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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:
January to March 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Dodecamethylcyclohexasiloxane
EC Number:
208-762-8
EC Name:
Dodecamethylcyclohexasiloxane
Cas Number:
540-97-6
Molecular formula:
C12H36O6Si6
IUPAC Name:
dodecamethylcyclohexasiloxane
Test material form:
other: liquid

Test animals

Species:
rat
Strain:
other: RccHan:WIST
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B.V., The Netherlands
- Age at study initiation: approximately 10 to 14 weeks
- Weight at study initiation: not stated
- Fasting period before study: no
- Housing: individually in Macrolon plastic cages (MIII type)
- Diet (ad libitum): SM R/M-Z (SSNIFF Spezialdiaten GmbH, Germany)
- Water (ad libitum): tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24
- Humidity (%): 40 to 70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 16 February 2015 To: 12 March 2015

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: dosed undiluted

Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
No chemical analysis was performed because the test substance was dosed undiluted.(i.e. without the use of a vehicle).
Details on mating procedure:
- Impregnation procedure: mated at supplier
- M/F ratio per cage: not stated
- Length of cohabitation: not stated
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
Duration of treatment / exposure:
Day 6 to Day 20 post-cotum, inclusive
Frequency of treatment:
Daily
Duration of test:
Day 0 to Day 21 post-coitum
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
Group 1
Dose / conc.:
100 mg/kg bw/day
Remarks:
Group 2
Dose / conc.:
330 mg/kg bw/day
Remarks:
Group 3
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
Group 4
No. of animals per sex per dose:
22 Females
Control animals:
other: yes, water
Details on study design:
- Dose selection rationale: Dose levels were selected based on results of the dose range finding study (Project 507522) in which no remarkable findings were noted at doses of 0, 100, 330 or 1000 mg/kg/day.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: Days 1, 3, 6, 9, 12, 15, 18 and 21 post-coitum

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Days 1, 3, 6, 9, 12, 15, 18 and 21 post-coitum
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: macroscopic examination, liver weighed and retained
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: In case implantations were not macroscopically visible, the uterus was stained using the Salewski technique
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test based on a pooled variance estimate was applied for the comparison of the treated groups and the control group. The mean of fetal weights per litter and mean of total litter weight was also analyzed using this method.
- The Steel-test was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Mann Whitney test was used to compare mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and sex distribution.
- Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.

All tests were two-sided and in all cases p<0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances.

No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.
Indices:
Pre-implantation loss (%) = ((number of corpora lutea - number of implantation sites) / number of corpora lutea) x 100

Post-implantation loss (%) = ((number of implantation sites - number of live fetuses) / number of implantation sites) x 100

The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison, calculating the number of affected fetuses as a mean litter proportion on a total group basis, where:

Viable fetuses affected/litter (%) = (number of viable fetuses affected per litter / number of viable fetuses per litter) x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
At 1000 mg/kg/day bw, maternal animals had higher absolute and relative liver weights. The difference from controls was slight but statistically significant. There were no test item-related microscopic findings observed during the histopathological evaluation therefore, the increases in liver weights were not considered toxicologically relevant.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
not specified
Pre- and post-implantation loss:
not specified
Total litter losses by resorption:
not specified
Early or late resorptions:
not specified
Dead fetuses:
not specified
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
not specified
Other effects:
not examined

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
not specified
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not specified
External malformations:
no effects observed
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Based on the initial description of findings (severely malaligned sternebrae, fused sternebrae, vertebral centra anomaly and bent limb bones), an external peer review was performed which concluded that there were no toxicologically relevant effects on skeletal morphology following treatment up to 1000 mg/kg bw/day.

Skeletal malformations only occurred in two fetuses only. At 1000 mg/kg bw/day, 1 fetus had a short rib and since this occurred once only and was seen previously in historical control fetuses, it was considered to not be test substance related. The other malformation was noted in a control fetus with polydactyly (not detected externally) and as such was not considered related to treatment.

