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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:
07 April 2014 to 28 August 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study 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 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, light
EC Number:
923-592-0
Molecular formula:
Not applicable. A generic molecular formula cannot be assigned to this UVCB substance.
IUPAC Name:
Shale oils, light
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: 194 - 261 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: 44 - 65 % (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: 02 April 2014 To: 28 August 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 left stirring using a propeller stirrer for at least 30 minutes. The formulation then was transferred to a suitable container.
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. Daily administration was performed under stirring with 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 concentrations of dose formulations administered were determined twice during the study and once (sampled from top/middle/bottom) for homogeneity in samples taken from the formulation to be administered to the 100 to 1000 mg/kg dose groups 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 results showed that test material concentrations did not deviate by more than 7 % 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. An additional stability trial was also prepared at room temperature for a period of 11 days and analysed.

INSTRUMENTAL PARAMETERS
Gas chromatograph: HP 6890N
Detector: MSD (HP 5975)
Column: DB-624 (J&W) 60 m x 0.25 mm i.d.; 1.4 µm film thickness
Inlet liner: Split liner with glass wool
Carrier gas: Helium
Mode: Constant flow
Velocity: 1 mL/min
Oven temperature: 50 °C for 1 minute, with 10 °C/minute to 200 °C, then with 50 °C/minute to 260 °C, for 3 minutes
Injection mode: Splitless
Inlet purge time: 1 minute
Injection volume: 1 µL
Injection temperature: 250 °C
Detector temperature: 260 °C
Ions measured: Group 1: SIM ions m/z: 67, 81, 91 and 110 from 5 to 11 minutes; Group 2 SIM ions m/z: 91, 94, 105, 106 and 120 from 11 to 20 minutes
Retention time: approximately 6 to 15 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 176 mg/L. The R² fit of the calibration curve to the data was 0.9999 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 107 %. 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. 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:
24 females were dosed at 0 (vehicle control) and 100 mg/kg bw/day.
25 females were dosed at 300 and 1000 mg/kg bw/day.
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 (RCC TOX Release 7.0) and soft tissue examination:
- 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.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
MATING PERFORMANCE
Two females from the control group were not pregnant and five, two and two females were not pregnant from the test material treated animals receiving doses of 100, 300 and 1000 mg/kg/ bw/day, respectively.

MORTALITY
None of the maternal animals died during the study.

CLINICAL SIGNS
Salivation after administration was recorded in all test material treatment groups. This clinical sign increased in frequency and number of occurrences with the dose level. Salivation was recorded after the administration in one female (one day) at 100 mg/kg bw/day, seven females at 300 mg/kg bw/day (one or two days) and all females at 1000 mg/kg bw/day from day 11 of pregnancy onwards.
In addition, female no. 34 at 100 mg/kg bw/day had remains of blood in the vagina on day 13 post coitum. This female showed nine implantation scars and one live foetus at caesarean section. Due to these observations, bleeding was considered most probably a sign of abortion.
One female (no. 5) from the control group and one female (no. 26) treated at 100 mg/kg bw/day occasionally had scabs on the scapular region.

BODY WEIGHTS
Slightly lower body-weight gain at 100 and 300 mg/kg bw/day was recorded from days 7 to 14 post coitum (1.8 and 1.4 %, respectively); afterwards a recovery was observed. No statistically significant differences were recorded. The gravid uterus weight and the corrected body-weight gain (body-weight gain from day 6 post coitum to the day of caesarean section corrected for the gravid uterus weight) were similar to the control group.
Lower body-weight gain (4.2 %) was recorded at 1000 mg/kg bw/day from day 7 post coitum onwards. The differences recorded were statistically significant from days 7 to 17 of pregnancy. In addition, the adjusted body weight was statistically lower; therefore the gravid uterus weight was similar to the control group.

FOOD CONSUMPTION
Slightly lower food consumption (87.3 %) was recorded from days 6 to 15 post coitum at 1000 mg/kg bw/day; this was statistically significant during the periods from 6-9 and 9-12 days post coitum.
No differences from the control group were recorded in relative values at 100 and 300 mg/kg bw/day.
The reduced food consumption and slightly lower body-weight gain recorded at 1000 mg/kg bw/day were not statistically significant and differed by less than 4 % from the control group; hence they were not considered adverse.

NECROPSY FINDINGS
No findings were recorded in control animals.
At 1000 mg/kg bw/day, female no. 68 had reddish pancreas and female no.77 had unilateral dilated renal pelvis.
At 300 mg/kg bw/day, female no. 45 had reddish gastric mucosa and females 54, 59, 62 and 95 had unilateral dilated renal pelvis.
At 100 mg/kg bw/day, female no. 44 had yellowish stomach content.
These findings could be considered within the range of normal background lesions that may be seen in rats of this strain.

