Silicon dioxide

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

The key study was carried out according to OECD TG 416 in rats under GLP. This study meets the criteria for Klimisch score 1. No adverse effects on fertility, development, maternal or paternal toxicity were observed after the administration of the substance by oral gavage. The NOEL was 1000 mg/kg bw/day, the highest dose tested.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

2001-01-22

Deviations:

yes

Remarks:

minor ones (s. attachement)

GLP compliance:

yes

Specific details on test material used for the study:

NM-200 Synthetic Amorphous Silica

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 or test system 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.

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.

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.

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.

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

Immunological findings:

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: 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.

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

Key result

Critical effects observed:

no

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.

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.

Developmental immunotoxicity:

no effects observed

There was no changes in the brain of F1 generation aninals that could indicated neurodevelopmental toxicity. In addition, in the available immune organs (spleen and thymus) of F1 animals, there was no indicator of immune toxicity.

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

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

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.

Reference 2

Endpoint:

one-generation reproductive toxicity

Remarks:

based on test type

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Jun. 1 - Dec. 3, 1962

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

number of pregnant animals low (5 females), mating ratio 1:5, only 1 male

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline available

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Deviations:

yes

Remarks:

no complete one generation study according to current standards: too low number of animals and examinations, one dose only, dose selection unclear (relatively low dose selected).

Principles of method if other than guideline:

Method:
5 instead of 20 females, 1 male per 5 females (mating ratio 1:5 instead of 1:2); mating over 14 d, only 1 male used per treated and control group

GLP compliance:

no

Specific details on test material used for the study:

Aerosil

Species:

rat

Strain:

Wistar

Sex:

male/female

Route of administration:

oral: feed

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

Exposure period: 6 months
Premating exposure period (males): 4.5 months
Premating exposure period (females): 4.5 months
Duration of test: 6 months

Frequency of treatment:

daily

Details on study schedule:

Number of generation studies: 1

Remarks:

Doses / Concentrations:
497 mg/kg bw (m); 509 mg/kg bw (f)
Basis:

No. of animals per sex per dose:

5 instead of 20 females, 1 male per 5 females

Control animals:

yes

Details on study design:

This sub-study was part of a complete chronic oral toxicity study: After 4.5 months fertility was also tested with 5 females and 1 male each in the test and control group.

Parental animals: Observations and examinations:

maternal fertility

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly at the same time

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- 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

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no


PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number of pups, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, clinical signs

GROSS EXAMINATION OF DEAD PUPS: no

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were sacrificed at the end of the 6 month study. The mating was after 18 weeks of dosing with the test substance.
- Maternal animals: All surviving animals were sacrificed at the end of the 6 month study. The mating was after 18 weeks of dosing with the test substance.

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

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively.

Reproductive performance:

no effects observed

Dose descriptor:

NOAEL

Effect level:

497 mg/kg bw/day

Sex:

male/female

Basis for effect level:

reproductive performance

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

497 mg/kg bw/day

Sex:

male/female

Basis for effect level:

clinical signs

body weight and weight gain

Reproductive effects observed:

no

Result: negative

Parents: No clinical symptoms; no mortality,no abnormalities in  body-weight gain and feed consumption, no haematological findings.
 
In pups during lactation [total: 45 and 37 (control),
resp.], no behavioral or developmental or structural
abnormalities.
  --------

 

Table 1 Fertility of Wistar rats after treatment with AEROSIL

	500 mg test substance/kg bw	control
Number of females	5	5
Number of pregnant dams	5	4
Number of live offspring	45	37
day of birth	11.-14.11.1962	11.-13.11-1962
Mean litter size	9,0	9,3
Mean weight of offspring at birth in g	5,8	5,7
Live animals 7 days p.p. (in %)	43 (96%)	36 (97%)
Live animals 21 days p.p. (in %)	43 (96%)	36 (97%)

 

Conclusions:

No effects to fertlity were observed in this study some limitations (as only 1 male rat was mated with 5 females).

Executive summary:

This is a sub-study to an oral chronic one with Aerosil in rats. There was no effect on fertility observed. The pubs had no prenatal damage and the development during the lactation periode was normal.

