Shale oils

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 April 2014 to 18 October 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:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Shale oils

EC Number:

269-646-0

EC Name:

Shale oils

Cas Number:

68308-34-9

Molecular formula:

Not applicable (a generic molecular formula cannot be provided for this specific UVCB substance)

IUPAC Name:

Shale oil

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: 190 - 280 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: 19 - 23 °C
- Humidity: 35 - 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: 24 April 2014 To: 20 May 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 top up the flask volume which was then left stirring using for at least 30 minutes (using a magnetic 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 into appropriate containers. The aliquots were stored at 5 ± 3 °C and protected from light. Daily administration was performed under stirring (using a magnetic 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 500 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 (at least 7 mL each) 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 results showed that test material concentrations did not deviate by more than 11 % from the nominal concentration in any of the formulations analysed.

PREPARATION OF CALIBRATION STANDARDS
The test material (nominal 100 mg) was dissolved in acetone (100 mL) to prepare a stock solution with a nominal concentration of 1mg/mL. This stock solution was further diluted with acetone to obtain a nominal 0.1 mg/mL calibration standard. A duplicate calibration standard was similarly prepared at 0.1 mg/mL. These duplicate calibration standards were used to determine the recovery and test sample concentrations. Test vehicle was included in the standard solutions so that they contained the equivalent amount of test vehicle to that of the sample at that test level.

PREPARATION OF LINEARITY STANDARDS
The test material (nominal 100 mg) was dissolved in acetone (100 mL) to prepare a stock solution with a nominal concentration of 1mg/mL. Defined volumes of this stock solution were serially diluted with acetone to obtain standards in the range of 0.05 to 0.15 mg/mL. These standards are used to evaluate the linearity of the analytical system.

PREPARATION OF SPIKED RECOVERY SAMPLES
Samples of test vehicle were accurately fortified with known amounts of test material equivalent to the lowest and highest anticipated dose concentrations.

PREPARATION OF TEST SAMPLES
The formulations were diluted with acetone. An aliquot of test material formulation was accurately weighed into a volumetric flask and brought to volume with acetone which was then shaken to dissolve. Where necessary, sample solutions were further diluted with acetone to achieve the final working concentration of 0.1 mg/mL.

PREPARATION OF HOMOGENEITY SAMPLES
Samples of vehicle were accurately fortified with known amounts of test material equivalent to the anticipated lowest and highest dose concentrations. These samples were then prepared for analysis, in triplicate, at the top, middle and bottom of the sample and analysed as for the test samples. The mean of these homogeneity samples were also used as Day 0 of a stability trial and were stored at 4 °C and analysed after 10 days.

INSTRUMENTAL PARAMETERS
Gas chromatograph: HP 6890N
Detector: MSD (HP 5975)
Column: Rxi - 1 ms (Restek) 15 m x 0.25 mm i.d.; 0.25 µm film thickness
Inlet liner: Split with glass wool
Carrier gas: Helium
Mode: Constant flow
Velocity: 1 mL/min
Oven temperature: 50 °C for 5 minutes, with 20 °C/minute to 300 °C, for 3 minutes
Injection mode: Splitless
Inlet purge time: 1 minute
Injection volume: 1 µL
Injection temperature: 250 °C
Detector temperature: 230 °C
Ions measured: Group 1: SIM ions m/z: 57, 91, 108 and 122 from 3 to 8 minutes; Group 2 SIM ions m/z: 69, 97, 121 and 131 from 8 to 9 minutes; Group 3 SIM ions m/z: 57, 71, 85, 123, 212, 218, 355 and 429 from 9 to 22 minutes
Retention time: approximately 6 to 16 minutes

RESULTS
- Specificity
The control dose samples and an analysed solvent blank showed no significant interfering response at the retention time of the test material. The standard solutions contained a peak specific to the test material whose area changed accordingly with known concentration; hence the specificity of the method by retention time was confirmed.
- Linearity
The data was found to have a linear correlation within the calibration range of 0 to 0.154 mg/L. The R² fit of the calibration curve to the data was 0.9988 and was considered to be acceptable.
- Accuracy (recovery)
The method was considered to sufficiently accurate and precise for the purposes of this test and was found to have recovery values within 100 ± 20 % of the fortification. The test sample results were not corrected for recovery.
- Test material formulations
The formulations were found to contain the test material in the range of 98 to 111 %. The test material was found to be stable in the formulation when kept for 10 days in the refrigerator (4 °C) or 11 days at room temperature. The formulations were found to be homogeneously prepared.