Skeletal variations occurred for 33.5%, 31.6%, 38.7% and 50.4% of the fetuses per litter in the control, 100, 330 and 1000 mg/kg bw/day groups, respectively.
Animals at 1000 mg/kg bw/day had a significantly higher percent of combined skeletal variations per litter than controls. None of the individual findings was significantly higher for animals at 1000 mg/kg bw/day though findings like (slight or moderate) malaligned sternebra(e), 14th rudimentary rib(s), caudal shift of the pelvic girdle, unossified metacarpal(s) and/or metatarsal(s), and sternebra(e) #1, 2, 3, 4, 5 and/or #6 unossified were seen at slightly higher incidences than controls. Taken together, these culminated in a statistically significant increase in skeletal variations overall, but was not regarded toxicologically relevant.

Fetuses at 1000 mg/kg bw/day (and at 330 mg/kg bw/day) had a higher incidence of slightly to moderately malaligned sternebra(e) (12.5% and 10.7%, respectively versus control value 8.4% per litter). Besides, three fetuses at the high dose level showed severe malaligned sternebra(e) (0.0% for controls versus 1.2% at 1000 mg/kg bw/d). However, the difference of both severities of malaligned sternebrae from controls was not statistically significant and only slightly higher than controls. Moreover, values of slightly to moderately malaligned sternebra(e) remained far below the historical control upper limit (21.3% per litter). As such, this was not considered to be adverse.

There were slightly higher incidences of unossified metacarpal(s) and metatarsal(s) seen for fetuses at 330 and 1000 mg/kg bw/day (6.4% and 8.3% per litter, respectively, versus control value 2.7% per litter). These were not considered to be toxicologically relevant as there were no other indications of developmental delay seen like decreases in other related ossification parameters and fetal body weights remained in the same range as controls.
Visceral malformations:
no effects observed
Other effects:
no effects observed

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed.

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

Table 1 - Summary of Maternal Survival and Pregnancy Status

Dose (mg/kg/day)

0

100

330

1000

 

Number (%)

Number (%)

Number (%)

Number (%)

Females on study

22

22

22

22

Females that aborted or delivered

0 (0.0)

0 (0.0)

0 (0.0)

0 (0.0)

Females that died
females that aborted
- nongravid
- gravid

0 (0.0)
0 (0.0)
0 (0.0)
0 (0.0)

0 (0.0)
0 (0.0)
0 (0.0)
0 (0.0)

0 (0.0)
0 (0.0)
0 (0.0)
0 (0.0)

0 (0.0)
0 (0.0)
0 (0.0)
0 (0.0)

Females that were euthanized
- nongravid

- gravid

0 (0.0)

0 (0.0)
0 (0.0)

0 (0.0)

0 (0.0)
0 (0.0)

0 (0.0)

0 (0.0)
0 (0.0)

0 (0.0)

0 (0.0)
0 (0.0)

Females examined at scheduled - necropsy
- nongravid
- gravid
- with resorptions only
- with viable fetuses

22 (100.0)

0 (0.0)
22 (100.0)
1 (4.5)
21 (95.5)

22 (100.0)

0 (0.0)
22 (100.0)
0 (0.0)
22 (100.0)

22 (100.0)

0 (0.0)
22 (100.0)
0 (0.0)
22 (100.0)

22 (100.0)

0 (0.0)
22 (100.0)
1 (4.5)
21 (95.5)

Total females gravid

22 (100.0)

22 (100.0)

22 (100.0)

22 (100.0)

Table 2 - Summary of Fetal Data at Scheduled Necropsy

Dose
(mg/kg/day)

 

Sex

Viable
fetuses

Dead fetuses

Resorptions

Post implantation loss

Implantation sites

Corpora lutea

Pre implantation loss

Fetal weight
 (g)

No. of gravid females

M

F

early

late


0

Total
mean
S.D.

122
5.5
2.22

107
4.9
2.08

229
10.4
3.36

0
0.0
0.00

16
0.7
0.77

0
0.0
0.00

16
0.7
0.77

245
11.1
3.20

268
12.2
1.99

23
1.0
1.59

NA
5.3
0.25


22


100

Total
mean
S.D.