Effects on developmental toxicity

Description of key information

6 Studies on prenatal developmental toxicity were carried out between 1973 and 2015 according to OECD TG 414 or similar protocols, 3 studies were conducted in rats and one each in mice, hamsters and rabbits. These 6 studies on different forms (BET range from 160 to 349 m²/g) of the substance meet the criteria of either Klimisch score 1 or 2. Neither maternal toxicity nor embryo-fetal toxicity was observed at the maximal dose tested in various species (mice, rats, hamsters and rabbits). Based on the available studies, the NOAEL of the substance is estimated as > 1000 mg/kg bw/day for each species. In the earlier studies slightly reduced maternal body weight were observed at the highest dose level tested (1350 mg/kg bw/day in rats and mice and 1600 mg/kg bw/day in hamsters and rabbits. Similarly no treatment related effects on the offspring were observed in the 2 Generation study with non-surface treated SAS (BET 160 m²/g).

Linked studies: all under developmental toxicity

Effect on developmental toxicity via oral route:

No effect observed

NOAEL 1000 mg/kg bw/d

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

4 dose levels, administered on GD 6-10 by oral gavage, examination for skeletal and external malformations on GD 14

GLP compliance:

not specified

Limit test:

no

Species:

hamster

Strain:

other: golden hamsters

Details on test animals or test system and environmental conditions:

Virgin adult female golden hamsters from an outbred strain were individually housed in mesh bottom cages in temperature and humidity controlled quarters with free access to food and fresh tap water at all times.

Route of administration:

oral: gavage

Vehicle:

not specified

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

Females hamsters were mated with young adult males and the appearance of motile sperm in the vaginal smear was considered Day 0 of gestation.

Duration of treatment / exposure:

GD 6-10

Frequency of treatment:

daily

Duration of test:

until GD 14

Dose / conc.:

16 mg/kg bw/day (actual dose received)

Dose / conc.:

74.3 mg/kg bw/day (actual dose received)

Dose / conc.:

345 mg/kg bw/day (actual dose received)

Dose / conc.:

1 600 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

20 - 22 pregnat hamsters

Control animals:

yes, sham-exposed

other: positve control: dosed with Aspirin (250 mg/kg bw/d)

Maternal examinations:

Body weights were recorded on. Days 0 , 8, 10, and 14 of the gestation period. All animals were observed daily for appearance and behavior with particular attention to food consumption in order to better recognize any abnonnmlities resulting from anorexic effects in the pregnant animal.

Fetal examinations:

All fetuses were examined grossly for the presence of external congenital abnormalities, One third of the fetuses of each litter underwent detailed visceral examinations employing 10X magnification. The remaining two-thirds were cleared in potassium hydroxide (KOH), stained with alizarin red S dye and examined for skeletal abnormalities.

Clinical signs:

not specified

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Maternal body weight after 14 days was 147.6 g in average in highest dose group, against a mean body weight of 157.4 in sham controls.

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

no significant difference between treated and sham control animals

Dead fetuses:

no effects observed

Changes in number of pregnant:

no effects observed

Key result

Dose descriptor:

LOAEL

Effect level:

>= 1 600 mg/kg bw/day (nominal)

Basis for effect level:

body weight and weight gain

Key result

Abnormalities:

no effects observed

Reduction in number of live offspring:

no effects observed

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

no significant difference between treated and sham control animals

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

no significant difference between treated and sham control animals

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 600 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

external malformations

skeletal malformations

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

effects observed, non-treatment-related

Localisation:

skeletal: skull

skeletal: sternum

skeletal: rib

skeletal: vertebra

other: external: extremities (unspecified)

Developmental effects observed:

yes

Lowest effective dose / conc.:

16 mg/kg bw/day (actual dose received)

Treatment related:

no

Relation to maternal toxicity:

developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

no

Relevant for humans:

no

Conclusions:

The administration of up to 1600 mg/kg (body weight) of the test material to pregnant hamsters for 5 consecutive days had no clearly discernible effect on nidation or on maternal or fetal survival. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring spontaneously in the sham-treated controls.

Executive summary:

The developmental toxicity of Syloid 244 was assessed in hamsters treated on GD 6 -10.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

4 dose levels, administered on GD 6-15 by oral gavage, examination for skeletal and external malformations on GD 17

GLP compliance:

not specified

Limit test:

no

Species:

mouse

Strain:

CD-1

Details on test animals or test system and environmental conditions:

Virgin adult female albino CD-1 outbred mice were ganghoused in disposable plastic cages in temperature and humidity-controlled quarters with free access to food and fresh tap water.

