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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 May 2014 to 25 November 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

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
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
Shale oils, heavy
EC Number:
930-690-7
Molecular formula:
Not applicable. A generic molecular formula cannot be assigned to this UVCB substance.
IUPAC Name:
Shale oils, heavy
Test material form:
not specified
Details on test material:
- Storage condition of test material: At room temperature (20 ± 5 °C) and in the dark

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 11 weeks
- Weight at study initiation: 189 - 289 g (day 0 post coitum)
- Housing: Animals were housed in cages with standard, granulated, softwood Lignocel S8/15 bedding. During acclimation females were housed up to 5 in a cage measuring 59 x 38.5 x 20 cm. During pregnancy females were housed individually in cages measuring 42.5 x 26.6 x 18 cm.
- Diet: ad libitum
- Water: tap water in bottles, ad libitum
- Acclimation period: at least 5 days prior to mating

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 24 °C
- Humidity: 40 - 70 % (relative)
- Air changes: 15-20 air changes per hour
- Photoperiod: 12 hours of darkness / 12 hours of light (07:00 to 19:00)

IN-LIFE DATES: From: 07 May 2014 To: 02 June 2014

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The necessary amount of test material was weighed in a volumetric flask. Vehicle was added to reach the flask volume and under stirring for at least 30 minutes (using a propeller stirrer). The formulation then was transferred to a suitable container. When there was vehicle left, it was used to wash away any formulation left on the walls of the volumetric flask and reach the final volume.
No correction factor for purity was applied.
For each day of administration and dose level, the necessary volume was taken from the respective stock solution under stirring and transferred to a suitable container. The aliquots were stored at 5 ± 3 °C and protected from light. Daily administration was performed under stirring (using a propeller stirrer).

DOSE VOLUME: 4 mL/kg
The amount of test material and administration volume for each animal was determined daily based on its body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of dose formulations administered was determined twice during the study and once (sampled from top/middle/bottom) for homogeneity. The samples analysed were taken from the 50 to 250 mg/kg dose groups and analysed by GC-MS analysis and data processing by Chemstation version D.02.00.275.
On each occasion, duplicate samples of the dosing solution were transferred to labelled vials. One aliquot was used to analyse the concentration and the remaining aliquot was retained for any possible subsequent needs until the study was completed. The vials were maintained at room temperature (20 ± 5 °C) and protected from light.
The formulations analysed during the study were found to contain test material in the range of 86 to 105 %; thus the required content limit of ± 20 % with reference to the nominal content was met. The test material was found to be stable in the formulation for 31 days in the refrigerator (4 °C).
Details on mating procedure:
After acclimatisation, females were housed with sexually mature males in a cage for a period of approximately 16 hours. At least 23 different males participated in each dose group. Vaginal smears were taken daily until mating was confirmed. The females were removed and housed individually when the vaginal smear was sperm positive, or when a copulation plug was observed. The day of mating was designated day 0 post coitum.
Duration of treatment / exposure:
Day 6 to Day 19 post coitum
Frequency of treatment:
Once daily during treatment period
Duration of test:
Females were sacrificed on day 20 post coitum
No. of animals per sex per dose:
23 females received the control dose and 33, 26 and 24 females were dosed at 50, 100 and 250 mg/kg bw/day, respectively.
Animal allocation is outlined in Table 1.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based upon a previous range finder toxicity study in Wistar rats.
- Rationale for animal assignment: Rats were assigned to the different groups using a randomisation procedure.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were observed for morbidity/mortality twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Daily

FOOD CONSUMPTION: Yes
- Time schedule: days 0-3, 3-6, 6-9, 9-12, 12-15, 15-18 and 18-20 post coitum.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20 by CO₂ inhalation.
Postmortem examination included gross macroscopic examination of all internal organs with emphasis on the uterus, uterine contents, position of foetuses in the uterus and the number of corpora lutea. The uterus (and contents) of all females were weighed during necropsy on day 20 post coitum to enable the calculation of the corrected bodyweight gain.
Specimens of any abnormal tissues were fixed in neutral phosphate buffered 4 % formaldehyde solution (10 % formalin) for possible microscopic examination.
When no implantation sites were evident, the uterus was placed in an aqueous solution of ammonium sulphide (Salewski, 1964) to accentuate possible haemorrhagic areas of implantation sites.
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
Fetal examinations:
FOETAL EXAMINATIONS
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes: [half per litter]