114
5.2
2.46

124
5.6
1.84

238
10.8
2.86

0
0.0
0.00

10
0.5
0.86

0
0.0
0.00

10
0.5
0.86

248
11.3
2.73

271
12.3
2.46

23
1.0
1.33

NA
5.3
0.29


22


330

Total
mean
S.D.

120
5.5
1.84

123
5.6
1.71

243
11.0
2.26

0
0.0
0.00

15
0.7
0.99

0
0.0
0.00

15
0.7
0.99

258
11.7
2.05

275
12.5
1.63

17
0.8
1.31

NA
5.3
0.35


22


1000

Total
mean
S.D.

125
5.7
2.97

117
5.3
2.08

242
11.0
3.59

0
0.0
0.00

13
0.6
0.80

0
0.0
0.00

13
0.6
0.80

255
11.6
3.11

279
12.7
2.63

24
1.1
1.87

NA
5.3
0.41


22

Table 3 - Summary of Fetuses amd Litters Data

 

Fetuses

Litters

Dose (mg/kg/day)

0

100

330

1000

0

100

330

1000

Number examined externally
Number with findings

229
0

238
0

243
0

242
0

21
0

22
0

22
0

21
0

Number examined viscerally
Number with findings

113
0

119
0

120
0

121
0

21
0

22
0

22
0

21
0

Number examined skeletally
- polydactyly

- rib anomaly
- sternebra, malaligned (slight/moderate)
- sternebra, malaligned (severe)

- ossification , reduced skull

- vertebral centra, unossified

- bent limb bones

229

1
0

18

0

0

0

0

238

0
0

14

1

1

0

0

243

0
0

27

0

1

0

1

242

0
1

30

3

0

1

1

21

1
0

12

0

0

0

0

22

0
0

11

1

1

0

0

22

0
0

15

0

1

0

1

21

0
1
14

3

0

1

1

Total number with malformations
- external
- soft tissue
- skeletal


0
0
1


0
0
0


0
0
0


0
0
1


0
0
1


0
0
0


0
0
0


0
0
1

Applicant's summary and conclusion

Conclusions:
In the prenatal developmental toxicity study with dodecamethylcyclohexasiloxane (D6), conducted according to OECD Test Guideline 414 and in compliance with GLP, no maternal or developmental toxicity was observed in rats at doses up to 1000 mg/kg bw/day. The maternal and developmental NOAEL for D6 were concluded to be at least 1000 mg/kg bw/day based on no adverse effects.
Executive summary:

A prenatal developmental toxicity study (OECD 414) of Dodecamethylcyclohexasiloxane (D6) was carried out in rats by oral gavage. Eighty-eight mated female Wistar Han rats were assigned to four dose groups. The test item was administered undiluted once daily by oral gavage from Days 6 to 20 post-coitum at doses of 0, 100, 330 and 1000 mg/kg bw/day (Groups 2, 3 and 4 respectively). The rats of the control group received water at the same dose volume as Group 4 animals. Females were checked daily for the presence of clinical signs. Food consumption and body weight were determined at periodic intervals.

On Day 21 post-coitum, all animals were subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. Terminal body and liver weights were recorded for each dam and body weights gains were calculated. A laparohysterectomy was performed on each dam of all groups. The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and corrected body weights (changes) were calculated. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations. All live fetuses were euthanized. One half of the fetuses were decapitated and the heads were fixed in Bouin’s fixative, these fetuses were dissected and examined for visceral anomalies. All fetuses were fixed in 96% aqueous ethanol and stained with Alizarin Red S and had skeletal examinations performed. Histopathology was performed on the livers from all dams of Groups 1 and 4.

RESULTS

At 1000 mg/kg bw/day, maternal animals had higher absolute and relative liver weights. The difference from controls was slight but statistically significant. There were no test item-related microscopic findings observed during the histopathological evaluation, therefore, the increases in liver weights were not considered toxicologically relevant.

No maternal toxicity was observed in the other parameters examined in this study with treatment up to 1000 mg/kg bw/day (mortality, clinical signs, body weights, food consumption and macroscopic examination). No developmental toxicity was observed up to 1000 mg/kg bw/day groups.

CONCLUSION

The maternal and developmental NOAEL for D6 were established as being at least 1000 mg/kg bw/day.

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