Route of administration:

oral: gavage

Vehicle:

not specified

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

Females mice were mated with young adult males, and observation of the vaginal sperm plug was considered Day 0 of gestation.

Duration of treatment / exposure:

GD 6-15

Frequency of treatment:

daily

Duration of test:

until GD 17

Dose / conc.:

13.4 mg/kg bw/day (actual dose received)

Dose / conc.:

62.3 mg/kg bw/day (actual dose received)

Dose / conc.:

289 mg/kg bw/day (actual dose received)

Dose / conc.:

1 340 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

21 - 26 pregnant dams

Control animals:

yes, sham-exposed

other: positive control: dosed with Aspirin (150 mg/kg bw/d)

Maternal examinations:

Body weights were recorded on Days 0, 6, 11, 15, and 17 of gestation. All animals were observed daily for appearance and behavior with particular attention to food consumption and weight, in order to rule out any abnormalities which may have occurred as a result of anorexic effects in the pregnant female animal.
On Day 17 all dams were subjected to Caesarean section under surgical anesthesia, and the numbers of implantation sites, resorption
sites, and live and dead fetuses were recorded. The body weights of the live pups were also recorded. The urogenital tract of each dam
was examined in detail for anatomical normality.

Fetal examinations:

All fetuses were examined grossly for the presence of external congenital abnormalities. One-third of the fetuses of each litter underwent detailed visceral examinations employing 10X magnification. The remaining two-thirds were cleared in potassium hydroxide (KOH), stained with alizarin red S dye and examined for skeletal defects.

Clinical signs:

not specified

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Maternal body weight after 17 days was 43.6 g in average in highest dose group, against a mean body weight of 50.1 in sham controls.

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

no significant difference between treated and sham control animals

Changes in number of pregnant:

no effects observed

Key result

Dose descriptor:

LOAEL

Effect level:

>= 1 340 mg/kg bw/day (actual dose received)

Basis for effect level:

body weight and weight gain

Abnormalities:

not examined

Reduction in number of live offspring:

no effects observed

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

no significant difference between treated and sham control animals

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

no significant difference between treated and sham control animals

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 340 mg/kg bw/day (actual dose received)

Sex:

not specified

Basis for effect level:

reduction in number of live offspring

external malformations

skeletal malformations

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

effects observed, non-treatment-related

Localisation:

skeletal: skull

skeletal: sternum

skeletal: rib

skeletal: vertebra

other: external: extremities (unspecified)

Developmental effects observed:

yes

Lowest effective dose / conc.:

13.4 mg/kg bw/day (actual dose received)

Treatment related:

no

Relation to maternal toxicity:

developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

no

Relevant for humans:

no

Conclusions:

The administration of up to 1340 mg/kg (body weight) of the test material to pregnant mice for 10 consecutive days had no clearly discernible effect on nidation or on maternal or fetal survival. The number of abnoimmaities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring spontaneously in the sham-treated controls.

Executive summary:

The developmental toxicity of Syloid 244 was evaluated in mice treated on GD 6 -15.

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

4 dose levels, administered on GD 6-18 by oral gavage, examination for skeletal and external malformations on GD 29

GLP compliance:

not specified

Limit test:

no

Species:

rabbit

Strain:

Dutch

Details on test animals or test system and environmental conditions:

Virgin, adult Dutch belted female rabbits were individually housed in mesh bottom cages in temperature and humidity-controlled quarters with free access to food and fresh tap water.

Route of administration:

oral: gavage

Vehicle:

not specified

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

Each dose was inseminated artificially with 0.3 ml of diluted semen from, a proven donor buck using approximately 20 x 10^6 motile sperm.