Details on mating procedure:

After acclimatisation, females were housed with sexually mature males in a cage for approximately 16 hours. At least 21 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:

22 females per dose level

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 uteri (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 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, 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. 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. 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

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation after administration was recorded at 100 and 500 mg/kg bw/day. This clinical sign increased in frequency and number of affected females with the dose level. Salivation was recorded after the administration in eight females at 100 mg/kg bw/day (one or two days) and all females at 500 mg/kg bw/day from day 8 of pregnancy onwards.
In addition, remains of blood in the vagina was recorded in female no. 18 from the control group on day 13 post coitum, and in females 69 and 81 at 500 mg/kg bw/day on days 13 and 15 post coitum, respectively.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

None of the maternal animals died during the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body-weight gain was statistically reduced at 100 and 500 mg/kg bw/day compared to the control group over the course treatment period (5 and 11 %, respectively). In addition, the gravid uterus weights and the corrected body-weight gains (body-weight gain from day 6 post coitum to the day of caesarean section corrected for the gravid uterus weight) were statistically lower than in the control group at 500 mg/kg bw/day.
There were no effects of treatment on body weight or adjusted body weight at 50 mg/kg bw/day.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Lower statistically significant relative food consumption compared to the control group was observed at 100 and 500 mg/kg bw/day from days 6-15 post coitum. The maximum differences were recorded on days 6-9 at 500 mg/kg bw/day (29 %) and on days 12-15 at 100 mg/kg bw/day (11 %).
Lower absolute food consumption was observed at 50 mg/kg bw/day; however, the relative food consumption was similar to the control group.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no findings at 50, 100 or 500 mg/kg bw/day.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Female no. 21 from the control group showed unilateral dilated renal pelvis and female no. 8 possessed a stomach with yellowish liquid content. These findings are considered of no toxicological relevance and within the range of normal background lesions that may be seen in rats of this strain.
No other findings were noted.

Histopathological findings: neoplastic:

no effects observed

Maternal developmental toxicity

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

A higher incidence of post-implantation loss was recorded at 500 mg/kg bw/day (10.6 % implantation loss vs. 2.9 % in the control). Consequently, the percentage of implantation sites with foetuses and mean number of foetuses per dam at termination were lower in this group with respect to the control animals. The differences recorded were statistically significant.

Dead fetuses:

effects observed, treatment-related

Description (incidence and severity):

At 100 and 500 mg/kg bw/day a slightly higher, statistically significant, percentage of embryonic/foetal deaths was recorded with respect to the control females (averaging 0.7 and 1 foetuses per litter, respectively, compared to 0.3 in the controls).

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

Two females from the control group were not pregnant and one, two and one females were not pregnant from the test material treated animals receiving doses of 50, 100 and 500 mg/kg/ bw/day, respectively.

Details on maternal toxic effects:

There were no effects of treatment on reproductive parameters at 50 mg/kg bw/day.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Maternal abnormalities

Abnormalities:

effects observed, treatment-related

Localisation:

other: general toxicity

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight of foetuses was lower (6 %) in the group treated at 500 mg/kg bw/day with respect to the control. Statistically significant differences were recorded when analysis was done on an individual basis.
There were no differences in foetal body weight at 50 or 100 mg/kg bw/day either on an individual or per litter basis.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There were no test material-related differences in sex ratio or distribution of males and females in the uterus content compared to the control group in any of the test material-treated groups. The sex ratio differences recorded at 50 and 500 mg/kg bw/day compared to the control group are related to the high percentage of males recorded in the control group.

Description (incidence and severity):

There were no findings in any of the groups.

Description (incidence and severity):

There was a slightly higher percentage of litters with large fontanelle and incomplete ossification of supraoccipital bones and sternebrae at 500 mg/kg bw/day compared to the control group. Although some differences were statistically significant, the findings were not considered of pathological relevance (secondary to maternal toxicity).
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.

Description (incidence and severity):

There was an increase in the occurrence of left-side umbilical artery (variation) at all the treatment doses (55, 70 and 81 %) at 50, 100 and 500 mg/kg bw/day, respectively, compared to the control group (32 %). Two litters at 500 mg/kg each showed one foetus with abnormalities; one foetus had total situs inversus and the other had no recognizable eye structures.
The incidence of common variations, such as slightly dilated renal pelvis, malpositioned kidney, 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.

