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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:
June 2014 - February 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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)
Version / remarks:
2001
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
1998
Deviations:
no
Principles of method if other than guideline:
Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), Part B: Methods for the determination of toxicity and other health effects: Prenatal Developmental Toxicity Study; Official Journal of the European Union, No. L 142.
GLP compliance:
yes (incl. QA statement)
Remarks:
2013
Limit test:
no

Test material

Constituent 1
Reference substance name:
63500-71-0; 65418-70-4 (trans); 65418-69-1 (cis)
IUPAC Name:
63500-71-0; 65418-70-4 (trans); 65418-69-1 (cis)
Constituent 2
Reference substance name:
Pyranol
IUPAC Name:
Pyranol
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Pyranol
- Physical state: liquid/colorless, clear
- Homogeneity: given (visually)
- Storage conditions: Room temperature, under light exclusion
- Stability under test conditions: The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility.

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Strain: Time-mated Wistar rats (CrIGIxBrIHan:WI)
- Source: Charles River Laboratories, Research Models and Services, Germany
- Animal identification: by ear tattoo
- Age at study initiation: 10-12 weeks
- Weight at study initiation: The body weight of the pregnant animals on day 0 varied between 143.8 – 189.4 g.
- Fasting period before study: no
- Housing: singly from day 0 - 20 p .c. in type DK III polycarbonate cages supplied by BECKER & CO., Castrop-Rauxel, Germany (floor area about 800 cm2). Dust-free wooden bedding was used in this study. For enrichment, wooden gnawing blocks were offered (Typ NGM E-022, supplied by Abedd® Lab. and Vet. Service GmbH, Vienna, Austria).
- Diet (e.g. ad libitum): ad libitum, ground Kliba maintenance diet mouse/rat meal (PROVIMI KLIBA SA, Kaiseraugst, Switzerland)
- Water (e.g. ad libitum): ad libitum, potable tap water in water bottles
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS (set to maintain)
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 02 July 2014 To: 17 July 2014

ANALYSES
- Food: assayed for chemical as well as for microbiological contaminants
- Water: regularly assayed for chemical contaminants by the municipal authorities of Frankenthal and by Technical Services of BASF Aktiengesellschaft as well as for the presence of microorganisms by a contract laboratory

Administration / exposure

Route of administration:
dermal
Vehicle:
corn oil
Details on exposure:
TEST SITE
- Area of exposure: dorsal and dorso-lateral parts of the trunk
- % coverage: not less than 10% of the body surface
- Type of wrap if used: semi-occlusive dressing
- Time intervals for shavings or clipplings: The rats were shaved on the application area at least 18 hours before the first treatment and if it was necessary additional times during the treatment period.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): After the daily 6-hour treatment period, the dressing was removed and the application site was washed off with lukewarm water and dried.

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2 mL/kg bw/day, based on the most recent individual body weight.
- Constant volume or concentration used: yes

VEHICLE
- Justification for use and choice of vehicle (if other than water): corn oil, no justification
- Amount(s) applied (volume or weight with unit): 2 mL/kg bw/day

USE OF RESTRAINERS FOR PREVENTING INGESTION: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical verifications of the stability of the test substance in corn oil at room temperature over a period of 8 days had been verified prior to the start of the study (Project No.: 01Y0639/09Y087). Concentrations were verified at the beginning of the application period.
Given that the test substance was completely miscible with corn oil, solutions were considered to be homogenous without further analysis.
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant:
The animals were mated by the breeder ("time-mated") and supplied on day 0 post coitum (GD 0) (= detection of vaginal plug / sperm). The animals arrived  on the same day (GD 0) at the experimental laboratory. The  following day was designed "day 1"  (GD 1). Animals were assigned to the test groups by taken random selection. The animals were acclimated to the laboratory conditions between start of the study (beginning of the experimental phase) and first application (GD 6).
Duration of treatment / exposure:
day 6 through day 19 post coitum (p.c.) (GD 6 to GD 19)
Frequency of treatment:
once daily for at least 6 hours
Duration of test:
On day 20 p.c. (GD 20) all surviving females were sacrificed
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: at request of the sponsor

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes, check for mortality
- Time schedule: twice a day on working days or once a day (Saturday, Sunday or on public holidays) (days 0-20 p.c.)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once a day, or more often when clinical signs of toxicity were elicited (days 0-20 p.c.)

BODY WEIGHT: Yes
- Time schedule for examinations: on days 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20 p.c.
The body weight change of the animals was calculated from these results

CORRECTED BODY WEIGHT GAIN (net maternal body weight change): Yes
- Time schedule: calculated after terminal sacrifice (terminal body weight on day 20 p.c. minus weight of the  unopened uterus minus body weight on day 6 p.c.)

