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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
25 Jun 2018
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Etocrilene
EC Number:
226-029-0
EC Name:
Etocrilene
Cas Number:
5232-99-5
Molecular formula:
C18H15NO2
IUPAC Name:
ethyl 2-cyano-3,3-diphenylprop-2-enoate
Specific details on test material used for the study:
- Physical state/appearance: Solid/ white
- Storage condition of test material: At room temperature

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Details on species / strain selection:
The rat is a frequently used laboratory animal, and there is comprehensive experience with this animal species. Moreover, the rat has been proposed as a suitable animal species by the OECD and the EPA for this type of study.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services Germany GmbH, Sulzfeld, Germany
- Age at study initiation: 42 ± 1 days
- Fasting period before study: no
- Housing: Group housing (5 animals per cage) in H-Temp polysulfonate cages type 2000P supplied by TECNIPLAST (floor area about 2065 cm2). Dust-free wooden bedding was used in this study. Wooden gnawing blocks (SAFE® block large; J. Rettenmaier & Söhne GmbH + Co KG, Rosenberg, Germany), and large play tunnels (art. No. 14153, supplied by PLEXX B.V., Elst, The Netherlands) were added for environmental enrichment.
- Diet (e.g. ad libitum): ground Kliba maintenance diet mouse/rat “GLP”, meal (Granovit AG, Kaiseraugst, Switzerland), ad libitum.
- Water (e.g. ad libitum): drinking water, ad libitum.
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY:
- The supplier assayed the food used in the study for chemical and microbiological contaminants.
- The drinking water was regularly assayed for chemical contaminants by the municipal authorities of Frankenthal and by the Environmental Analytics Water/Steam Monitoring Department of BASF SE as well as for the presence of microorganisms by a contract laboratory.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 09.02.2021 To: 14.05.2021

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The oral route was selected since this was proven to be suitable for the detection of a toxicological hazard.
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5%
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was applied as a suspension. To prepare this suspension, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, deionized water containing 0.5% sodium carboxymethyl cellulose was filled up to the desired volume and subsequently mixed with a magnetic stirrer. During administration, the test substance preparations were kept homogeneous by stirring with a magnetic stirrer.
The administration volume was 10 mL/kg body weight.

VEHICLE
- Concentration in vehicle: deionized water containing 0.5% sodium carboxymethyl cellulose.
- Amount of vehicle (if gavage): 10 mL/kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- The stability of the test substance in 0.5% sodium carboxymethyl cellulose was given over a period of minimum 7 days at room temperature.
- Homogeneity was verified in 3 samples of the highest and lowest concentration (it was used as a concentration control at the same time) at the beginning and towards the end of the administration period; additional concentration control analyses were done in the mid concentration.
Duration of treatment / exposure:
90 days
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Group 0 (vehicle)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Group 1
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Group 2
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Group 3
No. of animals per sex per dose:
10 animals/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose level rationale: In a combined repeated dose toxicity study with the reproductive/developmental screening test (OECD 422) in Wistar rats, the NOAEL (no observed adverse effect level) for general, systemic toxicity was 300 mg/kg bw/d based on decreased food consumption and decreased body weight parameters as well as clinical chemistry changes at 1000 mg/kg bw/d. The NOAEL for reproductive performance and fertility was 1000 mg/kg bw/d for the F0 parental rats. The NOAEL for developmental toxicity in the F1 offspring was 1000 mg/kg bw/d, the highest tested dose. Based on these data the dose level for this study was set to 100, 300 and 1000 mg/kg bw/d.

- Fasting period before blood sampling for clinical biochemistry: about 16 to 20 hours
Positive control:
none.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. All animals were checked daily for any abnormal clinically signs before the administration as well as within 2 hours and between 2 and 5 hours after the administration.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: performed in all animals before the beginning of the administration period (day 0) and subsequently once a week (in the morning).
- Parameters examined: refere to "Any other information on materials and methods incl. tables".

BODY WEIGHT: Yes
- Time schedule for examinations: performed in all animals before the start of the administration period and during the administration period on day 0 and thereafter at weekly intervals.

FOOD CONSUMPTION: Yes
- Time schedule: weekly

WATER CONSUMPTION: Yes
- Time schedule: daily

OPHTHALMOLOGICAL EXAMINATION: Yes
- Time schedule: Day 0 (all animals), Day 91 (control and high-dose animals)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: before necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters: refere to "Any other information on materials and methods incl tables".

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: before necropsy
- Animals fasted: Yes
- How many animals: all
- Parameters: refere to "Any other information on materials and methods incl tables".

