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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:
1st mating results: April 28, 2009; 1st administration: May 04, 2009; Termination of in-life phase: June 01, 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

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

Test material

Constituent 1
Chemical structure
Reference substance name:
Dioctyl ether
EC Number:
211-112-6
EC Name:
Dioctyl ether
Cas Number:
629-82-3
Molecular formula:
C16H34O
IUPAC Name:
1-(octyloxy)octane
Test material form:
liquid
Details on test material:
- State of aggregation: Colorless, clear liquid
Specific details on test material used for the study:
- Name of test material (as cited in study report): C-SAT 080029
- Physical state: colourless liquid
- Composition of test material, percentage of components: dioctylether: 99.1%
- Purity test date: February 27, 2008
- Lot/batch No.: CE72530027
- Expiration date of the lot/batch: September 09, 2009
- Storage condition of test material: at room temperature

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH, 97633 Sulzfeld, Germany
- Age at study initiation: 8 - 9 weeks (on day 0 of pregnancy)
- Weight at study initiation: 190 - 266 g (on day 0 of pregnancy)
- Fasting period before study: no
- Housing: singly
- Diet (e.g. ad libitum): conventional laboratory diet ad libitum
- Water (e.g. ad libitum): drinking water ad libitum
- Acclimation period: 6 adaptation days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 (maximum range)
- Humidity (%): 55 +/- 15 (maximum range)
- Air changes (per hr): 12 -18 times
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light


IN-LIFE DATES: From: April 28, 2009 To: June 01, 2009

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: sunflower oil
Details on exposure:
The test item was suspended in the vehicle (sunflower oil) to the appropriate concentrations and was administered orally at a constant volume of 5 mL/kg b.w. once daily from the 6th to the 19th day of pregnancy. The test item-vehicle mixtures were freshly prepared every day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The determination of the concentration of C-SAT 080029 in the vehicle sunflower oil was performed using a gas chromatography (GC) method with FID detection. The concentration of C-SAT 080029 in the mixture was quantified using a calibration curve calculated from defined peak areas of the test item. The analytical method used was developed and validated by LPT for linearity of the calibration curve, accuracy, precision, stability, specificity and sensitivity. The measured concentrations ranged from 107.3% to 108.4%. The results were well within the admissible limits of 90% to 110% of the normal concentration.
Details on mating procedure:
Sexually mature male rats of the same breed served as partners. The male and female breeding partners were randomly chosen. Mating was monogamous: 1 male and 1 female animal were placed together in one cage during the dark period. Each morning a vaginal smear was taken to check for the presence of sperm and the stage of oestrus cycle. If findings were negative, mating was repeated with the same partner. The day on which sperm was found was considered as the day of conception (day 0 of pregnancy). This procedure was repeated until enough pregnant dams were available for all groups. Rats which did not become pregnant were excluded from analysis of the results and replaced by other animals. A post-mortem negative staining according to SALEWSKI was carried out in the replaced animals in order to confirm the non-pregnancy status.
Duration of treatment / exposure:
from day 6 to 19 of pregnancy
Frequency of treatment:
once daily
Duration of test:
4 months (April 28, 2009 to August 31, 2009)
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
25 pregnant rats per dose (to obtain 20 litters per dose)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: in agreement with the Sponsor based on available toxicological data
- Rationale for animal assignment (if not random): The rat is a commonly used rodent species for embryotoxicity studies.
- Other: no

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
Clinical signs
- Time schedule: regularly throughout the working day from 7.00 a.m. to 3.45 p.m., on Saturdays and Sundays, starting from 7.00 a.m. to 11.00 a.m. with a final check at approx. 3.30 p.m.
- Cage side observations checked : behaviour, external appearance and nature of the faeces
Viability: early in each working day and again in the afternoon to look for dead or moribund animals
Body weight: on day 0 of gestation followed by daily weighings - always at the same time of the day. The body weight was also calculated in intervals (i.e. day 0-3, 3-6, 6-9, 9-12, 12-15, 15-18 and 18-20). Furthermore the net weight change from day 6 was given.
Food consumption: recorded daily with the exception of gestation day 20
Drinking water consumption: daily monitoring by visual appraisal of the drinking water bottles

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily


BODY WEIGHT: Yes
- Time schedule for examinations: on day 0 of gestation followed by daily weighings - always at the same time of the day. The body weight gain was also calculated in intervals (i.e. day 0-3,3-6, 6-9, 9-12, 12-15, 15-18 and 18-20). Furthermore the net weight change from day 6 was given.


