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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Jan. 5, 2011 - Feb. 27, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Version / remarks:
2001-01-22
Deviations:
yes
Remarks:
minor ones (s. attachement)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: nanoform, no surface treatment
Details on test material:
Precipitated, CAS-No.: 112926-00-8, Surface area / BET [m2/g]: 160
Specific details on test material used for the study:
NM-200 Synthetic Amorphous Silica

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
obtained from. Charles River, Deutschland, Sulzfeld, Germany
The Wistar strain was used because of their general acceptance and suitability for this type of studies.
Sex:
male/female
Details on test animals and environmental conditions:
ca. 5 weeks old, 4 animals/cage, feed: Rat & Rat No. 3 Breeding Díet RM3 and tap-water ad libitum, 22 +/- 2 °C, 45-65 % humidity, 12 h light/dark
Upon evidence of copulation the females were caged individually for the birth and rearing of their pups.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: MHPC (0.5% w/v)
Details on mating procedure:
At the end of premating each female was caged with one male from the same group. Animals were caged together until mating occurs or 2 weeks elapsed. ln case a male died before successful copulation it was replaced by another male from the same dose group (a male that already had successfully mated with another female). A rest period of at least 2 days was taken between the mating period of that male.
Mating pairs were clearly identified. Every consecutive morning during the mating period, vaginal smears were made for determination of the presence of sperm to determine whether mating had occurred.
The day on which a sperm-positive smear was detected was considered as gestation day 0.
Duration of treatment / exposure:
Male animals were dosed during a 10-week premating period and during mating and up to the day before sacrifice. The female animals were dosed with the test item during a 10-week premating period and durlng mating, gestation and lactation up to postnatal day 21. Selected F1-generation pups were dosed by gavage from postnatal day 22 until the day prior to sacrifice.
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
group B
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
group C
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
group D
No. of animals per sex per dose:
28
Control animals:
yes, concurrent vehicle
Details on study design:
The objective of this study was to provide data on the possible effects of the test item on reproductive performance of Wistar rats and on the development of pups consequent to daily oral administration of the test item by gavage to male and female rats during a premating period of at least 10 weeks and during mating (2 weeks), gestation and lactation until postnatal day 21.
At weaning, pups were selected for the F1-generation and were dosed at the same concentrations as their parents during their growth into adulthood.

On or shortly after postnatal day 21,the F1-pups were weaned and 28 males and 28 females were selected at random from as many litters as possible in each group to rear the next generation.

