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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Remarks:
DRF for OECD 414 study
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
18-11-2021 to Pending final QA report issue
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
DRF for OECD 414 study

Data source

Reference
Title:
Unnamed
Year:
2022

Materials and methods

Test guideline
Qualifier:
no guideline required
GLP compliance:
no
Remarks:
DRF study, GLP not required

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of 3,5-dimethylcyclohex-3-ene-1-carbaldehyde and 2,4-dimethylcyclohex-3-ene-1-carbaldehyde
EC Number:
943-728-2
Molecular formula:
C9H14O
IUPAC Name:
Reaction mass of 3,5-dimethylcyclohex-3-ene-1-carbaldehyde and 2,4-dimethylcyclohex-3-ene-1-carbaldehyde
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Sponsor; SHT036
- Purity, including information on contaminants, isomers, etc.: 99.48% (sum of isomers)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Store at laboratory conditions, protected from heat and light

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Wistar Crl
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: SPF breeding, VELAZ s.r.o., Dědinská 893/29, 161 00 Prague 6 - Ruzyně, Czech Republic, RČH CZ 11760500
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 12 weeks
- Housing: IVC TOUCH animal house of CETA; sterilized shavings of soft wood or Lignocel
- Diet: Maintenance pelleted diet for rats and mice - Altromin for rats/mice, Manufacturer: Altromin Spezialfutter GmbH & Co. KG, Germany
- Water: drinking water ad libitum, quality corresponding to the Regulation No. 252/2004 of Czech Coll. of Law
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30-70 %
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Analytical verification of doses or concentrations:
no
Remarks:
Stability and homogeneity were confirmed in a separate analytical study (refer to Key study record)
Details on mating procedure:
After acclimatization females were mated with males (1 male and 2 females). Vaginal smears were carried out daily in the morning to control fertilization (first time: 24 hours after placing the male to the females). Presence of sperm was examined. Day 0 of pregnancy was the day on which sperm in vaginal smears were observed. Pregnant females were randomly distributed to experimental groups.
Duration of treatment / exposure:
Test item administration lasted from implantation (the 5th day after fertilization) to one day prior to the day of scheduled euthanasia (the 19th day after fertilization).
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
6 females
Control animals:
yes, concurrent vehicle

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
Mortality/viability:twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice a day (except weekend, when clinical observation was performed once a day)

BODY WEIGHT: Yes
- Time schedule for examinations: 1st, 5th, 8th, 11th, 14th, 17th and 20th day of pregnancy

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: On the 20th day of pregnancy the females were euthanized. The revision of the external surface of the body was performed. During macroscopy, all orifices, the cranial, thoracic and abdominal cavities were examined and uterus (incl. the cervix) was removed and weighed.

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included: In gravid uteri, the number of viable foetuses, number of dead foetuses, number of early resorption (implantation without recognizable embryo/foetus) and number of late resorption (dead embryo or foetus with external degenerative changes) were recorded. The numbers of corpora lutea of both ovaries were recorded.
Blood sampling:
Before necropsy of animals the blood samples were collected from the orbital plexus by glass micropipette under the light diethyl ether narcosis into the PVC test tubes containing anticoagulation systems. Basic blood parameters (total erythrocyte count, total leucocyte count, mean corpuscule volume, haematocrit, haemoglobin concentration, total platelet count) was determined on haematology analyser.
Fetal examinations:
Sex, individual and body weights of foetuses were recorded. Each foetus was examined for external alterations and soft tissue alterations.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
No changes of animal health status or clinical symptoms of intoxication were observed in animals at the dose level 100 mg/kg bw/day. Piloerection was observed in one female at 500 mg/kg bw/day from the 1st to the 3rd day of application. Generally, piloerection and decreased reaction to stimuli (auditory, visual and proprioceptive) were recorded after application of the test item in females at the dose level 1000 mg/kg bw/day. The symptom of intoxication – decreased reaction to stimuli disappeared by the next day in females at 1000 mg/kg bw/day.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weights of pregnant females at all dose levels were comparable to the control group during the whole study period. The body weight increment was reduced at the dose level 1000 mg/kg bw/day.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The total leucocyte counts were increased at the dose levels 500 and 1000 mg/kg bw/day in comparison with the control. The haematocrit was slightly increased in all treated groups compared to the control group. Other haematological parameters did not show significant differences between dosed and control group.
Clinical biochemistry findings:
not examined
Endocrine findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic examination was performed in all females (including females without foetuses, except non-paired female No. 106). No finding related with treatment was noted at necropsy in all treated females.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
The number of implantations was slightly decreased at the dose level 100 mg/kg bw/day in comparison with the control group. The numbers of corpora lutea were comparable in all treated groups with the control group.
Total litter losses by resorption:
effects observed, treatment-related
Description (incidence and severity):
The number of resorptions was increased at the dose level 1000 mg/kg bw/day in comparison with the control group.
Early or late resorptions:
effects observed, treatment-related
Description (incidence and severity):

