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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:
No data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
Reference substance 001
Cas Number:
112926-00-8
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland (Sulzfeld, Germany)
- Age at study initiation: (P) no data (4-5 weeks at purchase); (F1): 22 days
- Weight at study initiation: No data
- Fasting period before study: Not relevant
- Housing: Macrolon cages with a bedding of wood shavings and strips of paper as environmental enrichment. During mating a single female and male were housed together. Once mated the females were housed individually, and later they were housed individually with their litters.
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: No data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 45-65%
- Air changes (per hr): approximately 10 changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

IN-LIFE DATES: No data

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: highly deionised water containing 10% foetal bovine serum
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Once per week throughout the study, seven bottles per dosing group were prepared, each containing the relevant amount of test substance. On each day, the required amount of vehicle was added to achieve concentrations of 0, 10, 30 and 100 mg/ml test substance and stirred for at least 60 minutes.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Up to 2 weeks
- Proof of pregnancy: Sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): Individually (no further details)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
At various weeks during the study, samples were taken from each of the dosing formulations for analytical investigation of hydrodynamic diameters of the silica particles.
Details on study schedule:
- F1 parental animals not mated until at least 10 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 22 days of age.
- Age at mating of the mated animals in the study: at least 10 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
28 (F0), 4 (F1, F2)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Random
Positive control:
None

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes, throughout the study, all animals were checked daily for clinical signs and abnormal behaviour.

DETAILED CLINICAL OBSERVATIONS: No data
- Limited information on this.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of all males and females was recorded weekly during premating, and for males, weekly thereafter. Mated females were weighed on gestation days 0, 4, 7, 10, 14, 17 and 21 and during lactation on post-natal days 1, 4, 7, 10, 14, 17 and 21. Animals were also weighed on their scheduled necropsy day.

FOOD CONSUMPTION: During the premating period, food consumption was measured weekly for each cage. Individual food consumption of all mated females was recorded from gestation days 0-4, 4-7, 7-10, 10-14, 14-17 and 17-21 and for all females with live pups on post-partum days 1-4, 4-7, 7-10, 10-14, 14-17 and 17-21.

WATER CONSUMPTION: No
Oestrous cyclicity (parental animals):
Three weeks prior to the end of the premating period of the F0 and F1 generation, vaginal smears were made from each female to evaluate the estrous cycle length and normality.
Sperm parameters (parental animals):
Parameters examined in F0 and F1 male parental generations: testes weight, epididymis weight, caudal epididymal sperm count, sperm motility and testicular sperm count
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- Maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS: a necropsy was performed on stillborn pups and pups that died during lactation.
Postmortem examinations (parental animals):
SACRIFICE
It is not clear from the publication when scheduled sacrifices occurred. The study is stated to be according to OECD TG 416, so timings are assumed to follow this guideline, i.e. F0 and F1 males dosed until they are no longer needed for assessment of reproductive effects and females are sacrificed after weaning of their litter.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations

HISTOPATHOLOGY / ORGAN WEIGHTS
The adrenals, brain, epididymides, kidneys, liver, ovaries, pituitary gland, prostate, seminal vesicles with coagulating glands, spleen, testes, thyroid, uterus with cervix (after counting of implantation sites), vagina and all gross lesions were weighed (except vagina) and preserved for microscopic examination for the control and the highest dose groups and on macroscopic abnormalities of all groups. Also, reproductive organs of F0 and F1 males who failed to sire, and of the non-mated/non-pregnant females from the low and mid dose groups were examined microscopically.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 21 days of age.
- Of the remaining F1 pups, one male and one female from each litter were selected for a thorough necropsy, and the brain, spleen and thymus were weighed. Sexual maturation was studied by scoring the day of vaginal opening in females and testes descent and preputial separation in males from post-natal days 31, 21 and 39, respectively.

HISTOPATHOLOGY / ORGAN WEIGHTS
As for parental animals.
Statistics:
For some offspring viability data, sexual maturation, some sperm parameters and organ weights: Anova followed by Dunnett's multiple comparison test.
For some offspring data: Fisher's exact test.
For precoital time, gestation time and post-implantation loss per animal: Kruskal-Wallis + Mann-Whitney U test.
For some offspring data: Kruskal-Wallis followed by Dunnett's multiple comparison.
For some sperm parameters: Kruskal-Wallis non-parametric analysis of variance followed by Mann-Whitney U test.
Reproductive indices:
Mating, fertility, fecundity, gestation
Offspring viability indices:
Live birth, viability (day 4), viability (day 21)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs were observed in any dose groups throughout the study.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred throughout the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No differences in the body weight occurred throughout the study.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No differences in the food consumption occurred throughout the study.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No histopathological findings were observed at any dose.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
The oestrous cycle length was not affected in female animals.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
No differences in epididymal sperm motility, epididymal sperm count, epididymal sperm morphology, testicular sperm count and daily sperm production were observed.
Reproductive performance:
no effects observed
Description (incidence and severity):
No differences in the reproductive performance occurred throughout the study. The mating index ranged from 96 to 100 %.

