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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:
19 February 2013 to 9 May 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014
Reference Type:
other: Appendix
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): dimethylsilanediol

Test animals

Species:
rat
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Inc., Raleigh, NC, US
- Age at study initiation: approx 79 days
- Housing:stainless steel wire-mesh cages suspended above cage-board
- Diet:PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 (ad libitum):
- Water: municipal water supply (ad libitum):
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.1 to 21.3
- Humidity (%): 42.1 to 46.5
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 19 February 2013 to 28 March 2013

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance formulations were prepared approximately weekly as single formulations for each dose level, divided into aliquots for daily dispensation, and stored refrigerated in plastic bags containing desiccant.


VEHICLE
- Concentration in vehicle: 0, 50, 100 or 200 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg
- Lot/batch no.: 2BD1117, 2BH0747, and 2AJ0197
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to the initiation of dose administration, two sets of duplicate samples for homogeneity determination were collected from the top, middle, and bottom strata of nondosing formulations prepared at 50, 100, and 200 mg/mL. In addition, two sets of duplicate samples for resuspension homogeneity and stability determinations were collected from the top, middle, and bottom strata of an aliquot taken from these same nondosing suspensions following refrigerated storage for 10 days; aliquots were mixed for a minimum of 30 minutes prior to sample collection. Two sets of duplicate samples for concentration analysis were collected from the middle stratum of each dosing formulation (including the control group) from the first and last formulations prepared during the in-life phase. One set of samples from each collection was subjected to the appropriate analyses. The remaining set of samples was stored refrigerated (approximately 2°C to 8°) as back-up. All analyses were conducted by using a validated gas chromatography method with flame ionization detection.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1/1
- Length of cohabitation: until positive evidence of mating
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
Gestation days 6 to 19
Frequency of treatment:
daily
Duration of test:
until gestational day 20
Doses / concentrationsopen allclose all
Dose / conc.:
250 mg/kg bw/day
Dose / conc.:
500 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
25 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels were selected based on results of an OECD 422 study performed with the same test substance. In that study, there were no obvious signs of
reproductive/developmental toxicity at a high-dose level of 500 mg/kg bw/day. Maternal toxicity was limited to higher liver weights and hepatocellular vacuolation. There was a potential for lower body weight gains at 1000 mg/kg bw/day based on a 14-day range-finding study.

The selected route of administration for this study was oral (gavage) because this is a potential route of exposure for humans. Historically, this route has been used extensively for studies of this nature.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, gestational day 0 to 20

BODY WEIGHT: Yes
- Time schedule for examinations: gestational day 0, 6-20

FOOD CONSUMPTION: Yes
- Time schedule for examinations: gestational day 0, 6-20
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: liver
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
- Other: placentae
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter by Wilson technique, half per litter by midcoronal slice
Statistics:
Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group. Each mean was presented with the standard deviation, standard error and the number of animals used to calculate the mean.

Maternal body weights (absolute and net), body weight changes (absolute and net), and food consumption, gravid uterine weights, absolute liver weights, numbers of corpora lutea, implantation sites, fetal body weights and viable fetuses were subjected to a parametric one-way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test substance-treated groups to the control group. In addition, fetal body weights were subjected to a parametric one-way ANCOVA (SAS Institute, Inc., 2002-2008) with litter size as the covariate, to determine intergroup differences. If the ANCOVA revealed significant (p<0.05) intergroup variance, Dunnett’s test (Dunnett, 1964) was used to compare the test article-treated groups to the control group. Mean litter proportions (percent per litter) of prenatal data (viable and nonviable fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and fetal sex distribution), total fetal malformations and developmental variations (external, visceral, skeletal, and combined) and each particular external, visceral, and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test (Kruskal and Wallis, 1952) to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunn’s test (Dunn, 1964) was used to compare the test substance-treated groups to the control group.
Historical control data:
Yes

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Low incidences of test substance-related yellow material on the anogenital area was noted in the 500 and 1000 mg/kg bw/day groups. This
finding was noted as early as gestation day 10 or 7 in these respective groups and continuing through gestation day 20. Low incidences of red material around the nose and/or read or clear material around teh mouth were also noted in these groups beginning on gestation day 7 or 6, respectively, through gestation day 20. No findings attributable to treatement noted at 250 mg/kg/day.

Mean maternal body weight gains in the 1000 mg/kg bw/day group were similar to the control group during gestation days 6-12. Significantly (p<0.05) lower mean body weight gains were noted in this group during gestation days 12-15 (27.8%) and 15-20 (13.7%) compared to the control group. As a result, mean body weight gain in the 1000 mg/kg bw/day group was lower than the control group when the entire treatment period (9.6%; gestation days 6-20) was evaluated. Mean gravid uterine weight in the 1000 mg/kg bw/day group was slightly lower (8.5%) than the control group. The lower mean body weight gain late in gestation and lower mean gravid uterine weight in this group were attributed to the lower mean fetal body weight and slightly lower mean number of viable fetuses and were not considered to be evidence of maternal toxicity. Lower mean food consumption was noted in the 1000 mg/kg bw/day group throughout the gestation treatment period (days 6-20).