At 1000 mg/kg bw/day, there was 1 early resorption in 1 female, 16 early resorptions in 1 female, and 6 early resorptions in 1 female.
Dead fetuses:
effects observed, treatment-related
Description (incidence and severity):
Four dead foetuses were recorded at the dose level 1000 mg/kg bw/day.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
effects observed, treatment-related
Description (incidence and severity):
One female at the dose level 1000 mg/kg bw/day became pregnant and then all implanted conceptuses in the uterus were totally resorbed (females without foetuses but with implantations and resorptions). The number of non-pregnant females was as follows: 2 – 0 – 0 – 0, respectively for the dose levels 0 – 100 – 500 – 1000 mg/kg bw/day.

Effect levels (maternal animals)

Dose descriptor:
other: No NOAEL; doses selected for main study

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weight of all foetuses was decreased at the dose level 1000 mg/kg bw/day compared to the control foetuses. Male foetuses were heavier than females in all groups.
Reduction in number of live offspring:
not examined
Changes in sex ratio:
effects observed, non-treatment-related
Description (incidence and severity):
The mean number of foetuses per litter was slightly decreased at dose levels 100 mg/kg bw/day compared to the control group. Also the mean number of female foetuses was markedly decreased at this dose level.
Changes in litter size and weights:
not examined
Anogenital distance of all rodent fetuses:
not examined
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Cranial meningocele, lip cleft and exophthalmos were recorded in one foetus at the dose level 100 mg/kg bw/day. This was considered as a sporadic finding and probably not related to the test item treatment. No other macroscopic changes of soft tissues and external alteration were found during the pathological examination of the foetuses at all dose levels.
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed

Effect levels (fetuses)

Dose descriptor:
other: No NOAEL; doses selected for main study

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

Refer to Tables 3 - 9 attached.

Applicant's summary and conclusion

Conclusions:
After evaluation of the results of the DRF, the following dose levels – 0, 100, 300 and 1000 mg/kg bw/day will be used for the main Prenatal Developmental Toxicity Study.

Executive summary:

In a dose range finding study (no guideline) for a prenatal developmental toxicity in rats (OECD 414/GLP), the test item (99.48% (sum of isomers)) was administered to pregnant Wistar Crl rats (6/dose) by oral gavage in olive oil at dose levels of 0, 100, 500 and 1000 mg/kg bw/day daily from GD 5-19.



Twenty-four females (6 for each group) were mated in the study. The number of confirmed pregnant evaluated dams was 4 in the Control and 6 in all test item dose groups. No unscheduled death of maternal animals was recorded during the DRF.


 


No changes of animal health status or clinical symptoms of intoxication were observed in animals at the dose level 100 mg/kg bw/day. Piloerection was observed in one female at 500 mg/kg bw/day from the 1st to the 3rd day of application. Generally, piloerection and decreased reaction to stimuli (auditory, visual and proprioceptive) were recorded after application of the test item in females at the dose level 1000 mg/kg bw/day with the latter resolving the next day.The body weights of pregnant females at all dose levels were comparable to the control group during the whole study period. The body weight increment was reduced at the dose level 1000 mg/kg bw/day.


 


The total leucocyte counts were increased at the dose levels 500 and 1000 mg/kg bw/day in comparison with the control. The haematocrit was slightly increased in all treated groups compared to the control group. Other haematological parameters did not show significant differences between dosed and control group.


 


Macroscopic examination was performed in all females (including females without foetuses). No finding related with treatment was noted at necropsy in all treated females.


 


During necropsy on the 20th day of pregnancy, foetuses and implantations were not found in all females. One female at the dose level 1000 mg/kg bw/day became pregnant and then all implanted conceptuses in the uterus were totally resorbed (females without foetuses but with implantations and resorptions). The number of implantations was slightly decreased at the dose level 100 mg/kg bw/day in comparison with the control group. The numbers of corpora lutea were comparable in all treated groups with the control group. At 1000 mg/kg bw/day, there was 1 early resorption in 1 female, 16 early resorptions in 1 female, and 6 early resorptions in 1 female.



The mean body weight of all foetuses was decreased at the dose level 1000 mg/kg bw/day compared to the control foetuses. Male foetuses were heavier than females in all groups. The mean number of foetuses per litter was slightly decreased at dose levels 100 mg/kg bw/day compared to the control group. Also the mean number of female foetuses was markedly decreased at this dose level. Four dead foetuses were recorded at the dose level 1000 mg/kg bw/day. Cranial meningocele, lip cleft and exophthalmos were recorded in one foetus at the dose level 100 mg/kg bw/day. This was considered as a sporadic finding and probably not related to the test item treatment. No other macroscopic changes of soft tissues and external alteration were found during the pathological examination of the foetuses at all dose levels.


 


Based on the results of the DRF, the following doses were selected for the main study: 0, 100, 300 and 1000 mg/kg bw/day.