FOOD CONSUMPTION : Yes
- 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: No


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data
- Time schedule for examinations: daily monitoring by visual appraisal of the drinking water bottles


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20 of gestation
- Organs examined: internal organs and placentae


OTHER:
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: location of fetuses in the uterus
Fetal examinations:
- External examinations: Yes: [ all per litter ] : for damages, especially for malformations
- Soft tissue examinations: Yes: [ half per litter ] : for soft tissue anomalies. Body sections were made and examined according to WILSON.
- Skeletal examinations: Yes: [half per litter ]: for skeletal anomalies. The thorax and peritoneal cavity (without damage to ribs and sternum) were opened and the location, size and condition of the internal organs were determined. Then the skeleton was double-stained with Alcian blue for the examination of cartilage and with Alizarin red to reveal ossifications (according to DAWSON). The skeletal system was examined (determination of the number and type of retardations, variations as well as malformations).
- Head examinations: Yes: [ half per litter ] included in soft tissue examination of body sections
Statistics:
For all numerical values, homogeneity of variances was tested using the BARTLETT chi-square test. When the variances were homogeneous, the DUNNETT test (p <= 0.01) was used to compare the experimental groups out, limit of significance was p <= 0.01.
For comparison of classification measurements (for example malformation-, resorption-, retardation- and variation rate) the FISHER's exact test (n < 100) or chi-square test with YATES' correction for continuity (n >= 100) (p <= 0.05 and p <= 0.01) was employed.
Indices:
Corpora lutea: number per dam, absolute number per group, mean per group
Implantations: number per dam, distribution in the uterine horns, absolute number per group, mean per group
Reorptions: number per dam, % per litter; distribution in the uterine horns, absolute number per group, mean per group, mean % per group, early resorptions < 2 mm, number and % per litter; late resorptions > 2 mm, number and % per litter; % litters with resorptions per group
Resorptions rate [%]: resorptions / implantations x 100
Weight of placentae: individual data per fetus, mean per litter, mean per group, litter mean per sex and group
Weight of fetuses: individual data per fetus, mean per litter, mean per sex and litter, litter mean per group, litter mean per sex and group
Fetuses: number and % per dam (alive and dead), number of fetuses per sex and dam, sex ratio per litter, distribution in the uterine horns, absolute number of fetuses alive per group, mean number of fetuses alive per group, mean % of fetuses alive per group, mean % per sex and group
Dead fetuses: number per dam, mean per group
Runts: number per dam, mean per group
Malformed fetuses: individual data per fetus, mean per group and type of malformation
Malformation rate [%]: malformed fetuses / fetuses x 100
Fetuses with variations: individual data per fetus grouped according to litter and dose, number and % offspring with external, visceral or skeletal variations per litter; mean % per group and type of variation
Variation rate [%]: fetuses with variations / fetuses x 100
Fetuses with retardations: individual data per fetus, mean per goup and type of retardation
Retardation rate [%]: fetuses with retardations / fetuses x 100
Pre-implantation loss [%]: corpora lutea - implantations / corpora lutea x 100
Post-implantation loss [%]: implantations - living fetuses /implantations x 100
Historical control data:
Summarized results of 45 last embryotoxicity studies in Sprague-Dawley rats performed at LPT in the years 2000 to June 2009 (1. General reproductive incidences, 2. Skeletal retardations, 3. Variations a) skeletal, b) visceral, 4. Malformations)