REPRODUCTION DATA
Slightly higher post-implantation losses were recorded at 100 mg/kg bw/day. Consequently, the percentage of implantation sites with foetuses and mean number of foetuses per dam at termination were lower in this group.
No differences were recorded in the remaining groups.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
BODY WEIGHTS
Body weight of live foetuses (both sexes) per individual basis was statistically lower at 1000 mg/kg bw/day (6 %) and in female foetuses at 300 mg/kg bw/day (6 %) than in the control group.
No relevant differences were recorded in foetal body weight at 100 mg/kg bw/day, individually or per litter.
The statistically significant lower foetal body weights were less than 6 % lower than controls and were not considered adverse.

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

EXTERNAL EXAMINATIONS
At the external examination, one litter from 100 mg/kg bw/day showed one foetus with tail abnormalities and one foetus at 300 mg/kg bw/day had general oedema with head abnormalities, small/absent digits/toes, bent tail and small hind limb (abnormalities). There was no evidence of a compound-related increase in the occurrence of these findings.

SKELETAL EXAMINATIONS
The skeletal examination of the foetuses did not reveal any toxicologically relevant alterations.

VISCERAL EXAMINATIONS
The visceral examination revealed slight dilation of 4th brain ventricle and oval shaped lens in one, two and one foetuses, respectively, from control, 300 and 100 mg/kg bw/day groups. These foetuses had lower body weight (<2 g). Furthermore, one foetus from control group had head abnormalities such as slightly dilated cerebral aqueduct and lateral ventricles and small ocular structures.
Although some abnormalities were found in test material treated groups, there was no evidence of a compound-related increase in the occurrence of these findings.
Furthermore, 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 (distended stomach, left-sided umbilical artery, additional small lobe in the liver and bilateral azygos vein) were recorded in all the groups including the control. These alterations should be regarded as spontaneous variations with limited pathological relevance.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
1 000 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

0 (vehicle control)

100 mg/kg bw/day

300 mg/kg bw/day

1000 mg/kg bw/day

0-3

23.1

22.9

23.2

23.4

3-6

22.0

22.5

23.7*

22.7

6-9

21.1

20.7

21.7

18.7**

9-12

22.1

21.7

22.4

19.3**

12-15

22.2

22.1

23.1

20.4

15-18

23.7

23.8

24.5

23.5

18-20

20.3

20.4

20.1

20.8

*/** Dunnett's test based on pooled variance 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, developmental toxicity and teratogenicity was 1000 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), 100, 300 and 1000 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 in all dose groups in a dose-dependent manner. Slightly lower food consumption and lower body-weight gain were observed at 1000 mg/kg bw/day but these values did not deviate more than 4 % from the control group.

In the observations deriving from the hysterectomies, no test material related embryofoetal toxicity was observed at any of the administered doses. Although slightly lower mean foetal weights were recorded at 300 and 1000 mg/kg bw/day, the differences observed compared to control group were less than 6 %.

At the external examination, one litter from 100 mg/kg bw/day showed one foetus with tail abnormalities and one foetus at 300 mg/kg bw/day had general oedema with head abnormalities, small/absent digits/toes, bent tail and small hind limb (abnormalities). There was no evidence of a compound-related increase in the occurrence of these findings.

The skeletal examination of the foetuses did not reveal any toxicologically relevant alterations.

The visceral examination revealed slight dilation of 4th brain ventricle and oval shaped lens in one, two and one foetuses, respectively, from the control, 300 and 100 mg/kg bw/day groups. These foetuses had lower body weight (<2 g). Furthermore, one foetus from the control group had head abnormalities such as slightly dilated cerebral aqueduct and lateral ventricles and small ocular structures.

Although some abnormalities were found in test material treated groups, there was no evidence of a compound-related increase in the occurrence of these findings.

Therefore, based on the results of this study, the dose of 1000 mg/kg bw/day (the highest dose tested) is considered the No Observed Adverse Effect Level (NOAEL) for pregnant females. The reduced food consumption and slightly lower body-weight gain recorded at 1000 mg/kg bw/day were not statistically significant and differed by less than 4 % from the control group; hence they were not considered adverse.

With respect to the effects on embryofoetal development, 1000 mg/kg bw/day is considered the NOAEL. The statistically significant lower foetal body weights were less than 6 % lower than controls and were not considered adverse.

There was no treatment related teratogenicity at 1000 mg/kg bw/day (the highest dose tested); therefore 1000 mg/kg bw/day is considered the NOAEL.