Mean liver weights in the 250, 500, and 1000 mg/kg bw/day groups were 20.2%, 35.7% and 65.6% higher, respectively, than the control group value. Test substance-related microscopic findings were noted in the liver of the 250, 500, and 1000 mg/kg bw/day groups. Centrilobular hypertrophy was considered an adaptive response. Periportal vacuolation, characterized by primarily macrovesicular change, was morphologically consistent with lipid accumulation. Although hepatocellular lipid accumulation may be an adaptive response, in this study the vacuolation was considered an adverse finding at ≥250 mg/kg bw/day due to the increased incidence and severity in the test substance-treated groups, along with the increases in liver weight.

Effect levels (maternal animals)

Dose descriptor:
LOAEL
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Maternal abnormalities

Abnormalities:
not specified

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
A higher mean litter proportion of postimplantation loss (primarily due to a higher incidence of late resorptions), with a corresponding lower mean litter proportion of viable fetuses was noted in the 1000 mg/kg bw/day group. Statistically significant lower mean fetal body weights were observed in all test substance-treated groups. Although the mean fetal body weight in the 250 mg/kg bw/day group was only 5.3% lower than the concurrent control group value (0.2 g less) and within the range of thehistorical control data, the difference from the concurrent control group was statistically significant when analyzed using both an analysis of variance (ANOVA; p<0.05) and an analysis of covariance (ANCOVA; p<0.01) with litter size as the covariate. However, these methods assume litter size is unaffected by treatment.

The lower mean fetal weights in these groups coincided with increased incidences of skeletal variations in a dose-related manner related to lesser degree of ossification and bent ribs or limb bones (see below). Mean fetal sex ratios in all test substance-treated groups and intrauterine survival in the 250 and 500 mg/kg bw/day groups were unaffected by maternal test substance administration.

Test substance-related adverse effects on fetal morphology were noted in all test substance-treated groups, with some of the findings interpreted as test substance-related having incidences in the 250 and 500 mg/kg bw/day groups being within the range of the historical control data and some of the findings only represent 1 fetus in the group, and consisted of the following:

- External malformations (fetal anasarca, localized fetal edema, tarsal flexure, and cleft palate) were noted at 1000 mg/kg bw/day.
- Visceral malformations (retroesophageal, right-sided, or coarctation of the aortic arch and absent cartilaginous rings of the trachea) were noted at 1000 mg/kg bw/day; one fetus in the 500 mg/kg bw/day group also had a retroesophageal aortic arch.
- Skeletal malformations consisted of costal cartilage anomaly (500 and 1000 mg/kg bw/day), bent scapula (500 and 1000 mg/kg bw/day; statistically significant at 1000 mg/kg bw/day), and bent limb bone(s) and sternoschisis (1000 mg/kg bw/day).
- Visceral developmental variations consisted of small thyroid gland(s) (250 mg/kg bw/day and higher), pale spleen (500 mg/kg bw/day and higher), and major blood vessel variations and pale or swollen liver (1000 mg/kg bw/day). One of the fetuses in the 1000 mg/kg bw/day group with a swollen liver also had white areas in the liver.

The following skeletal developmental variations were also noted in the indicated test substance-treated groups and generally corresponded to the test substance-related lower mean fetal weights at all dose levels.
- A lower incidence of cervical centrum no. 1 ossified (250, 500, and 1000 mg/kg bw/day); higher incidences of bent ribs and 14th rudimentary rib(s) (250 mg/kg bw/day and higher); higher incidences of pubis unossified and 27 presacral vertebrae (250 mg/kg bw/day and higher); and higher incidences of reduced ossification of the rib(s) (500 mg/kg bw/day and higher).
- Higher incidences of sternebra(e) nos. 5 and/or 6 unossified (500 and 1000 mg/kg bw/day); higher incidences of 7th cervical rib(s), reduced ossification of the vertebral arches, and sternebra(e) nos. 1, 2, 3, and/or 4 unossified (500 and 1000 mg/kg bw/day); and higher incidences of unco-ossified vertebral centra and reduced ossification of the skull and rib(s) (500 and 1000 mg/kg bw/day).
- Higher incidences of vertebral centra unossified, metacarpals and/or metatarsals unossified, entire sternum unossified, and 14th full rib(s), (1000 mg/kg bw/day).