Examinations

Parental animals: Observations and examinations:
- Each animal was observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity.
- Body weights of male and female rats were recorded shortly before the start of the treatment at randomization and at the start of the study (premating).
Males were weighed weekly until sacrifice. Females were weighed weekly during the premating and mating period. Mated females were weighed on days 0, 4,7,10, 14, 17 and 21 during presumed gestation and on days 1 , 4,7 , 10, 14, 17 and 21 of lactation.
- Food consumption of male rats were measured weekly, except during the mating per¡od. Food consumption of female rats were measured weekly during the premating period. Food consumption of mated females were recorded during pregnancy over successive periods (day 0-4, 4-7,7-10,10-14,14-17 and 17-21), and during lactation on days 1-4,4-7,7-10, 10-14, 14-17, 17-21.
- At the end of the gestation period (gestation day 21), females were examined twice daily for signs of parturition. Any difficulties occurring during parturition were recorded.
- During the last part of the lactation period of the FO-generation females, milk of ca. 12 lactating females per group was collected in order to obtain insight in the exposure of the F1-generation pups during lactation.
Oestrous cyclicity (parental animals):
Vaginal smears to evaluate the estrus cycle length and normality were made daily for about 3 weeks prior to mating. Smears were made and stained of all females but only the smears of the control group and the high-concentration group were evaluated.
Sperm parameters (parental animals):
At scheduled necropsy, epididymal sperm was derived from the left cauda epididymis of all males of all groups. For this purpose the cauda epididymis was dissected, weighed and thereafter minced in M199 medium containing 0.5% bovine serum albumin. Sperm motility and, after sonification and DNA staining, the cauda epididymal sperm reserves (sperm count) were measured for all males of all groups, using the Hamilton Thorne lntegrated Visual Optical System (IVOS). ln addition, a smear of the sperm solution was prepared and stained for all males, but only the smears of the control and the
high-dose group males were examined for morphology. lf treatment-related changes were observed in the high-dose group, the examination of sperm morphology was extended to the intermediate-dose groups in consultation with the sponsor's monitor.
Litter observations:
- To keep nest disturbance to a minimum the litters were examined only once daily for dead pups.
- The total litter size and numbers of each sex as well as the number of stillbirths, live and dead pups and grossly malformed pups were evaluated on days 1,4,7,10, 14, 17 and 21 of lactation. The pups were weighed individually on days 1, 4,7, 10, 14, 1T and 21 ot lactation. Mean pup weíghts were calculated per sex and both sexes combined.
Postmortem examinations (parental animals):
- Males were sacrifìced after successful mating and females were sacrificed at or shortly after weaning on postnatal day 21.
- Microscopic examination was performed on the following organs of all rats of the control and high-dose groups: epididymides, ovaries, pituitary gland,
prostate, seminal vesicles and coagulating qlands, spleen, testes, uterus, vagina
Postmortem examinations (offspring):
- All stillborn pups and pups found dead during the study were stored in a freezer (<-18.C) for macroscopical examination for structural and pathological changes. Gross necropsy was also performed on pups of dams that died during lactation (these pups were sacrificed at the time of the dam's death) and on pups that showed external abnormalities at weaning.
- After selection of the pups for the next generation, from the remaíning pups 1 male and 1 female pup of each litter was subjected to a thorough necropsy. Special attention was paid to the organs of the reproductive system.
- At scheduled necropsy, epididymal sperm was derived from the left cauda epididymis of all males of all groups. For this purpose the cauda epididymis was dissected, weighed and thereafter minced in M199 medium containing 0.5% bovine serum albumin. Sperm motility and, after sonification and DNA staining, the cauda epididymal sperm reserves (sperm count) were measured for all males of all groups, using the Hamilton Thorne lntegrated Visual Optical System (IVOS). ln addition, a smear of the sperm solution was prepared and stained for all males, but only the smears of the control and the
high-dose group males were examined for morphology. lf treatment-related changes were observed in the high-dose group, the examination of sperm morphology was extended to the intermediate-dose groups in consultation with the sponsor's monitor.
Statistics:
The resulting data were analyzed using the methods given below. p < 0.05 was considered as the level of significance.
- Clinical findings were evaluated by Fisher's exact probability test.
- Body weight, body weight gain, food consumption and organ weights data were subjected to one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison tests.
- Fisher's exact probability test was used to evaluate the number of mated and pregnant females, the number of pregnant females with ímplants but no pups, females with live pups, females with stillborn pups, live and dead fetuses or pups and the numbers of litters lost entirely.
- Pre-coital time (mean number of days), the duration of gestation, the number of corpora lutea and implantation sites, the total number of pups delivered (mean), the mean number of live pups per litter and pre- and post-implantation loss (%) were evaluated by Kruskal-Wallis nonparametric analysis of variance and by the Mann-Whitney U test.
- Mortality data and data of the pathology of parent animals were evaluated by the Fisher's exact probability test.
- Sperm parameters were evaluated by one-way analysis of variance followed by Dunnett's multiple comparison test (epididymal and testicular sperm count and numerical sperm motility parameters) or by Kruskal-Wallis non parametric analysis of var¡ance and by Mann-Whitney U test (motility parameters expressed as a percentage and sperm morphology).
- Estrus cyclic¡ty was evaluated by Fisher's exact test (number of acyclic animals and number of animals with prolonged estrus per¡od), ANOVA followed by Dunnett's multiple comparison tests (number of cycles per animal) and Kruskal-Wallis nonparametric ANOVA followed by Mann-Whitney U test (length of the longest cycle).
- Sexual developmental parameters (preputial separation, vaginal opening and testes descending) were subjected to one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison tests.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The clinical findings observed in the animals of the F0-generation are common findings in rats of this strain and age or occurred as individual fortuitious
findings. The distribution of all findings was equally amongst the various groups or occurred in only one or a few animals. Therefore, they were not considered to be related to treatment.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Animal B98 was killed in moribund condition on Day 58. Macroscopical findings were a intra-abdominal nodule of 5x3 cm. At sacrifice it was shown that this nodule originated from the right kidney. Microscopically, the nodule appeared to be a nephroblastoma
Animal A21 was killed in moribund condition during the first week of the lactation period. This animal showed a prolaps of the uterus. Moreover, some other animals were sacrificed during the first week of the lactation because they lost their complete litters.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean body weight changes of the male animals of all treatment groups of the F0-generation were stat¡stically significant different from the control group during several intervals. However, the effects were inconsistent. ln the low-dose group, mean body weight changes were decreased between days 7-14, 28-35 and 49-56 and increased between days 21-28 and 42-49. ln the mid-dose group, mean body weight changes were decreased between days 7-14, 28-35,49-56 and increased between days 0-7, 14-21,35-42 and 56-63. ln the high-dose groups, mean body weight changes were decreased between days 21-28 and 42-49. No statistically significant effects on overall body weight changes were observed between the various groups during the entire period.
Mean body weight changes of the female animals of all treatment groups of the F0-generation were statistically significant different from the control group during several intervals of th epremating period. However, the effects were inconsistent: ln the low-dose group, mean body weight changes were decreased between days 28-35 and 49-56 and increased between days 42-49. ln the mid-dose group, mean body weight changes were decreased between days 28-35, 49-56 and 63-70 and increased between 35-42 and 56-63. ln the high-dose groups, mean body weight changes were decreased between days 21-28 and 63-70. No statistically significant effects on overall body weight changes were observed between the various groups during the entíre period (day 0-70).
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Microscopic exam¡nation of the sampled organs and tissues did not reveal treatment related histopathological changes. The histopathological changes observed were about equally distributed amongst the different treatment groups or occurred in one or a few animals only. They are common findings in rats of this strain and age or occurred as individual chance findings. Therefore, they were not considered to be related to treatment.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Description (incidence and severity):
No statistically significant effects were observed on the mating-, female fecundity, male fertility-, female fertility and gestion indices. There was no statistically significant difference between post-implantation loss in the control group and treatment groups.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: P1 (second parental generation)

