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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
DRF for OECD 408 test
Adequacy of study:
supporting study
Study period:
01.12.2021 - Pending final QA report issue
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: DRF for OECD 408 study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2022

Materials and methods

Test guideline
Qualifier:
no guideline required
GLP compliance:
no
Remarks:
DRF study so no GLP 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
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River SPF breeding, supplied via VELAZ s.r.o.,
Czech Republic, RČH CZ 11760500
- Age at study initiation: 8 weeks on arrival
- 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
- Air changes: 15 per hour

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The concentrations of suspension at all dose levels were adjusted to ensure the administration of 1 mL per 100 g of body weight. For each dose level concentration, the suspension was prepared separately. The administration of the test item to animals was performed during one hour after preparation of application form. The stirring of suspension continued during administration.

VEHICLE
The test item was administered to the stomach by gavage as a suspension in an olive oil (olive oil (batch number: 20D09-T10; CAS number: 8001-25-0)).The vehicle control group was administered olive oil in the same volume.
Analytical verification of doses or concentrations:
no
Remarks:
Stability and homogeneity were confirmed in a separate analytical study (refer to Key study record)
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
62.5 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
6 males and 6 females per dose
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
Control of the health condition was performed once a day during animal handling before the application of the test item.

DETAILED CLINICAL OBSERVATIONS: Yes
During the whole study in scheduled intervals the clinical observation were made in order to record possible clinical effect of the test item after application and all changes in behaviour of animals. Animals were observed in natural conditions in their cages.

BODY WEIGHT: Yes
The body weight of animals was recorded on automatic balances with group average computing module.
First weighing was performed before the first application and then weekly. Weight increment will be computed as an average per group per week (in grams).

HAEMATOLOGY: Yes
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) will be determined on haematology analyser.
Sacrifice and pathology:
The animals (on the 28th day of study) was humanely killed under ether anaesthesia by breaking the cervical spinal cord. During the necropsy a revision of the external surface of the body, of all orifices and the cranial, thoracic and abdominal cavities were carried out. All macroscopic changes found were recorded in protocols.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No changes of animal health status or clinical symptoms of intoxication were observed in animals at the dose levels 62.5 and 250 mg/kg bw/day and in animals of the control group. In males dosed at 1000 mg/kg bw/day, decreased response to stimuli (auditory, visual and proprioceptive) was observed from the 4th to 8th day of the application period. In male No. 34 piloerection and apathy were also observed on the 9th and 10th days of the application period. No changes of animal health status or clinical symptoms of intoxication from the 9th day to the end of study in males at the dose level 1000 mg/kg bw/day were observed.


No changes of animal health status or clinical symptoms of intoxication were observed in animals at the dose levels 62.5 and 250 mg/kg bw/day and in animals of the control group. In females dosed at 1000 mg/kg bw/day, decreased response to stimuli (auditory, visual and proprioceptive) was observed from the 4th to 8th day of the application period. No changes of animal health status or clinical symptoms of intoxication from the 9th day to the end of study in females at the dose level 1000 mg/kg bw/day were observed.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
During the DRFE one male rat (at the dose level 1000 mg/kg bw/day) died on the 10th day of the study. The cause of death was not identified during necropsy.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Upon necropsy, the body weights of all treated males were lower compared to the control group. The decrease in body weight was marked in the first week of application but then recovered throughout the remaining weeks of the study.

Upon necropsy, the body weights of all treated females were slightly higher compared to the control group. Body weight of females at the dose level 1000 mg/kg bw/day was slightly increased every week of the study, compared to the 62.5 and 250 mg/kg bw/day dose groups.
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, non-treatment-related
Description (incidence and severity):
The values of total leucocyte count, total erythrocyte count and haematocrite were slightly decreased in males at the dose level 1000 mg/kg bw/day, in comparison with the control group.

Other parameters did not show differences among dose levels.
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):
No macroscopic changes were observed during necropsy of treated males and control males. The cause of death of male rate No. 34 (1000 mg/kg bw/day, Day 10) was not identified during necropsy.

No macroscopic changes were observed during necropsy of treated females and control females.
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Effect levels

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

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Refer to Tables 3-5 attached.

Applicant's summary and conclusion

Conclusions:
On the basis of the results given above the following dose levels – 62.5, 250 and 1000 mg/kg bw/day were proposed for the main Repeated Dose 90-day Oral Gavage Toxicity Study.
Executive summary:

In a dose range finding study (no guideline), for a repeated dose 90 day oral toxicity study (OECD 408/GLP), the test item (99.48% (sum of isomers)) was administered to
Wistar Crl rats (6/sex/dose) by gavage in olive oil at dose levels of 0, 62.5, 250 and 1000 mg/kg bw/day for 28 days.

One male at the high dose (1000 mg/kg bw/day) died on Day 10. In all male and female animals at the high dose (1000 mg/kg bw/day), decreased response to stimuli (auditory, visual and proprioceptive) was observed from the 4th to 8th day of the application period. Clinical observation of animals at the mid dose level (250 mg/kg bw/day) and low dose level (62.5 mg/kg bw/day) did not detect any impact of the test item on the health condition of the animals.


 


Upon necropsy, the body weights of all treated males were lower compared to the control group. The decrease in body weight was marked in the first week of application but then recovered throughout the remaining weeks of the study. Upon necropsy, the body weights of all treated females were slightly higher compared to the control group. Body weight of females at the dose level 1000 mg/kg bw/day was slightly increased every week of the study, compared to the 62.5 and 250 mg/kg bw/day dose groups.
Haematological examination showed slightly decreased values of total leucocyte count, total erythrocyte count and haematocrit in males at the high dose (1000 mg/kg bw/day) in comparison with the control group. Other examined parameters in all other males and females did not show differences among dose levels.


 


No macroscopic changes were observed during necropsy of treated male and female animals. The cause of death of one male rat (1000 mg/kg bw/day, Day 10) was not identified during necropsy.




On the basis of the results given above the following dose levels – 62.5, 250 and 1000 mg/kg bw/day were proposed for the main Repeated Dose 90-day Oral Gavage Toxicity Study.