of implantation sites or prenatal losses, total numbers of pups born, stillborn pups, the live birth index, percentage of males born, the litter size at birth and the viability index.

Overall, no indication for systemic availability could be concluded from the study. The NOAELs were set 1000 mg/kg bw, both for general toxicity of F0 animals and for reproduction toxicity.


Justification for selection of Effect on fertility via oral route:
Only one reproduction/developmental toxicity screening study (OECD TG 421) available

Effects on developmental toxicity

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Data waiving:
other justification
Justification for data waiving:
other:
Abnormalities:
not specified
Developmental effects observed:
not specified
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Additional information

The screening for developmental and reproduction toxicity (OECD TG 421) as described above revealed no toxicological relevant findings in F1 pups during the five days lactation period, on pup weights at birth or on day 4 or any macroscopical alterations with a remarkable incidence.

Thus, the NOAEL for developmental toxicity was set 1000 mg/kg bw.


Justification for selection of Effect on developmental toxicity: via oral route:
Only one reproduction/developmental toxicity screening study (OECD TG 421) available

Justification for classification or non-classification

Not classified for reproduction toxicity according to Regulation (EC) No 790/2009 (Amendment to Regulation (EC) No 1272/2008) and based on the criteria set out in Annex I to Regulation (EC) No 1272/2008 or in Annex VI to Council Directive 67/548/EEC (June 1967).

Additional information