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Description (incidence and severity):
No statistically significant effects were observed on the mating-, female fecundity, male fertility-, female fertility and gestion indices. There was no statistically significant difference between post-implantation loss in the control group and treatment groups.

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The clinical findings observed in the animals of the F1-generation are common findings in rats of this strain and age or occurred as individual fortuitious
findings. The distribution of all findings was equalty amongst the various groups or occurred in only one or a few animals. Therefore, they were not considered to be related to treatment.
Undistended lungs as is observed in many pups of the control, mid-and high-dose groups indicates that these pups were still born.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Animal C653 was killed in moribund condition at the end of the gestation period. This female was in labour but was unable to deliver the pups. The dystocia exhausted the animal and it was therefore humanely killed. The uterus contained I dead fetuses.
Animal D673 was found dead on gestation day 12, The main macroscopical findings were incompletely collapsed lungs with a firm appearance
Moreover, some other animals were sacrificed during the first week of the lactation because they lost their litters completely

No statistically significant effects were observed on liveborn index and on pup mortality
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean body weight changes of the male animals of all treatment groups of the F1-generation were statistically significant different from the control group during several intervals. However, the effects were inconsistent: ln the low-dose group, mean body weight changes were decreased between days 35-42 and 56-63 and increased between days 14-21, 28-35, 49-56 and 70-76. ln the mid-dose group, mean body weight changes were decreased between days 35-42 and 56-63 and increased between days 7-14,42-49 and 63-70. ln the high-dose groups, mean body weight changes were increased between days 70-76. No statistically significant effects on overall body weight changes were observed between
the various groups during the entire period (day 0-76).
Mean body weight changes of the female animals of all treatment groups of the F1-generation were statistically significant different from the control group during several intervals of the premating period. However, the effects were inconsistent: ln the low-dose group, mean body weight changes were decreased between days 35-42 and 56-63 and increased between days 49-56. ln the mid-dose group, mean body weight changes were decreased between days 0-7, 35-42 and 56-63 and increased between 42-49 and 63-70. ln the high-dose groups, mean body weight changes were decreased between days 14-21 and increased between days 21-28. No statistically significant effects on overall body weight changes were observed between the various groups during the entire period (day 0-76).
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption (expressed as g/animal/day) of the male animals of the mid- and high-dose groups was statistically significantly increased between days 21-28 and 70-76. No statistically significant effects were observed on food consumption of the male anlmals of the F1-generat¡on when expressed as g/kg/day.
Food consumption (expressed as g/animal/day) of the female animals of the mid-dose group was statistically significantly decreased between days 28-35, 42-49,56-63 and 63-70 of the premating period. ln the high-dose group, food consumption (expressed as g/animal/day) was statistically significantly increased between days 14-21 and 21-28. Food consumption (expressed as g/kg/day) of the female animals of the mid-dose group was
statistically significantly decreased between days 28-35, 4249 and 63-70 of the premating period. No effects were observed in the high-dose group.
During the gestation and lactation periods of the F1-generation, no statistically significant effects were observed on food consumption (both
expressed as g/animal/day and g/kg/day). ln conclusion, no treatment-related effects on food consumption were observed.
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Except for a statistically significant decreased relative weight of the thyroid of the male animals of the mid-dose group, no statistically significant differences were observed between absolute and relative organ weights of male and female animals of the various groups.
Gross pathological findings:
no effects observed
Histopathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Microscopic exam¡nation of the sampled organs and tissues did not reveal treatment related histopathological changes. The histopathological changes observed were about equally distributed amongst the different treatment groups or occurred in one or a few animals only. They are common findings in rats of this strain and age or occurred as individual chance findings. Therefore, they were not considered to be related to treatment.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
viability
sexual maturation
clinical signs
mortality
body weight and weight gain
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
ln conclusion, in this study, oral administration of NM-200 Synthetic Amorphous Silica up to 1000 mg/kg body weight had no adverse effect on the reproductive performance of rats or on the growth and development of the offspring into adulthood for two consecutive generations.
Executive summary:

The toxicity potential of NM-200 to reproduction was assessed in rats.