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
No test item-related changes in behaviour or external appearance were noted in the dams treated with 100, 300 or 1000 mg/kg bw/day of the test substance.
The faeces of all animals were of normal consistency throughout the experimental period.
Reddened anus and thin hair were noted in 3 of 20 dams treated with 1000 mg/kg bw/day of the test substance from gestation day 16 or 18 onwards until laparotomy on gestation day 20. However, these changes are considered to be within the normal variability and not test item-related.
Mortality:
no mortality observed
Description (incidence):
None of the dams died prematurely.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
At 100, 300 and 1000 mg/kg bw/day, no test item-related influence was noted on the body weight. The body weight gain was similiar to that of the control at all tested dose levels. The increase in the mean body weight from the start value (day 0 of pregnancy) was plus 58.8 %, 59.2 % or 61.1 %, respectively, at the time point of laparotomy (control: plus 57.7 %).
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No test item-related influence was noted on absolute and relative food consumption compared to the control at 100, 300 or 1000 mg/kg bw/day. The marginal changes observed are within the normal variability of food consumption for rats.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Drinking water consumption showed no test item-related changes at any tested dose level as observed during daily visual appraisal.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The gravid uterus weight, the carcass weight and the net weight change from day 6 onwards (carcass weight minus day 6 body weight) of the low-, intermediate- and high-dosed dams (100, 300 or 1000 mg/kd bw/day) were not influenced by the exposure to the test item.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Necropsy on gestation day 20 revealed no test item-related pathological changes in any of the dams treated with either 100, 300 or 1000 mg/kg bw/day of the test substance.
Necropsy revealed bilateral dilatation of renal pelvis (diameter approx. 5 mm) in the intermediate-dosed dam no. 53 treated with 300 mg/kg bw/day. This finding was considered as incidental.

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
No test item-related influence on the prenatal fetal development was detected at either 100, 300 or 1000 mg/kg bw/day, with respect to the number of corpora lutea, implantation sites, resorptions and live fetuses or the values calculated for the pre- and post-implantation losses.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No test item-related influence on the prenatal fetal development was detected at either 100, 300 or 1000 mg/kg bw/day, with respect to the number of corpora lutea, implantation sites, resorptions and live fetuses or the values calculated for the pre- and post-implantation losses.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
No test item-related influence on the prenatal fetal development was detected at either 100, 300 or 1000 mg/kg bw/day, with respect to the number of corpora lutea, implantation sites, resorptions and live fetuses or the values calculated for the pre- and post-implantation losses.
Early or late resorptions:
no effects observed
Description (incidence and severity):
No test item-related influence on the prenatal fetal development was detected at either 100, 300 or 1000 mg/kg bw/day, with respect to the number of corpora lutea, implantation sites, resorptions and live fetuses or the values calculated for the pre- and post-implantation losses.
Dead fetuses:
no effects observed
Description (incidence and severity):
No test item-related influence on the prenatal fetal development was detected at either 100, 300 or 1000 mg/kg bw/day, with respect to the number of corpora lutea, implantation sites, resorptions and live fetuses or the values calculated for the pre- and post-implantation losses.
Details on maternal toxic effects:
Maternal toxic effects:no effects

Effect levels (maternal animals)

Dose descriptor:
NOEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Basis for effect level:
other: maternal toxicity

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
The mean fetal weights were not influenced by the administration of 100, 300 or 1000 mg/kg bw/day of the test substance as compared to the control group.
One runt was noted in the litter of the low-dosed dam no. 35 (treated with 100 mg/kg bw/day of the test substance). This finding is regarded to be spontaneous and is within the normal range of variation.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex distribution of the fetuses in test groups 2, 3 and 4 (100, 300 or 1000 mg/kg bw/day of the test substance) was comparable to the control fetuses. The slight differences observed in comparison to the control are without any biological relevance.
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
Laparotomy revealed no dead fetuses at any tested dose level.
A pair of female twins was noted in control dam no. 20. This finding is regarded to be spontaneous.
External malformations:
no effects observed
Description (incidence and severity):
No macroscopically visible malformations or variations were noted at either 100, 300 or 1000 mg/kg bw/day during external examination at laparotomy of the fetuses.
Skeletal malformations:
no effects observed
Description (incidence and severity):
Skeletal examination according to DAWSON revealed no malformations at 100, 300 or 1000 mg/kg bw/day.
No test item-related influence was noted for the incidence of skeletal variations at any tested dose level (100, 300 or 1000 mg/kg bw/day).
No test item-related influence was noted for the incidence of skeletal retardations at 100, 300 or 1000 mg/kg bw/day.
Visceral malformations:
no effects observed
Description (incidence and severity):
The examination of the fetal organs (according to WILSON) revealed no malformations at 100, 300 or 1000 mg/kg bw/day.
No test item-related influence was noted in the incidence of soft tissue variations at 100, 300 or 1000 mg/kg bw/day.
Other effects:
no effects observed
Description (incidence and severity):
The dissection and internal examination of the fetuses revealed no test item-related findings for any of the tested dose levels or for the control group.
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Basis for effect level:
other: teratogenicity
Dose descriptor:
NOEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Basis for effect level:
other: embryotoxicity
Dose descriptor:
NOEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Basis for effect level:
other: fetotoxicity