FOOD CONSUMPTION: Yes
The consumption of food was recorded for the intervals GD 0-1, 1-3, 3-6, 6-8, 8-10, 10-13, 13-15, 15-17, 17-19 and 19-20.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: uteri and ovaries (weight of the unopened uterus, number of corpora lutea, number and distribution of implantation sites classified as live fetuses or dead implantations (early resorptions, late resorptions, dead fetuses)

SKIN EXAMINATION: Yes
The application site was examined twice (before and after the 6-hour exposure). Possible skin reactions were classified following OECD Guideline for Testing of Chemicals No. 404: Acute Dermal Irritation/Corrosion (adopted 24 Apr 2002). Classification was documented for each animal.
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
- Number of dead fetuses: Yes
- Calculations of conception rate and pre- and postimplantation losses
Fetal examinations:
At necropsy each fetus was weighed, sexed, and external tissues and all orifices were examined macroscopically. The sex was determined by observing the distance between the anus and the base of the genitalia. Furthermore, the viability of the fetuses and the condition of the placentae, the umbilical cords, the fetal membranes, and fluids were examined. Individual placental weights were recorded.
- 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
Statistics:
Simultaneous comparison of all dose groups with the control group using the DUNNETT-test (twosided) for the hypothesis of equal means was performed for: food consumption, body weight, body weight change, corrected body weight gain (net maternal body weight change), carcass weight, weight of unopened uterus, number of corpora lutea, number of implantations, number of resorptions, number of live fetuses, proportions of preimplantation loss, proportions of postimplantation loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight.

Pairwise comparison of each dose group with the control group using FISHER'S EXACT test (one-sided) for the hypothesis of equal proportions was performed for: female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings.

Pairwise comparison of each dose group with the control group using the WILCOXON- test (one-sided) for the hypothesis of equal medians was performed for: proportions of fetuses with malformations, variations and/or unclassified observations in each litter.
Indices:
The conception rate (in %): (number of pregnant animals/ number of fertilized animals) x 100

The preimplantation loss (in %): ((number of corpora lutea – number of implantations)/ number of corpora lutea) x 100

The postimplantation loss (in %): ((number of implantations – number of live fetuses)/ number of implantations) x 100
Historical control data:
included in report.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

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

Details on maternal toxic effects:
Clinical examinations of the dams:
Only pregnant dams were used for the study. Therefore, the following females were excluded:
- Test group 0 (0 mg/kg bw/day): one female – not pregnant
- Test group 3 (1000 mg/kg bw/day): one female – not pregnant

Mortality:
There were no substance-related or spontaneous mortalities in any females of all test groups.

Clinical symptoms:
No clinical signs or changes of general behavior, which may be attributed to the test sub-stance, were detected in any female at dose levels of 100, 300 or 1000 mg/kg bw/day during the entire study period.
One low-dose (100 mg/kg bw/day), three mid-dose (300 mg/kg bw/day) as well as one high-dose animal (1000 mg/kg bw/day) occasionally showed vaginal hemorrhage either before daily dermal application or after the daily 6-hour exposure time. This finding was observed in these individuals on GD 15-17 only, and is most likely related to handling and semiocclusive dressing, but not to the test substance itself. The assessment was also supported by the fact that no impairment of reproduction parameters became obvious.

Skin examination:
The treated skin of all female animals of all test groups was free from any notable findings during the entire study period.

Food consumption:
The mean food consumption of the dams in all test groups was comparable to the concurrent control throughout the entire study period.

Body weight data:
The mean body weights and the average body weight gains of all test substance-treated dams in test groups 1-3 were comparable to the controls throughout the entire study period. However, on GD 6-8, the mean body weight gain value of the high-dose dams (1000 mg/kg bw/day) was significantly lower (-37%), but recovered afterwards and was comparable throughout the remaining treatment period.

Corrected (net) body weight gain:
The corrected body weight gain of animals in all test groups was not influenced by treatment. The differences between these groups and the control group revealed no dose-dependency and were assessed to be without biological relevance.

Uterus weight:
The mean gravid uterus weights of the animals of the test groups were not influenced by the test substance. The differences between these groups and the control group revealed no dose-dependency and were assessed to be without biological relevance.

Necropsy findings:
No necropsy findings which could be attributed to the test substance were seen in any dam.