Duration of treatment / exposure:

GD 6-18

Frequency of treatment:

daily

Duration of test:

until GD 29

Dose / conc.:

16 mg/kg bw/day (actual dose received)

Dose / conc.:

74.3 mg/kg bw/day (actual dose received)

Dose / conc.:

345 mg/kg bw/day (actual dose received)

Dose / conc.:

1 600 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10 - 15 pregnat rabbits

Control animals:

yes, sham-exposed

other: positve control: dosed with 6-AN (2.5 mg/kg) on GD 9

Details on study design:

On Day 0, each doe was given an injection of 0.4 ml of human chorionic gonadotropin (400 IU) via the margins 1 ear vein. Three hours later, each
doe was inseminated artificially with 0.3 ml of diluted semen from, a proven donor buck using approximately 20 x 10^6 motile sperm. Beginning on Day 6 and continuing daily through Day 18 the females were dosed with the indicated dosages by oral intubation. The controls were sham treated with the vehicle at a level equivalent to the group receiving the highest test dose.

Maternal examinations:

Body weights were recorded on Days 0, 6, 12, 18, and 29 of gestation. All animals were observed daily for appearance and behavior, with particular attention to food consumption and body weight in order to rule out any abnormalities which may have occurred as a result of anorexic effects in the pregnant female animal.

Fetal examinations:

All fetuses underwent a detailed gross examination for the presence of external congenital abnommlities. The live fetuses of each litter were then placed in an incubator for 24 hours for the evaluation of neonatal survival. All surviving pups were sacrificed, and all pups examined for visceral abnormalities (by dissection) . All fetuses were then cleared in potassum hydroxide (KOH), stained with alizarin red S dye and examined for skeletal defects.

Clinical signs:

not specified

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Maternal body weight after 29 days was 3.34 kg in average in highest dose group, against a mean body weight of 3.84 kg in sham controls.

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

no significant difference between treated and sham control animals

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

no significant difference between treated and sham control animals; the highest mortality was in the lowest dose group, but not in shame control animals.

Changes in number of pregnant:

no effects observed

Key result

Dose descriptor:

LOAEL

Effect level:

>= 1 350 mg/kg bw/day (nominal)

Basis for effect level:

body weight and weight gain

Key result

Abnormalities:

no effects observed

Reduction in number of live offspring:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

no significant difference between treated and sham control animals

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 600 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

external malformations

skeletal malformations

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

effects observed, non-treatment-related

Localisation:

skeletal: skull

skeletal: sternum

skeletal: rib

skeletal: vertebra

Developmental effects observed:

yes

Lowest effective dose / conc.:

16 mg/kg bw/day (actual dose received)

Treatment related:

no

Relation to maternal toxicity:

developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

no

Relevant for humans:

no

Conclusions:

The administration of up to 1600 mg/kg (body weight) of the test material to pregnant rabbits for 13 consecutive days had no clearly discernible effect on nidation or on maternal or fetal survival. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring spontaneously in the sham-treated controls.

Executive summary:

The developmental toxicity of Syloid 244 was assessed in rabbits treated on GD 6 -18.

Reference 4

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

4 dose levels, administered on GD 6-15 by oral gavage, examination for skeletal and external malformations on GD 20

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Virgin adult female albino rats (Wistar derived stock) were individually housed in mesh bottom cages in temperature and humidity controlled quarters with free access to food and fresh tap water.

Route of administration:

oral: gavage

Vehicle:

not specified

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

Females rats were mated with young adult males, and observation of the vaginal sperm plug was considered Day 0 of gestation.

Duration of treatment / exposure:

GD 6-15

Frequency of treatment:

daily

Duration of test:

until GD 20

Dose / conc.:

13.5 mg/kg bw/day (actual dose received)

Dose / conc.:

62.7 mg/kg bw/day (actual dose received)

Dose / conc.:

292 mg/kg bw/day (actual dose received)

Dose / conc.:

1 350 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

20 - 25 pregnat rats

Control animals:

yes, sham-exposed

other: positve control: dosed with Aspirin (250 mg/kg bw/d)

Maternal examinations:

Body weights were recorded on Days 0, 6, 11, 15, and 20 of gestation. All animals were observed daily for appearance and behavior with particular attention to food consumption and weight, in order to rule-out any abnormalities which may have occurred as a result of anorexic effects in the pregnant female animal.

Fetal examinations:

All fetuses were examined grossly for the presence of external congenital abnormalities, One-third of the fetuses of each litter underwent detailed visceral examinations employing 10X magnification. The remaining two-thirds were cleared in potassium hydroxide (KOH), stained with alizarin red S dye and examined for skeletal defects.