FOETAL PATHOLOGY
Foetuses were removed from the uterus by caesarean section at maternal necropsy, sexed, weighed individually, examined for gross external abnormalities Markis, 2009), sacrificed by intraperitoneal injection of sodium pentobarbital and allocated to one of the following procedures:
1. One-half of the foetuses from each litter was fixed in Bouin's fixative Wilson, 1965). Foetuses were serially sectioned and examined (evaluation of the internal structures of the heads, thoracic and abdominal organs). The tissue was preserved in a solution of 96 % ethyl alcohol (one foetus per container). Descriptions of any abnormalities and variations were recorded.
2. The remaining foetuses were eviscerated and fixed in 70 % alcohol. Then they were placed in a solution of potassium hydroxide with Alizarin red S (for clearing and staining ossified bone) and a solution of glycerin/alcohol for preservation and storage Dawson, 1926). The skeletons were examined and all abnormal findings and variations were recorded. The specimens were preserved individually.
Statistics:
The following statistical methods were used to analyse food consumption, body weights, reproduction data, skeletal data and soft tissue examination data (RCC TOX LIMS computer system Version 7.0):
- The Dunnett-test (Dunnett, 1955) (many to one t-test) based on a pooled variance estimate was applied when the variables can be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (Fisher, 1950) (many-one rank test) was applied instead of the Dunnett test when the data could not be assumed to follow a normal distribution.
- Fisher's exact-test (Miller, 1981) for 2x2 tables was applied when the variables could be dichotomised without loss of information.
- In the case of the soft tissues, Fisher’s test from Microstat Copyright Version 4.1.08 was used to analyse the results.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
MATING PERFORMANCE
Three females from the control group were not pregnant and seven, five and one females were not pregnant from the test material treated animals receiving doses of 50, 100 and 250 mg/kg/ bw/day, respectively.

MORTALITY
None of the maternal animals died during the study.

CLINICAL SIGNS
Female no. 106 at 50 mg/kg bw/day, female no. 65 at 100 mg/kg bw/day and females 69, 78, 79 and 87 at 250 mg/kg bw/day showed remnants of blood in the vagina around day 15 post coitum. These females had live foetuses at caesarean section.
Salivation after administration was recorded at 50 and 250 mg/kg bw/day. This clinical sign was recorded in two females at 50 mg/kg bw/day (occasionally) and in all females at 250 mg/kg bw/day from day 9 post coitum onwards.
In addition, two females (no. 9 and 10) from the control group occasionally showed a scab in the scapular region.

BODY WEIGHTS
Body-weight gain was slightly lower in all test material-treated groups, but not in a dose dependent manner.
In addition, gravid uterus weights in all test material-treated groups were similar to the control group. However, the adjusted body weights (body-weight gain from day 6 post coitum to the day of caesarean section corrected for the gravid uterus weight) were slightly lower than that of the control group in all test material-treated groups.

FOOD CONSUMPTION
Occasional statistically significant lower relative food consumption than the control group was observed in all test-material-treated groups from days 6 to 15 post coitum and 18 to 20 post coitum but was not dose-dependent. The maximum differences were recorded on days 6 to 9 at 250 mg/kg bw/day (13 %), on days 18 to 20 at 50 mg/kg bw/day (15.9 %) and similarly in all periods at 100 mg/kg bw/day (8 %).

NECROPSY FINDINGS
No findings were recorded in the control group or at 250 mg/kg bw/day.
At 50 mg/kg bw/day, female no. 37 showed dilated renal pelvis and small right uterine horn. This female was not pregnant at caesarean section.
At 100 mg/kg bw/day, female no. 53 (not pregnant) showed dilated renal pelvis.

REPRODUCTION DATA
Statistically higher incidence of post-implantation losses was recorded at 250 mg/kg bw/day (17 % vs. 1.8 % in the control) as well as of the number of litters affected when compared to the control group. These losses were at foetal and embryonic level. Consequently, the percentage of implantation sites with foetuses and mean number of foetuses per dam at termination were lower in this group.
There were no effects of treatment on reproductive parameters at 50 and 100 mg/kg bw/day.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
250 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:yes

Details on embryotoxic / teratogenic effects:
BODY WEIGHTS
The body weight of foetuses was significantly lower in all test-material-treated groups. Differences were mainly recorded at 100 and 250 mg/kg bw/day (approximately 14 %).

SEX RATIO
No differences were recorded in sex ratio or distribution of males and females in the uterus compared to the control group.

EXTERNAL EXAMINATIONS
No treatment-related findings were recorded.
Foetus no. 524 from litter no. 60 at 100 mg/kg bw/day had placenta with haemorrhagic content.

SKELETAL EXAMINATIONS
There was a slightly higher percentage of litters with incomplete ossification of supraoccipital bones, caudal vertebrae, fore/hind limb phalanx and sternebrae at 100 and 250 mg/kg bw/day compared to the control group. Although some differences were statistically significant, the findings were not considered of pathological relevance.
The skeletal examination revealed a range of variations in all groups. There was no indication of a test-material-related trend in the type or incidence of these findings.