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Increased incidence of fetal variations and malformations and lower fetal weight at 500 and 1000 mg/kg/day.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Table - Incidence of Selected Histopathologic Findings,
                       Gestation Day 20 Scheduled Necropsy

 

Females

Dosage (mg/kg bw/day):

0

250

500

1000

 

 

 

 

 

Livera

25

25

25

25

  Hypertrophy, hepatocellular, centrilobular

0

25

24

23

      Minimal

-

1

9

0

      Mild

-

23

15

18

      Moderate

-

1

0

5

 

 

 

 

 

  Vacuolation, hepatocellular, periportal

14

21

24

22

      Minimal

6

15

12

5

      Mild

8

5

11

13

      Moderate

-

1

1

4

a - Number of tissues examined from each group.

Table - Mean Litter Proportions of Test Substance-Related External
Malformations (% per Litter)

Finding

0
mg/kg bw/day

250
mg/kg bw/day

500
mg/kg bw/day

1000 mg/kg bw/day

HC Mean (Range)

Fetal Anasarca

0.0

0.0

0.0

4.3 

0.0 (0.0-1.3)

Localized Fetal Edema

0.0

0.0

0.0

0.9

0.0 (0.0-0.9)

Tarsal Flexurea

0.0

0.0

0.0

0.6

0.0 (0.0-0.3)

Cleft Palate

0.0

0.0

0.0

0.3

0.0 (0.0-0.3)

a- Noted as Carpal and/or Tarsal Flexure on Tables
Values are bolded for findings considered to be test substance-related

Table - Mean Litter Proportions of Test Substance-Related Visceral
Malformations (% per Litter)

Finding

0
mg/kg bw/day

250
mg/kg bw/day

500
mg/kg bw/day

1000 mg/kg bw/day

HC Mean (Range)

Retroesophageal Aortic
    Arch

0.0

0.0

0.3

1.4

0.0 (0.0-0.3)

Coarctation of the Aortic
    Arch

0.0

0.0

0.0

1.4

Not observed

Trachea- Absent
    Cartilaginous Rings

0.0

0.0

0.0

1.3

Not observed

Right-Sided Aortic Arch

0.0

0.0

0.0

0.3

0.0 (0.0-0.2)

Values are bolded for findings considered to be test substance-related.

Table - Mean Litter Proportions of Test Substance-Related Visceral
Developmental Variations (% per Litter)

Finding

0
mg/kg bw/day

250
mg/kg bw/day

500
mg/kg bw/day

1000 mg/kg bw/day

HC Mean (Range)

Major Blood Vessel
   Variation

0.0

1.8

0.6

4.7

0.1 (0.0-0.8)

Thyroid Gland(s)- Small

0.0

0.3

0.5

0.8

Not observed

Spleen- Pale

0.0

0.0

0.6

5.6

0.0 (0.0-0.6)

Liver- Swollen

0.0

0.0

0.0

0.6

0.0 (0.0-0.3)

Liver- Pale

0.0

0.0

0.0

0.5

0.0 (0.0-0.3)

Values are bolded for findings considered to be test substance-related.

Table - Mean Litter Proportions of Test Substance-Related Skeletal
Malformations (% per Litter)

Finding

0
mg/kg bw/day

250
mg/kg bw/day

500
mg/kg bw/day

1000 mg/kg bw/day

HC Mean (Range)

Bent Scapula

0.0

0.0

3.7

17.8**

0.0 (0.0-0.3)

Bent Limb Bone(s)

0.0

0.0

0.0

6.1

0.0 (0.0-0.3)

Costal Cartilage Anomaly

0.0

0.2

0.8

8.3

0.0 (0.0-0.3)

Sternoschisis

0.0

0.3

0.0

0.8

0.0 (0.0-0.6)

Values are bolded for findings considered to be test substance-related.
** = Significantly different from the concurrent control group at p<0.01.

Table - Mean Litter Proportions of Test Substance-Related Skeletal
Variations (% per Litter)

Finding

0
mg/kg bw/day

250
mg/kg bw/day

500
mg/kg bw/day

1000 mg/kg bw/day

HC Mean (Range)

Cervical Centrum No. 1
   Ossified

19.1

0.3**

0.3**

0.0**

20.2 (6.6-35.8)

Bent Rib(s)

0.0

3.7

31.0**

56.9**

0.2 (0.0-2.1)

Sternebra(e) Nos. 5 and/or 6
   Unossified

11.0

10.4

26.7*

44.1**

6.4 (0.2-26.1)

14th Rudimentary Rib(s)

13.1

27.5

37.9**

44.7**

7.1 (0.0-18.9)

Pubis Unossified

0.0

2.4

8.1

13.0**

0.1 (0.0-2.3)

7th Cervical Rib(s)

2.3

3.5

8.4

21.2**

0.8 (0.0-3.7)

Reduced Ossification of
   the Skull

0.3

0.9

2.1

7.6

0.1 (0.0-1.0)