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

Table 2: Summary of animals examined

 

Group 1

Group 2

Group 3

Group 4

Treated dams

25

25

25

25

Non-pregnant dams

0

3

2

1

Dams without viable fetuses

0

0

0

0

Not examined dams

5

2

3

4

Evaluated litters

20

20

20

20

Table 3: Relevant reproduction data

Parameter

Group 1

Group 2

Group 3

Group 4

Corpora lutea

 

 

Implantation sites

 

Total

Per dam

 

Total

Per dam

264

13.2

 

254

12.7

259

13.0

 

249

125

274

13.7

 

250*

12.5

280

14.0

 

273

13.7

Resorptions

 

Total

Per dam

2

0.1

1

0.1

4

0.2

5

0.3

Early resorptions

 

 

Late resorptions

 

Total

Per dam

 

Total

Per dam

2

0.1

 

0

0.0

0

0.0

 

1

0.1

1

0.1

 

3

0.2

4

0.2

 

1.

0.1

Live fetuses

 

Total

Per dam

253

12.7

248

12.4

246

12.3

268

13.4

Dead fetuses at laparotomy

total

0

0

0

0

Pre-implantation loss

Mean %

3.3

6.2

9.8

2.4

Post-implantation loss

Mean %

0.7

0.4

1.6

1.7

*significantly different from the controls at p≤0.05

**significantly different from the controls at p≤0.01

Table 4: External / Internal Malformations

Parameter

Group 1

Group 2

Group 3

Group 4

Fetal incidence

 

N

%

0

0.0

0

0.0

0

0.0

0

0.0

Litter incidence

 

N

%

0

0.0

0

0.0

0

0.0

0

0.0

Table 5: Skeletal Malformations

Parameter

Group 1

Group 2

Group 3

Group 4

Fetal incidence

 

N

%

0

0.0

0

0.0

0

0.0

0

0.0

Litter incidence

 

N

%

0

0.0

0

0.0

0

0.0

0

0.0

Table 6: Soft Tissue Malformations

Parameter

Group 1

Group 2

Group 3

Group 4

Fetal incidence

 

N

%

0

0.0

0

0.0

0

0.0

0

0.0

Litter incidence

 

N

%

0

0.0

0

0.0

0

0.0

0

0.0

Table 7: External / Internal Variations

Parameter

Group 1

Group 2

Group 3

Group 4

Fetal incidence

 

N

%

0

0.0

0

0.0

0

0.0

0

0.0

Litter incidence

 

N

%

0

0.0

0

0.0

0

0.0

0

0.0

Table 8: Skeletal Variations

Parameter

Group 1

Group 2

Group 3

Group 4

Fetal incidence

 

N

%

3

2.4

5

4.0

2

1.6

1

0.7

Litter incidence

 

N

%

2

10.0

4

20.0

2

10.0

1

5.0

Table 9: Soft Tissue Variations

Parameter

Group 1

Group 2

Group 3

Group 4

Fetal incidence

 

N

%

24

19.0

17

13.7

14

11.4

26

19.4

Litter incidence

 

N

%

14

70.0

11

55.0

10

50.0

14

70.0

Table 10: Skeletal Retardations

Parameter

Group 1

Group 2

Group 3

Group 4

Fetal incidence

 

N

%

127

100.0

122

98.4

122

99.2

132

98.5

Litter incidence

 

N

%

20

100.0

19

95.0

20

100.0

20

100.0

Table 11: Summary of maternal clinical signs

 

Group 1

Group 2

Group 3

Group 4

No remarkable observations

 

N

%

20

100.0

20

100.0

20

100.0

17

85.0

Reddened anus, thin hair

 

