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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
No data
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
powder

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Germany GmbH
- Age at study initiation: 12-15 weeks
- Weight at study initiation: 195.3- 271g
- Fasting period before study: Not relevant
- Housing: Individually in Macrolon type III cages, with a bedding of dust-free wood shavings and wooden gnawing blocks as environmental enrichment.
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 30-70%
- Air changes (per hr): approximately 15 changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: No data

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: highly deionised water containing 10% fetal bovine serum
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Dosing formulations of synthetic amorphous silica were pre-pared with highly deionized water containing 10% fetal bovine serum in order to avoid agglomeration.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The formulations were analysed by scanning electron microscopy after shock freezing and in situ analytical ultracentrifugation.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1-2 untreated females with 1 untreated male
- Length of cohabitation: Not stated but mating occurred between 15:30 and 7.30 on the day after cohousing started.
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
Gestation day 6-19
Frequency of treatment:
One dose per day
Duration of test:
Approximately 21 days
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
25 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Highest dose was chosen as the limit dose according to the test guideline.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: described as 'regular'

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: described as 'regular'

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes (described as 'regular')

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: uterus and ovaries
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes, but no data on number examined
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 analysed 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 analysed 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.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
Results are summarised in Tables 1 and 2. There was no treatment-related mortality or clinical signs of toxicity, and there was no effect on body weight gain or food consumption. Gravid uterine weights were not influenced by treatment with SAS. No treatment-related macroscopic findings were observed at necropsy. 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, a compound-related effect can be excluded as treatment started after implantation.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Results are summarised in Table 1. No dead fetuses were noted. The sex distribution of the fetuses was comparable in all groups. Mean fetal weights did not show any biologically relevant differences between the test substance-treated groups and the control. 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 soft tissue malformation (i.e. supernumerary liver lobes unilateral) was observed in the control group. Skeletal malformations were noted in single fetuses at 0, 100, and 1000 mg/kgbw/day. 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.

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/kgbw/day 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. For all groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilage. The observed skeletal variations were related to several parts of fetal skeletons and appeared without a relation to dosing. The overall incidences of skeletal variations were comparable to the historical control data. The incidence of the variations observed showed no dosing-related statistical significance and were within historical control ranges. Therefore these observations are not considered toxicologically relevant.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: There were no adverse effects on developmental parameters.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Table 1 Summary of reproductive and developmental results

 Dose (mg/kg bw/d)    0 (vehicle control)  100  300  1000
 Females mated  N 25 25 25 25
  Pregnant  N 22 21 25  23 
  Conception rate  % 88  84  100  92 
  Aborted  N 0 0 0 0
  Premature births  N
  Dams with viable fetuses  N 22 21  25  23 
  Dams with total litter loss  N
Female mortality N 0
Pregnant at terminal sacrifice  N 22  21  25  23 
   % 88 84  100  92 
Corpora luteaD mean ± SD  14.2 ± 2.4  13.9 ± 2.08  14.0 ± 1.99  14.7 ± 2.03
Implantation sitesD mean ± SD  13.5 ± 2.56  12.5 ± 2.8  12.8 ± 1.62  13.4 ± 2.27
Pre-implantation loss (%)D mean ± SD  4.9 ± 7.31  9.4 ± 17.69 7.7 ± 11.81  8.4 ± 12.31 
Post-implantation loss (%)D mean ± SD  8.3 ± 9.96 5.2 ± 5.22  9.9 ± 16.2  6.0 ± 3.56 
  Early resorptions (%)D    7.6 ± 9.27 5.2 ± 5.22  9.9 ± 16.2  5.6 ± 3.75 
  Late resorptions (%)D    0.7 ± 2.26 0.3 ± 1.49 
  Dead fetuses    0 0 0
 Dams with viable fetuses  N 22 21 25  23 
  Live fetusesD  mean ± SD  12.4 ± 2.59 11.8 ± 2.71  11.5 ± 2.47  12.7 ± 2.23 
  Live fetuses (% implantation)D  mean ± SD  91.7 ± 9.96 94.8 ± 5.22  90.1 ± 16.2  94.0 ± 3.56 
  MalesD  mean ± SD  5.9 ± 1.7 5.5 ± 1.91  6.5 ± 1.85  5.7 ± 1.79 
  Males (%)D  mean ± SD  43.7 ± 10.64 46.5 ± 18.79  50.9 ± 14.23  43.2 ± 13.09 
  FemalesD  mean ± SD  6.5 ± 1.95 6.3 ± 2.76  5.0 ± 2.34  6.9 ± 2.21 
  Females (%)D  mean ± SD  48.0 ± 11.17 48.3 ± 18.55 39.2 ± 15.98  50.9 ± 11.85 
 Sex ratio (% males)    47.4 46.8  56.3  45.4 
 Placental weightsD  mean ± SD  0.47 ± 0.040 0.47 ± 0.042  0.46 ± 0.038  0.47 ± 0.038 
   N 22  21  25  23 
  Of male fetusesD  mean ± SD 0.49 ± 0.038  0.48 ± 0.036  0.47 ± 0.043  0.48 ± 0.037 
   N 22 21  25  23 
  Of female fetusesD  mean ± SD 0.46 ± 0.042  0.46 ± 0.052  0.46 ± 0.044  0.46 ± 0.041 
   N 22 20  25  23 
 Fetal weightsD  mean SD 3.4 ± 0.26  3.4 ± 0.15  3.4 ± 0.24  3.4 ± 0.14 
   N 22  21  25  23 
  MalesD  mean SD 3.5 ± 0.27  3.4 ± 0.18  3.5 ± 0.24  3.5 ± 0.14 
   N 22  21  25  23 
  FemalesD  mean SD 3.3 ± 0.25 3.3 ± 0.17  3.4 ± 0.28  3.3 ± 0.15 
   N 22  20  25  23 

*p<0.05; **p<0.01 (D, two-sided Dunnett's test)

Table 2 Summary of fetal malformations

 Dose (mg/kg bw/d)   0 (vehicle control)  100  300  1000
Litters evaluated  N 22 21  25  23 
Fetuses evaluated  N 272  248  288  291 
                
Total Malformations               
Fetal incidence  N 3 1
 % 1.1  0.4  0.0  0.3 
 Litter incidenceF  N
   % 9.1 4.8  0.0  4.3 
 Affected fetuses per litterW  mean% ± SD 1.3 ± 4.07  0.4 ± 1.98  0.0 ± 0.00  0.3 ± 1.60 

*<0.05; **p<0.01 (F, one-sided Fisher's exact test; W, one-sided Wilcoxon)

Applicant's summary and conclusion

Conclusions:
Based on a prenatal developmental toxicity study in Wistar rats conducted in accordance with OECD TG 414 and to GLP the NOAEL for developmental toxicity was ≥1000 mg/kg bw/day as no adverse effects on developmental parameters were observed up to the highest dose tested. The NOAEL for general systemic toxicity was also ≥1000 mg/kg bw/day as there were no signs of adverse effects in any of the maternal animals up to the highest dose tested.