BLOOD AND SERUM: Yes
- Time of blood sample collection: In the morning
- Animals fasted: Yes
- How many animals: all
- Thyorid hormone determination: Yes (including T3, T4, TSH determination)

URINALYSIS: Yes
- Time schedule for collection of urine: overnight
- Animals fasted: Yes
- Parameters examined: refere to "Any other information on materials and methods incl. tables".

NEUROBEHAVIOURAL EXAMINATION: Yes
- Functional observation battery (FOB) was performed in all animals at the end of the administration period starting at about 10:00 h.
Parameters examined: refere to "Any other information on materials and methods incl. tables".
-The motor activity (MA) was measured from 13:00 h onwards on the same day as the FOB was performed.

ESTROUS CYCLE DETERMINATION: Yes
- Time schedule for examinations: Vaginal smears for terminal vaginal cytology examinations were prepared in the morning of the day of sacrifice.


Sacrifice and pathology:
PATHOLOGY: Yes (organs examined: refere to "Any other information on materials and methods incl. tables").
- Organ weights
- Fixation
- Histopathology
Statistics:
Please refere to table 1-3 in the section "Any other information on materials and methods incl. tables".

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Male animal No. 21 of test group 3 (1000 mg/kg bw/d) showed skin lesion to the ear region from study day 21 to study day 48.
For the remaining animals no clinical findings were observed for male and female animals of test groups 0-3 (0, 100, 300 and 1000 mg/kg bw/d).
On study days 24 and 86 the post dosing symptomatic (2-5 h after treatment) was entered into the system after the 5 hours. The clinical observations in the animal house took place within the 2 to 5 hours and was therefore only entered late into the system.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female animal (animal 47, test group 0; 0mg/kg bw/d; study day 27) died prematurely in the present study.
Body weight and weight changes:
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of the administration period, in males of test group 2 (300 mg/kg bw/d) absolute reticulocyte counts were significantly increased. In females of test group 1 (100 mg/kg bw/d) relative neutrophil counts were significantly increased whereas relative lymphocyte counts were significantly decreased. All mentioned alterations were not dose dependent. Therefore, these changes were regarded as incidental and not treatment related.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the administration period, in males of test groups 2 and 3 (300 and 1000 mg/kg bw/d) total bilirubin values were significantly decreased. In absence of any signs of anemia, this change was most probably due to an increased conjugation rate of bilirubin followed by an accelerated excretion via the bile. This effect is considered as treatment related but adaptive rather than adverse.
Endocrine findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
The significant increase of the absolute adrenal weight in males of test group 1 was not dose dependent and showed no histopathological correlate and was therefore regarded as incidental.

The significant increase in relative liver weight in males of test groups 2 and 3 (2.226%, +4.4%; 2.31%, +8.3%) was within the historical control range (2.045%, - 2.34%). The significant increase in relative liver weights of females of test group 3 (2.492%, +6.2%) was only marginally above the historical control range (2.28% - 2.49%). The absolute liver weights were not significantly changed, and the relative weight changes were not correlated to macroscopical or histopathological findings and additionally no alteration of liver enzyme values were detected in clinical chemistry. Therefore, the changes were regarded as not treatment-related.
For details please refere to Table 4 and 5 in section "Any other information on results incl tables".
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: systemic toxicity

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

 


Table 4: Absolute organ weights



























 Male animals
Test group
(mg/kg)

1


(100)




(300)



3


(1000)


Final body weight-2.3%-6.9%-2.8%
Adrenal glands+13.1%*-3.7%+11.6%

* : p <= 0.05, **: p <= 0.01


 


Table 5: Relative organ weights




























 Male animalsFemale animals
Test group
(mg/kg)

1


(100)



2


(300)



3


(1000)



1


(100)



2


(300)



3


(1000)


Liver+2.6%+4.4%*+8.3%**-1.5%+1.6%+6.2%*

* : p <= 0.05, **: p <= 0.01

Applicant's summary and conclusion

Conclusions:
The administration of the test substance by gavage for 3 months to male and female Wistar rats did
not cause any test substance-related, adverse signs of toxicity in male and female animals up to a concentration of 1000 mg/kg bw/d. Therefore, under the conditions of the present study the NOAEL was 1000 mg/kg bw/d in male and female Wistar rats.
Executive summary:

The following test substance-related, relevant findings were noted:
Test group 3: 1000 mg/kg bw/d
Clinical Examinations, Clinical Pathology and Pathology
• No treatment-related, adverse effects were observed.
Test group 2: 300 mg/kg bw/d
Clinical Examinations, Clinical Pathology and Pathology
• No treatment-related, adverse effects were observed.
Test group 1: 100 mg/kg bw/d
Clinical Examinations, Clinical Pathology and Pathology
•No treatment-related, adverse effects were observed.