Clinical signs:

not specified

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Maternal body weight after 20 days was 334 g in average in highest dose group, against a mean body weight of 356 in sham controls.

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

no significant difference between treated and sham control animals

Dead fetuses:

no effects observed

Changes in number of pregnant:

no effects observed

Key result

Dose descriptor:

LOAEL

Effect level:

>= 1 350 mg/kg bw/day (nominal)

Basis for effect level:

body weight and weight gain

Key result

Abnormalities:

no effects observed

Reduction in number of live offspring:

no effects observed

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

no significant difference between treated and sham control animals

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

no significant difference between treated and sham control animals

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 350 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

external malformations

skeletal malformations

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

effects observed, non-treatment-related

Localisation:

skeletal: skull

skeletal: sternum

skeletal: rib

skeletal: vertebra

other: external: extremities (unspecified)

Developmental effects observed:

yes

Lowest effective dose / conc.:

13.5 mg/kg bw/day (actual dose received)

Treatment related:

no

Relation to maternal toxicity:

developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

no

Relevant for humans:

no

Conclusions:

The administration of up to 1350 mg/kg (body weight) of the test material to pregnant rats for 10 consecutive days had no clearly discernible effect on nidation or on maternal or fetal survival. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring spontaneously in the sham-treated controls.

Executive summary:

The developmental toxicity of Syloid 244 was assessed in rats treated on GD 6 -15.

Reference 5

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Specific details on test material used for the study:

NM-200

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Obtained from: Charles River Laboratories, Research Models and Services Germany GmbH, Germany; individually caged, feed: Kilba GLP and bottled drinking water ad libitum, 20-24 °C, 30-70 % humidity, 12 h light/dark. The body weight of the pregnant animals on GD0 varied between 195.3 and 271.0 g.

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

with highly deionized water containing 10% fetal bovine serum in order to avoid agglomeration

Details on mating procedure:

After an acclimatization period of at least 5 days,1–2 untreated female ratswere mated with one untreated male animal of the same breed. The male mating partners were kept under the same conditions (air conditioning, feed, water) as the female rats of this study. Mating took place from about 15:30 h to about 7:30 h on the following day.If sperm were detected microscopically in a vaginal smear in the morning, the female was considered impregnated and transferred into the study. This day was referred to as gestation day 0 (GD0, beginning of the experimental phase) and the following day as GD1.

Duration of treatment / exposure:

GD 6-19

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

25 (females only)

Control animals:

yes, concurrent vehicle

Maternal examinations:

Clinical signs, body weights and food consumption were recorded regularly throughout the study,

Ovaries and uterine content:

On GD 20,females were sacrificed and examined macroscopically. Uteri and ovaries were removed. Gravid uterus weights and corrected body weight gain of the dams (terminal body weight−gravid uterus weight−body weight on GD 6) were determined. The number of corpora lutea, as well as the number, status and distribution of implantation sites (live fetuses, early resorptions, late resorptions, dead fetuses) were also determined.
Conception rate, pre-implantation loss (%), and post-implantation loss (%)were calculated using the litter as unit in the latter two parameters. Placental diameters were measured during gross pathological assessment.

Fetal examinations:

At necropsy each fetus was weighed, sexed, and external tissues and allorificeswere examined macroscopically.The sex was determined by observing the distance between the anus and the base of the genital tubercle. Furthermore, the viability of the fetuses and the condition of placentae, umbilical cords, fetal membranes, and fluids were examined. Individual placental weights were recorded.

Statistics:

The data for food consumption, maternal, litter weight and body weight change,carcass and gravid uterus weight, number of corpora lutea and implantations, number of resorptions, number of live fetuses, percent pre- and post-implantation loss, percent live fetuses per litter, number of pups per litter, post-implantation loss, and mean placental weights were analyzed by the two-sided Dunnett’s test for the hypothesis of equal means.
The female mortality, number of pregnant females, and number of litters with fetal findings,were analyzed by pairwise comparison of each dose
group with the control group using the one-sided Fisher’s exact test for the hypothesis of equal proportions.
Proportions of fetuses per litter with findings were analyzed by pairwise comparison of each dose group with the control group by a one-sided Wilcoxon
test for the hypothesis of equal medians.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

The conception rate,mean number of corpora lutea, implantation sites, and post-implantation loss were comparable in all groups. The only minor difference to control values was pre-implantation loss (slightly higher in the groups treated with SAS); however, acompound-related effect can be excluded as treatment started after implantation.