Reproduction data:
The conception rate was 96% in controls and in 1000 mg/kg bw/day animals, and 100% in test groups 100 and 300 mg/kg bw/day.
No test substance-related and/or biologically relevant differences between controls and the test groups were observed with regard to conception rate, mean number of corpora lutea and implantation sites or values calculated for the pre- and the postimplantation losses, the number of resorptions as well as viable fetuses. All observed differences were considered to reflect the normal range of fluctuations for animals of this strain and age. This includes the apparently lower mean number of viable female fetuses at 300 mg/kg bw/day.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: not determinable due to absence of adverse toxic effects

Results (fetuses)

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

Details on embryotoxic / teratogenic effects:
Sex distribution of the fetuses:
The sex distribution of the fetuses in the test groups was comparable to the control fetuses. The significantly lower mean number of viable female fetuses at 300 mg/kg bw/d was considered to be incidental and without any toxicological relevance.

Weight of the placentae:
The mean placental weights of the test groups were comparable to the corresponding control group.

Weight of the fetuses:
The mean fetal weights were comparable between the control and the test groups. Biologically relevant differences were not observed. The significantly lower mean weight of female fetuses in test group 100 mg/kg bw/d did not show a dose-response relationship and was clearly within the historical control data.

Fetal external malformations:
No external malformations were recorded.

Fetal external variations:
One external variation, i.e. limb hyperextension, was detected for one high-dose fetus (1000 mg/kg bw/d). This single finding was considered to be spontaneous in nature.

Fetal external unclassified observations:
No unclassified external observations were recorded.

Fetal soft tissue malformations:
No soft tissue malformations were recorded.

Fetal soft tissue variations:
Two soft tissue variations were detected, i.e. large liver lobe and dilated renal pelvis. The incidences of these variations were neither significantly nor dose-dependently increased. Therefore, all differences were not considered biologically relevant.

Fetal soft tissue unclassified observations:
No unclassified soft tissue observations were recorded.

Fetal skeletal malformations:
One skeletal malformation was detected in the high dose group (1000 mg/kg bw/day) affecting the fore-limb. This single finding can be found in the historical control data in a comparable frequency. An association to the treatment was not assumed.

Fetal skeletal variations:
For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeleton and appeared without a relation to dosing. The overall incidences of skeletal variations were comparable to the historical control data.
The increased incidence of the skeletal variation “wavy rib” was only marginally above the historical control data. Therefore, the finding was regarded to be incidental and not related to treatment.

Fetal skeletal unclassified cartilage observations
Some isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in all test groups. The observed unclassified cartilage findings were related to the skull, the sternum and ribs. They did not show any relation to treatment.

Effect levels (fetuses)

Key result
Dose descriptor:
other:
Remarks on result:
not determinable due to absence of adverse toxic effects

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Based on the analytical results it is concluded, that Pyranol is stable in corn oil over a period of 8 days at ambient temperature. The determined concentrations were in the range of 90 % - 110 % of the nominal concentration: between 98.7 and 100.5%.

 

The values of Pyranol in corn oil were found to be in the range of 90 % – 110 % of the nominal concentration (96.8-103.0%)

No test substance could be detected in the vehicle control sample with a concentration of ≥ 30 % of the lowest calibration solution.

These results demonstrated the correctness of the concentrations of Pyranol in corn oil.

Applicant's summary and conclusion

Conclusions:
Dermal application of Pyranol to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) at a dose level of 1000 mg/kg bw/day did not cause any evidence of maternal toxicity or prenatal developmental toxicity.
Based on the absence of substance- related adverse effects, the no observed adverse effect level (NOAEL) for maternal toxicity and for prenatal developmental toxicity was 1000 mg/kg bw/day, the highest tested dose.
In addition, as no local effects were observed , the NOAEL for local dermal effects in this study is 1000 mg/kg bw/day, the highest dose tested.
Executive summary:

In a prenatal developmental toxicity study the test substance Pyranol was applied dermally (6 hours/day) to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) for the evaluation of its potential maternal and prenatal developmental toxicity (according to OECD 414). With regard to clinical examinations, no signs of general systemic toxicity including food consumption and body weight gain were observed. No toxicologically relevant differences between the animals receiving the test substance and control animals were observed. No differences of toxicological relevance between the control and the treated groups (100, 300 or 1000 mg/kg bw/day) were determined for any reproductive parameters, such as conception rate, mean number of corpora lutea, mean number of implantations, as well as pre- and postimplantation loss. Similarly, no influence of the test substance on fetal weight and sex distribution of the fetuses was noted at any dose level. Overall, there was no evidence for toxicologically relevant, adverse effects of the test substance on fetal morphology at any dose level.

Based on the absence of substance- related adverse effects, the no observed adverse effect level (NOAEL) for maternal toxicity and for prenatal developmental toxicity was 1000 mg/kg bw/day, the highest tested dose.

In addition, as no local effects were observed , the NOAEL for local dermal effects in this study is 1000 mg/kg bw/day, the highest dose tested.