N

%

0

0.0

0

0.0

0

0.0

3

15.0

Table 12: Mean maternal body weights during gestation [g]

 

Group 1

Group 2

Group 3

Group 4

Day 0

Mean

SD

N

221.0

14.7

20

219.7

16.5

20

223.7

15.2

20

218.2

12.4

20

Day 3

Mean

SD

N

238.6

19.5

20

238.2

18.2

20

241.4

15.8

20

235.4

13.8

20

Day 6

Mean

SD

N

251.0

18.8

20

250.3

16.8

20

255.2

15.2

20

248.9

13.9

20

Day 9

Mean

SD

N

259.3

16.9

20

256.9

20.2

20

264.9

16.6

20

254.7

13.3

20

Day 12

Mean

SD

N

272.0

17.3

20

272.4

17.6

20

279.2

16.7

20

268.7

14.0

20

Day 15

Mean

SD

N

286.4

18.3

20

288.2

19.3

20

294.8

19.1

20

285.5

13.0

20

Day 18

Mean

SD

N

320.8

23.3

20

322.8

26.1

20

328.8

23.8

20

321.4

17.1

20

Day 20

Mean

SD

N

348.5

29.4

20

348.8

33.1

20

356.2

29.4

20

351.6

19.6

20

Table 13: Summary of Reproduction Data

 

Group 1

Group 2

Group 3

Group 4

Females pregnant

-         Aborted

-         Premature birth

-         Dams with viable fetuses

-         Dams with all resorptions

N

N

N

N

N

20

0

0

20

0

20

0

0

20

0

20

0

0

20

0

20

0

0

20

0

Female mortality

 

N

%

0

0

0

0

0

0

0

0

Pregnant at C-section

 

N

%

20

100

20

100

20

100

20

100

Corpora lutea

 

 

Mean

SD

Total

13.2

3.7

264

13.0

3.9

259

13.7

2.7

274

14.0

1.8

280

Implantation sites

 

 

Mean

SD

Total

12.7

3.5

254

12.5

4.2

249

12.5

3.6

250*

13.7

1.7

273

Pre-implantation loss

 

Mean %

SD

3.3

5.7

6.2

16.7

9.8

15.0

2.4

3.4

Post-implantation loss

 

Mean %

SD

0.7

2.1

0.4

1.9

1.6

3.3

1.7

6.1

Pregnant at C-section

N

20

20

20

20

Resorption

-         Total

 

 

 

 

 

-         Early

 

 

 

 

 

-         Late

 

 

 

 

 

Mean

SD

Total

 

Mean %

SD

Mean

SD

Total

 

Mean %

SD

Mean

SD

Total

 

Mean %

SD

 

0.1

0.3

2

 

0.7

2.1

0.1

0.3

2

 

0.7

2.1

0.0

0.0

0

 

0.0

0.0

 

0.1

0.2

1

 

0.4

1.9

0.0

0.0

0

 

0.0

0.0

0.1

0.2

1

 

0.4

1.9

 

0.2

0.4

4

 

1.6

3.3

0.1

0.2

1

 

0.4

1.9

0.2

0.4

3

 

1.2

2.9

 

0.3

0.9

5

 

1.7

6.1

0.2

0.7

4

 

1.3

4.6

0.1

0.2

1

 

0.3

1.5

Dead fetuses

N

0

0

0

0

*significantly different from the controls at p≤0.05

**significantly different from the controls at p≤0.01

Applicant's summary and conclusion

Conclusions:
Not teratogenic or embryotoxic up to 1000 mg/kg bw/day.
Executive summary:

In this prenatal developmental toxicity study, the test substance was administered to female rats at dose levels of 100, 300 and 1000 mg/kg bw orally, by gavage from the 6th to 19th day of pregnancy. Under the present test conditions, the no-observed-effect level (NOEL) was above 1000 mg/kg bw for the dams. The NOEL for the fetuses was also above 1000 mg/kg bw. No test item-related malformations or variations were noted during external/internal examination of the fetuses or soft tissue examination (according to Wilson); skeletal examination (according to Dawson) revealed no test item-related malformations, variations or retardations.

In conclusion, the test substance possessed no teratogenic properties. No test item-related increase was noted in the incidence of malformations, variations and retardation tested until the dose of 1000 mg/kg bw.