Dead fetuses:

no effects observed

Description (incidence and severity):

No dead fetuses were noted.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Three rats of the control group, four rats of the low dose group and two animals of the high dose group were not pregnant.

Other effects:

no effects observed

Description (incidence and severity):

Gravid uterus weights were not influenced by the treatment.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Basis for effect level:

body weight and weight gain

changes in number of pregnant

clinical signs

dead fetuses

food consumption and compound intake

mortality

necropsy findings

pre and post implantation loss

other:

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Changes in sex ratio:

no effects observed

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

External malformations were recorded for one fetus each in the low- and high-dose group. Both fetuses concerned had multiple malformations. Mandibular micrognathia mirrored the severely malformed skull bones found during skeletal examination in one fetus; these findings were considered related to each other. The cleft palate observed in the other fetus is present in the historical control data at a comparable incidence. Both findings were considered to be spontaneous in nature and without a relation to dosing. The total incidence of external malformations in treated animals did not differ significantly from that of the control group. One sof ttissue malformation (i.e. super numerary liver lobes – unilateral) was observed in the control group.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal malformations were noted in single fetuses at 0, 100 and 1000 mg/kg body weight/d. Each of them affected individual fetuses and neither statistically significant differences between the test groups nor a dose–response relationship was observed. The overall incidences of skeletal malformations were comparable to those found in the historical control data.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Fetal morphology examinations
Three soft tissue variations, i.e. short innominate, enlarged atrial chamber of the heart and uni- or bilateral dilation of renal pelvis, were detected. These findings observed in 1–5 fetuses of 1–4 litters at 0, 100, 300 and 1000 mg/kg bw/d showed no dose–response relationship. The observable differences between the groups reflect the usual fluctuation for this parameter and were clearly with in the range of the historical control data.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

changes in sex ratio

fetal/pup body weight changes

external malformations

skeletal malformations

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

As no maternal toxicity was observed, and the test compound did not alter cesarean section parameters and did not influence fetal or placental weights and no compound-related increase in the incidence of malformations or variations was observed in the fetuses, both the maternal and fetal NOAEL were 1000 mg/kg bw/d.

Executive summary:

The developmental toxicity of NM-200 was assessed in rats treated on GD 6 -19.

Reference 6

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Sep. 14, 2000 - 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2001-01-22

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

Aug. 1998

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

NM-200 (synthetic amorphous silica)

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

supplied by Charles River Laboratories, Research Models and Services, Germany GmbH; 10-12 weeks old, individually caged, feed: Kilba and bottled drinking water ad libitum, 20-24 °C, 30-70 % humidity, 12 h light/dark

Route of administration:

oral: gavage

Vehicle:

other: highly deionized water containing 10% FBS

Details on exposure:

To prepare the dose formulations, the specific amount of test substance (1, 3 and 10 g) was weighed and placed into a calibrated glass beaker, topped up to 90 ml with highly deionized water and gently homogenized manually.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analyses of the test-substance preparations were carried out at Competence Center Analytics, BASF SE, Ludwigshafen, Germany, not in full compliance with GLP under the supervision of Dr. W. Wohlleben.
The test substance is assumed to be stable in the formulation for at least the time of administration.
Samples of the test substance preparations were sent to the analytical laboratory twice during the study period (at the beginning and towards the end) for verification of the size stability of the test material in the vehicle.

Details on mating procedure:

After an acclimatization period of at least 5 days, 1 - 2 untreated female rats were mated with one untreated male animal of the same breed. The male mating partners were kept under the same conditions (air conditioning, feed, water) as the female rats of this study.
Mating took place from about 15:30 h to about 7:30 h on the following day. If sperm were detected microscopically in a vaginal smear in the morning, the female was considered impregnated and transferred into the study. This day was referred to as gestation day 0 (GD 0, beginning of the experimental phase) and the following day as GD 1.