Details on results (P0)

Post-implantation loss ranged from 8.6 to 10.5 for the F0 generation.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects were observed in parental animals.

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
One female of the low-dose group of the F0 generation delivered only dead pups. This finding was incidental and not considered as treatment-related. There were no other findings.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No differences in the body weight occurred for the pups.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
No difference in the sexual maturation was found in the F1 animals among NM-200 groups and the control group.
Anogenital distance (AGD):
not specified
Nipple retention in male pups:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No effects were observed on pup body weights and on the absolute and relative weight of the brain, thymus and spleen of male and female pups. A statistically significant decreased relative weight of the thyroid of the male animals of the mid-dose group was observed. However, no statistically significant differences were observed between absolute and relative organ weights of male and female animals and there were no findings in the histopathology. Consequently, the finding was considered as not treatment-related.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment-related effects were observed at necropsy.
Histopathological findings:
no effects observed
Description (incidence and severity):
No histopathological findings were observed at any dose.
Other effects:
no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

The gestation index for the F1 generation was 89-98%. Post-implantation loss ranged from 8.9 to 17.7 for the F1 generation.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects were observed in the F1 offspring.

Target system / organ toxicity (F1)

Critical effects observed:
no

Results: F2 generation

Effect levels (F2)

Dose descriptor:
NOAEL
Generation:
F2
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects were observed in any of the F2 offspring.

Target system / organ toxicity (F2)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

Table 1 Reproductive Performance of female rats receiving NM-200

              NM-200 dose (mg/kg bw/day)
 Parameter of reproductive performance  Generation  0  100  300  1000
 Mating index (%)  F0  96  100  100  100
   F1  100  100  100  100
 Fertility index (%)  F0  96  100  96  96
   F1  98  93  93  89
 Fecundity index (%) F0   100  100  96  96
   F1  89  93  93  89
 Gestation index (%)  F0  100  96  100  100
   F1  100  100  96  96
 Precoital time (days)  F0  2.7±0.21  2.8±0.25  2.3±0.19  2.5±0.21
   F1  2.8±0.27  2.5±0.2  2.4±0.2  2.4±0.26
 Gestation time (days)  F0  21.7±0.09  21.6±0.11  21.6±0.1  21.6±0.1
   F1  21.3±0.09  21.2±0.08  21.4±0.12  21.3±0.09
 Postimplantation loss per animal (%)  F0  8.6±2.66  10.5±3.72  9.5±2.54  10.1±2.52
   F1  8.9±1.48  13.6±3.68  17.6±5.47  14.8±4.91

Table 2 - Offspring data from rats receiving NM-200

                NM-200 dose (mg/kg bw/day)
   Generation  0  100  300  1000
 Pups delivered (total)  F0  10.3±2.9  10.7±2.3 11.3± 2.0  11.0±1.5
   F1 11.5 ±1.6 10.7±2.8   10.4±2.8 11.0± 2.6
 Live birth index (%)  F0  97.3±9.9 94.8± 19.1  96.5±12.7 98.7± 4.9
   F1  96.2±6.1 90.7± 17.3  94.2±12.7 93.5± 16.1
 Pup mortality day 1 (%)  F0  2.7±9.9 5.2± 19.1  3.5±12.7 1.3± 4.9
   F1  3.8±6.1 9.3± 17.3 5.8 ±19.0 6.5± 16.1
 Viability index day 4 (%)  F0  99.3±2.5 99.6± 2.0  98.9±4.2 98.8± 3.5
  F1   84.7±28.0 83.8± 34.6 95.3 ± 20.0  73.8±42.3
 Viability index day 21 (%)  F0  100±0  100±0  100±0  100±0 
   F1  100±0  99.5 ±2.6  98.6 ±6.8  100±0
 Whole litter losses (litters lost/total number of litters)  F0  2/27  2/28  2/27  0/27
   F1  1/25  3/26  1/25  4/24
 Sex ratio day 1 (% males)  F0  51.9±17.6  43.9± 12.2  50.8 ±15.6  51.1± 10.9
   F1  47.4± 16.2   52.6±21.4  45.3± 19.6  42.3± 15.4

Applicant's summary and conclusion

Conclusions:
In a two-generation reproductive toxicity study, conducted according to OECD Test Guideline 416 and in compliance with GLP, the NOAEL for systemic and reproductive toxicity of synthetic amorphous silica was concluded to be ≥1000 mg/kg bw/day in Wistar rats based on no adverse effects up to the highest dose tested, 1000 mg/kg bw/day.