VISCERAL EXAMINATIONS
There was an increase in the occurrence of left-side umbilical artery (variation) per foetus and litter basis at all doses. The increases were 77, 62 and 91 % on a per foetus basis compared to the control group (35 %), and 21, 20 and 38 % on a per litter basis compared to control group (8 %) at 50, 100 and 250 mg/kg bw/day, respectively.
There was an increase in the occurrence of bilateral azygous vein (variation) on a per litter basis at all doses (12, 10 and 14 % at 50, 100 and 250 mg/kg bw/day, respectively) compared to control group (0 %).
The incidence of common variations, such as slightly dilated renal pelvis, slightly dilated and/or convoluted ureter, long cranial thymus, malpositioned testis and kidney, additional small lobe and dark area in the liver, was similar in all groups including the control group.

The visceral examination revealed the following: at 250 mg/kg bw/day there were five litters with one foetus each with abnormalities: three foetuses with diaphragmatic hernia, one foetus with asymmetric nasal septum and one foetus with oval-shaped eyes; at 100 mg/kg bw/day one foetus had abnormality with dilation of cerebral aqueduct; at 50 mg/kg bw/day one foetus had abnormalities, such as severe dilation of cerebral ventricles, no recognisable ocular structures and asymmetry of nasal structures. Finally, two litters from the control group showed abnormalities, i.e. one foetus with oval shaped lens and one foetus with interrupted ureter and renal pelvis severely dilated.
The diaphragmatic hernia could be considered related to the test material as it was only observed at 250 mg/kg bw/day. There was no evidence of a compound-related effect in the number of occurrences in the remaining abnormalities found in the test material-treated groups.

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: embryotoxicity
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: teratogenicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Table 1: Food Consumption of Females (Mean Values in g/animal/day)

Days

Dose Group (mg/kg bw/day)

0

50

100

250

0-3

21.4

20.8

21.3

22.1

3-6

21.1

21.1

21.5

21.0

6-9

19.7

18.2

17.7*

17.1**

9-12

21.8

20.5

19.4**

19.1**

12-15

22.4

20.3*

19.8**

19.3**

15-18

22.4

22.2

21.6

21.5

18-20

19.3

16.8

17.2

17.7

Mean of Means

21.2

20.0

19.8

19.7

*/** Dunnett's test based on pooled variance significant at the 5 % (*) or 1 % (**) level

 

Table 2: Percentage Body Weight Gain (Mean Values)

Day

Dose Group (mg/kg bw/day)

0

50

100

250

0

-7.7

-6.3

-8.6

-7.8

1

-6.9

-5.6

-8.1

-7.0

2

-5.4

-4.4

-6.2

-6.1

3

-4.0

-2.8

-4.1

-3.6

4

-2.2

-1.5

-2.8

-2.1

5

-0.5

-0.6

-1.5*

-0.9

6

0.0

0.0

0.0

0.0

7

1.4

0.9

0.7

1.0

8

3.3

2.0

1.7

2.1

9

5.0

3.3

2.7*

3.5

10

6.7

5.1

4.5*

4.9

11

8.8

7.5

6.6

6.9

12

10.5

8.7

8.2

8.1

13

12.0

9.9

9.5

9.3

14

14.8

11.8

11.4*

11.6

15

17.6

14.3*

13.8

13.3**

16

20.9

18.0

17.4

17.1*

17

25.2

22.6

22.1

21.3

18

30.1

27.3

27.0

25.7*

19

33.4

30.5

30.6

29.1

20

37.5

32.6

33.0

31.7*

*/** Dunnett's test based on pooled variance significant at the 5 % (*) or 1 % (**) level

 

Table 3: Selected Reproduction Data Parameters

 

Dose Group (mg/kg bw/day)

0

50

100

250

Number of Dams

20

26

21

23

Corpora lutea

Mean

Standard deviation

252

12.6

1.7

346

13.3

1.7

292

13.9

1.5

310

13.5

1.8

Pre-implantation loss

% of corpora lutea

Mean

Standard deviation

No. dams affected

32

12.7

1.6

2.0

12

33

9.5

1.3

1.8

12

28

9.6

1.3

1.2

14

39

12.6

1.7

2.6

10

Implantation sites

% of corpora lutea

Mean

Standard deviation

220

87.3

11.0

3.1

313

90.5

12.0

2.2

264

90.4

12.6

1.9

271

87.4

11.8

2.7

Post-implantation loss

% of implantation sites

Mean

Standard deviation

No. dams affected

4

1.8

0.2

0.4

4

3

1.0

0.1

0.3

3

12

4.5

0.6

1.0

7

46

17.0**

2.0†

1.7

18

Implantation site scars

% of implantation sites

Mean

Standard deviation

0

1

0.3

0.0

0.2

0

1

0.4

0.0

0.2

*/** Fisher’s Exact Test significant at the 5 % (*) or 1 % (**) level

†Steel test significant at the 5 % level

 