Effect levels (fetuses)

1

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: embryotoxicity

2

Dose descriptor:

NOAEL

Effect level:

500 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	500
0-3	21.8	21.3	21.4	21.9
3-6	22.9	22.7	22.9	23.1
6-9	22.3	20.7	20.1**	15.1**
9-12	23.1	21.6	21.8	18.8**
12-15	24.8	22.3**	21.4**	19.9**
15-18	25.0	23.3*	23.5	21.8**
18-20	21.8	21.1	21.1	20.1

*/** 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	500
0	-9.0	-9.9	-9.7	-8.9
1	-7.3	-7.1	-7.9	-7.2
2	-5.5	-5.7	-6.1	-5.5
3	-3.4	-.3.	-4.0	-3.6
4	-2.4	-1.8	-2.9	-2.6
5	-0.8	-1.3	-1.6	-1.5
6	0.0	0.0	0.0	0.0
7	1.6	1.5	0.7	-0.2**
8	3.0	2.6	1.3**	0.3**
9	4.9	4.3	2.9**	1.1**
10	6.5	6.5	4.8**	2.9**
11	8.7	8.6	7.0**	4.6**
12	10.5	10.3	8.6**	5.6**
13	12.6	11.9	9.6**	7.5**
14	15.0	13.8	11.7**	9.2**
15	17.8	16.7	14.3**	11.5**
16	21.4	20.2	17.6**	14.4**
17	26.2	24.8	22.5**	18.7**
18	31.2	30.3	27.4*	23.1**
19	36.0	34.2	31.8*	26.8**
20	39.7	37.5	35.1*	29.0**

*/** 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	500
Number of Dams	20	21	20	21
Corpora lutea Mean Standard deviation	280 14.0 3.2	280 13.3 3.0	264 13.2 1.9	291 13.9 2.2
Pre-implantation loss % of corpora lutea Mean Standard deviation No. dams affected	35 12.5 1.8 2.4 14	37 13.2 1.8 1.8 16	22 8.3 1.1 1.4 11	55 18.9* 2.6 2.5 15
Implantation sites % of corpora lutea Mean Standard deviation	245 87.5 12.3 4.1	243 86.8 11.6 3.4	242 91.7 12.1 2.3	236 81.1* 11.2 2.9
Post-implantation loss % of implantation sites Mean Standard deviation No. dams affected	7 2.9 0.4 0.7 5	7 2.9 0.3 0.5 7	13 5.4 0.7 1.1 6	25 10.6** 1.2 1.5 11
Implantation site scars % of implantation sites Mean Standard deviation	2 0.8 0.1 0.4	1 0.4 0.0 0.2	0	3 1.3 0.1 0.5

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

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study the No Observed Adverse Effect Level (NOAEL) for maternal toxicity was 100 mg/kg bw/day. The NOAEL for embryofoetal development was 100 mg/kg bw/day and the NOAEL for teratogenic effects was 500 mg/kg bw/day, the highest dose level tested.

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 500 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.

Salivation after administration was recorded at 100 and 500 mg/kg bw/day in a dose-dependent manner. Lower food consumption and slightly lower body-weight gain were observed at 100 and 500 mg/kg bw/day; however, these values did not deviate from the control group by more than 11 and 29 % for food consumption and 5 and 11 % for body weight, respectively.

In the observations deriving from the hysterectomies, test material-related embryofetal toxicity was observed at 500 mg/kg bw/day (higher number of post-implantation losses and, consequently, lower mean number of foetuses per litter).

Treatment showed no effects on mean foetal weight evaluated on a per litter basis but lower body weight (6 %) with respect to the control group was recorded at the dose of 500 mg/kg bw/day when analysis was done on an individual basis.

There were no findings at the external examination.

The skeletal examination of the foetuses did not reveal any toxicologically relevant alterations. The slight delay in ossification (skull bones and sternebrae) recorded in some litters from 500 mg/kg bw/day compared to the control group should be considered a variation without pathological significance and secondary to the maternal toxicity recorded.

The visceral examination at 100 and 500 mg/kg bw/day/day showed an increase in foetus and litters with left-sided umbilical artery. Two litters at 500 mg/kg each showed one foetus with abnormalities; one foetus had total situs inversus and the other had no recognisable eye structures.

Furthermore, common variations mainly involving urogenital morphology (dilated renal pelvis, and dilated and/or convoluted ureter, malpositioned kidney, malpositioned cranial testis) and other findings (dilated stomach, long cranial thymus, additional small lobe in the liver and bilateral azygos vein) were recorded in all the 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 dose of 100 mg/kg bw/day (the middle dose tested) was considered the No Observed Adverse Effect Level (NOAEL) for pregnant females, based on the significant decrease in food consumption and body-weight gain recorded at 500 mg/kg bw/day; there was a 10 % difference compared to the control group.

With respect to the effects on embryofoetal development, 100 mg/kg bw/day is considered the NOAEL, based on the statistically significant higher post-implantation losses recorded at 500 mg/kg bw/day.

Regarding the potential for teratogenic effects, 500 mg/kg bw/day is considered to be the NOAEL.