Reduced Ossification of
   the Vertebral Arches

0.3

0.9

2.1

17.1**

0.1 (0.0-1.3)

Sternebra(e) Nos. 1, 2, 3,
   and/or 4 Unossified

0.2

0.3

4.2

11.8**

0.2 (0.0-1.5)

Unco-Ossified Vertebral
   Centra

0.0

0.0

0.9

3.6

0.0 (0.0-0.5)

27 Presacral Vertebrae

0.2

4.1

6.1

14.0**

0.2 (0.0-1.8)

Vertebral Centra
   Unossified

0.0

0.0

0.0

2.7

0.0 (0.0-0.4)

Metacarpal(s) and/or
   Metatarsals Unossified

0.0

0.0

0.0

1.3

Not observed

Entire Sternum Unossified

0.0

0.0

0.0

4.6

0.0 (0.0-0.4)

Reduced Ossification of the
   Rib(s)

0.0

0.6

1.8

1.5

0.0 (0.0-1.2)

14th Full Rib(s)

0.0

1.5

0.3

1.4

0.1 (0.0-0.9)

Values are bolded for findings considered to be test substance-related.
* = Significantly different from the concurrent control group at p<0.05.

** = Significantly different from the concurrent control group at p<0.01.

Table - Results of additional statistical analysis: Number of dams (N), mean litter size, mean fetal weight (Analysis 1) and litter weight (Analysis 3) by DMSD dose. Relative differences to controls and p-values from linear regression on categories of dose.

Dose

/

mg/kg/day

N

Mean
Litter Size

Mean Fetal
Weight

 / g

Relative Difference
Mean Fetal Weight*

/ %

Relative Difference
Mean Fetal Weight*

P -Value

Litter Weight

/ g

Relative Difference
Litter Weight*

/ %

Relative Difference
Litter Weight*

P-Value

0

25

14.2

3.83

0.00

-

54.47

0.00

-

250

25

15.2

3.64

-4.91

.0037

55.38

1.67

.7029

500

25

14.3

3.44

-10.14

<0.0001

48.91

-10.21

.0218

1000

25

13.2

2.92

-23.82

<0.0001

38.84

-28.70

<0.0001

*reference: dose = 0 mg/kg/day

See attachments for result tables.

Applicant's summary and conclusion

Conclusions:
Oral administration of dimethylsilanediol to pregnant rats at 250, 500 or 1000 mg/kg/day resulted in a maternal No-Observed-Adverse-Effect-Level (NOAEL) of < 250 mg/kg/day based on hepatic findings (increased weight, hypertrophy and vacuolation) at all doses.

The fetal developmental NOAEL was considered to be 250 mg/kg/day based on lower fetal body weight and increased incidences of fetal malformations and variations at 500 or 1000 mg/kg/day.
Executive summary:

Administration of dimethylsilanediol to pregnant rats at doses of 250, 500 or 1000 mg/kg/day resulted in a maternal No-Observed-Adverse-Effect-Level (NOAEL) of less than 250 mg/kg/day based on higher mean liver weights and corresponding microscopic findings of hepatocellular hypertrophy and periportal hepatocellular vacuolation at all doses.

A higher mean litter proportion of post implantation loss, with a corresponding lower mean litter proportion of viable fetuses, was considered attributable to treatment and adverse in the 1000 mg/kg bw/day group.

Mean fetal body weights at the three doses were 5.3%, 10.5%, and 23.7% lower, respectively, than the concurrent control group and these differences were statitically significant at all doses when analysed by ANOVA or ANCOVA. However, these statistical methods assume litter size is unaffected by treatment. The mean fetal weight in the 250 mg/kg bw/day group was within the historical control data range and was only 0.2 g lower than the concurrent control group mean. Furthermore, litter size can be affected by treatment so additional statistical analysis of the fetal weight data using an appropriate statistical method specific for this type of data, which takes litter size into account (Morfeld, 2013), demonstrated that fetal weight at 250 mg/kg/day was comparable with control and not statistically significant and therefore unaffected by treatment at this dose.

Fetal malformations were noted in the 500 and 1000 mg/kg bw/day groups, but not in the 250 mg/kg bw/day group. Fetal variations were recorded in all dose groups with some visceral (small thyroid gland) and skeletal variations in the 250 mg/kg bw/day group showing a dose-response relationship. However, many of the fetal variations noted in the 250 mg/kg/day group were either recorded at an incidence that was not statistically significant and/or within the historical control incidence. Furthermore, as many, but not all, of these variations (skeletal developmental delays) correlate with fetal body weight, and fetal body weight was not affected by treatment, then the variations noted at the low dose of 250 mg/kg/day are also considered not to be attributable to treatment.

Therefore, the NOAEL for prenatal developmental toxicity was considered to be 250 mg/kg/day.