Table 4: Body Weights of Live Foetuses (Mean Values in g)

 

Dose group (mg/kg bw/day)

0

50

100

250

Males and Females

3.6

3.4

3.1

3.1

Males

3.7

3.4

3.2

3.3

Females

3.5

3.3

3.1

3.1

Applicant's summary and conclusion

Conclusions:
Under the conditions of the study the No Observed Adverse Effect Level (NOAEL) for maternal toxicity was 250 mg/kg bw/day, the NOAEL for embryofoetal development was 50 mg/kg bw/day and the NOAEL for teratogenic effects was 100 mg/kg bw/day.
Executive summary:

The developmental toxicity of the test material was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 414 and EU Method B.31.

During the study, the test material was administered by oral gavage to groups of mated female Wistar rats at dose levels of 0 (corn oil vehicle control), 50, 100 and 250 mg/kg bw/day. The females received treatment from day 6 to day 19 post coitum. During the treatment period the females were observed for mortality and morbidity as well as for any adverse clinical signs. Body weights and food consumption were recorded. On day 20 post coitum the females were sacrificed, subjected to necropsy and the foetuses removed by caesarean section. Post mortem examination included a gross macroscopic examination of all internal organs with emphasis on the uterus, uterine contents, position of foetuses in the uterus and the number of corpora lutea were counted and recorded.

Foetuses were removed from the uterus, sexed, weighed individually, examined for gross external abnormalities and then sacrificed. Half of the litter was subjected to skeletal examination while the other half was subjected to visceral examination.

Treatment caused salivation at 50 and 250 mg/kg bw/day, mainly at 250 mg/kg bw/day in a dose-dependent manner. Slightly lower food consumption and lower body-weight gain were observed in all test material-treated groups. These values did not deviate by more than 11, 8 and 13 % from the control group for food consumption and by more than 5, 5 and 6 % for body weight, at 50, 100 and 250 mg/kg bw/day.

In the observations deriving from the hysterectomies, test material-related embryofoetal toxicity was observed at 250 mg/kg bw/day with an increase in post-implantation losses.

Treatment led to a decrease in mean foetal body weight in all test material-treated groups. These values did not deviate by more than 6 % at 50 mg/kg bw/day and 14 % at 100 and 250 mg/kg bw/day. At the external examination, no findings were recorded.

The skeletal examination of the foetuses did not reveal any toxicologically relevant alterations. The slight delay in ossification in some litters at 100 and 250 mg/kg bw/day compared to the control group can be considered a variation without pathological meaning and secondary to lower foetal body weight.

The visceral examination revealed the following: at 250 mg/kg bw/day there were five litters with one foetus each with abnormalities: three foetuses with diaphragmatic hernia, one foetus with asymmetric nasal septum and one foetus with oval-shaped eyes; at 100 mg/kg bw/day one foetus had abnormality with dilation of cerebral aqueduct; at 50 mg/kg bw/day one foetus had abnormalities, such as severe dilation of cerebral ventricles, no recognisable ocular structures and asymmetry of nasal structures. Finally, two litters from the control group showed abnormalities, i.e. one foetus with oval shaped lens and one foetus with interrupted ureter and renal pelvis severely dilated.

The diaphragmatic hernia could be considered related to the test material as it was only observed at 250 mg/kg bw/day. There was no evidence of a compound-related effect in the number of occurrences in the remaining abnormalities found in the test material-treated groups.

Furthermore, there was an increase in the incidence of common variations, such as left-sided umbilical artery and bilateral azygos vein, in the test material-treated groups. Other common variations, mainly involving urogenital morphology (dilated renal pelvis, and dilated and/or convoluted ureter, malpositioned kidney, malpositioned cranial testis) in addition to other findings (additional small lobe and dark-area in the liver) were recorded in all groups including the control group. These alterations should be regarded as spontaneous variations with limited pathological relevance.

Therefore, based on the results of this study, the top dose tested of 250 mg/kg bw/day is considered the No Observed Adverse Effect Level (NOAEL) for maternal toxicity. Although there were decreases in food consumption and body weight at 250 mg/kg bw/day, the decreases in body weight gain were not greater than 5 - 6 %.

With respect to the effects on embryofoetal development, 50 mg/kg bw/day is considered to be the NOAEL based on the lower foetal body weight (14 %) at 100 and 250 mg/kg bw/day and the increase in the post-implantation losses at 250 mg/kg bw/day.

Regarding the potential for teratogenic effects, 100 mg/kg bw/day is considered to be the NOAEL based on the diaphragmatic hernia recorded at 250 mg/kg bw/day.