Duration of treatment / exposure:

GD 6-19

Frequency of treatment:

once daily

Duration of test:

until GD 20

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

25 (females only)

Control animals:

yes, concurrent vehicle

Details on study design:

The test substance was administered as an aqueous solution to groups of 25 presumed pregnant female Wistar rats by gavage at doses of 100; 300 and 1000 mg/kg body weight/day (mg/kg bw/d) on gestation days (GD) 6 through 19.

Maternal examinations:

Food consumption and body weights of the animals were recorded regularly throughout the study period. The state of health of the animals was checked each day.

Fetal examinations:

On GD 20, all females were sacrificed and assessed by gross pathology (including weight determinations of the unopened uterus and the placentae). For each dam, corpora lutea were counted and number and distribution of implantation sites (differentiated between resorptions, live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed, weighed and further investigated for external findings. Thereafter, one half of the fetuses of
each litter were examined for soft tissue findings and the remaining fetuses for skeletal (inclusive cartilage) findings.

Statistics:

DUNNETT-test: Food consumption, body weight, body weight change, corrected body weight gain (net maternal body weight change), carcass weight, weight of unopened uterus, number of corpora lutea, number of implantations, number of resorptions, number of live fetuses, proportions of preimplantation loss, proportions of postimplantation loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight
FISHER'S EXACT test: Female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings
WILCOXON-test: Proportions of fetuses with malformations, variations and/or unclassified observations in each litter

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in number of pregnant:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

changes in number of pregnant

clinical signs

dead fetuses

early or late resorptions

food consumption and compound intake

gross pathology

mortality

number of abortions

organ weights and organ / body weight ratios

pre and post implantation loss

total litter losses by resorption

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Changes in sex ratio:

no effects observed

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

External malformations were recorded for one fetus each in the low- and high-dose group (100 and 1000 mg/kg bw/d). Both fetuses concerned had multiple malformations. In one fetus the mandibular micrognathia mirrored the severely malformed skull bones found during skeletal examination; these findings are not considered independent of each other. Both findings were considered to be spontaneous in nature and without a relation to dosing. The total incidence of external malformations in treated animals did not differ significantly from that of the control group.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal malformations were noted in single fetuses of test groups 0, 1 and 3 (0, 100 and 1000 mg/kg bw/d). Although some of these findings are not in the historical control data, each of them affected individual fetuses and neither statistically significant differences between the test groups nor a dose-response relationship was observed. The overall incidences of skeletal malformations were comparable to those found in the historical control data.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Three soft tissue variations, i.e. short innominate, enlarged atrial chamber of the heart and uni- or bilateral dilation of renal pelvis, were detected. These findings observed in 1 to 5 fetuses of 1 to 4 litters in test groups 0, 1, 2 and 3 (0, 100, 300 and 1000 mg/kg bw/d) showed no dose-response relationship. The observable differences between the groups reflect the usual fluctuation for this parameter and were clearly within the range of the historical control data

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

changes in sex ratio

fetal/pup body weight changes

external malformations

skeletal malformations

visceral malformations

Key result

Abnormalities:

effects observed, non-treatment-related

Localisation:

external: eye

external: face

skeletal: skull

skeletal: pelvic girdle

visceral/soft tissue: cardiovascular

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

0 mg/kg bw/day (actual dose received)

Treatment related:

no

Relation to maternal toxicity:

developmental effects in the absence of maternal toxicity effects

Dose response relationship:

no

Relevant for humans:

no

No treatment related devopmental toxicity was observed. The observed effects were also seen in the control group (0 mg/kg bw/d).

Conclusions:

Under the conditions of this prenatal developmental toxicity study, the oral administration of NM-200 Synthetic Amorphous Silica to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) elicited no test substance-related adverse effects at any tested dose.
In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity and prenatal developmental toxicity is 1000 mg/kg body weight/day. No adverse maternal and fetal findings of toxicological relevance were evident at any dose.

Executive summary:

The developmental toxicity of NM-200 was evaluated in rats.

Justification for classification or non-classification

A valid and reliable study according OECD guideline 416 did not identify any effects on development and did not evidence any maternal or paternal toxicity. 6 Studies carried out according to OECD TG 414 or similar protocols did not display any developmental toxicity. According to Annex I of the Regulation (EC) 1272/2008 and GHS (Globally Harmonized Classification System), the test substance requires no classification and has no mandatory label requirement.