Ethyl 4-oxovalerate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

other: study in progress

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

Justification for Read Across is given in Section 13 of IUCLID

Data source

Reference

Reference Type:

other: details from study plan

Title:

Unnamed

Year:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

Adopted 25th June 2018

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

Council Regulation (EC) No. 440/2008, Published in O.J. L142, 2008.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Similar Substance 02_EL

IUPAC Name:

Similar Substance 02_EL

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Wistar Crl (SPF quality)

Details on species / strain selection:

Selection of animal species: laboratory rat has been chosen because the testing laboratory has a long experience with this species and has kept historical data base of examination results. Rat is also the preferred species as per the OECD TG Guideline 408.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Supplier: SPF breeding, VELAZ s.r.o., Únětice, Czech Republic, RČH CZ 11760500
Age at the beginning of the study: 6 – 7 weeks
Acclimatization: at least 5 days for the dose range finding study: 5 males and 5 females at each dose level
for the main study 10 males and 10 females at each dose level, 6 males and 6 females in satellite groups
Housing conditions:
- Grouping: 2 animals of the same sex in one cage
- Food: complete pelleted diet for rats
- Water: drinking water ad libitum,
- Light cycle:12 hour light / 12 hour dark ature: 22 ± 3°C
- Relative humidity: 30-70 %
- Air changes: 15 per hour
- Bedding: sterilised Lignocel or sterilized shavings of soft wood
- Selection of animals: random selection according to the internal rule – at the beginning of the study the weight variation of animals in groups of each sex should not exceed + 20 % of the mean weight
- Identification of animals: the animals will be identified by the colour marks on their fur each cage will be marked with the number of animals, sex, number of cage, name and dose level of the test item
- Animal housing: The study will proceed in SPF conditions according to internal SOP

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

Preparation of test formulation and Administration: The formulation of the test item will be administered to the stomach by gavage. The test item concentration at single dose level will be adjusted so that the administered volume will be constant at all dose levels – 1 ml/100 g body weight. The administration form of the test item will be prepared daily before administration and during administration it will be mixed by the magnetic stirrer.
Vehicle: Aqua pro iniectione

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and Homogeneity of test formulation: Before the start of the study stability and homogeneity of test formulation will be determined at the test facility by HPLC.

Duration of treatment / exposure:

90 days exposure
Post-exposure recovery period in satellite groups: 28 days

Frequency of treatment:

7 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

DRF

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

DRF

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

DRF

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

DRF

No. of animals per sex per dose:

10 females/males for main groups
6 females/males for satellite groups

Control animals:

yes, concurrent vehicle

Details on study design:

DOSE RANGE FINDING EXPERIMENT
- Duration: 28-days
- number of animals: 5 males and 5 females
- doses: 0-100-300-1000 mg/kg bw/day. Dose levels were determined by study monitor considering the available results of the available OECD 423 conducted on the substance. The acute toxicity estimate (ATE) was greater than 300 but lower than 2000 mg/kg body weight
Animals will be dosed daily by the oral gavage at 1 ml/100 g body weight.
Animals will be acclimatised for 5 days. The same housing conditions as in the main study apply for the DRFE. Wistar Crl (SPF quality – guaranteed) of 6-7 weeks will be used for the study.
- Examination methods
Health condition control: Control of the health condition will be performed once a day during animal handling before the application of the test item.
Body weight: The body weight of animals will be recorded on automatic balances with group average computing module. First weighing will be performed before the first application and then weekly. Weight increment will be computed as an average per group per week (in grams).
Clinical observations: During the whole study in scheduled intervals the clinical observation will be made in order to record possible clinical effects of the test item after application and all changes in behaviour of animals. Animals will be observed in natural conditions in their cages. Clinical observation: twice a day (except weekend when clinical observation will be performed once a day)
Mortality observation: twice a day (except weekend when mortality observation will be performed once a day)
Haematological examination: Before necropsy of animals the blood samples will be 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 corpuscular volume, haematocrit, haemoglobin concentration, total platelet count) will be determined on haematology analyser.
Pathological examination: During the necropsy (on the 29th day of study) a revision of the external surface of the body, of all orifices and the cranial, thoracic and abdominal cavities will be carried out. All macroscopic changes found will be recorded in protocols.
Biometry of organs: Terminal body weight and absolute weights of liver, kidneys, adrenal glands, brain, spleen, thymus, thyroid gland, stomach and heart will be recorded from all animals.
- Experimental data collection
Body weight: before the first application and then weekly
Health condition control: daily before application
Clinical observations: during the whole dose-range finding study the clinical observation will be practised in repeated intervals after application by the reason of finding a maximal effect of the test
item
Laboratory examinations: haematological examination (basic blood parameters) – 29th day; pathological examination (macroscopic changes) – 29th day; biometry of organs - 29th day

Examinations

Observations and examinations performed and frequency:

morbidity/mortality/viability: twice daily
body weight: weekly
food consumption: weekly
water consumption: three times per week
health condition control: daily - in acclimatization, administration and recovery period
general clinical observations: daily - during the administration period (after application) and recovery period
detailed clinical observations: once prior to start of administration, then in week intervals
functional observations: in the last week of study
ophthalmoscopic observation: before administration and in the last week of administration
period – at least vehicle control and the highest dose level
in the last week of recovery period
recovery period is 28 days
laboratory examinations (number of animals per group):
- urinalysis: 90th day of study 10M + 10F and 118th day of study 6M + 6F
- haematological examination: 91st day of study 10M + 10F and 119th day of study 6M + 6F
- biochemical examination: 91st day of study 10M + 10F and 119th day of study 6M + 6F
- biometry of organs: 91st day of study 10M + 10F and 119th day of study 6M + 6F
- pathological examination: 91st day of study 10M + 10F and 119th day of study 6M + 6F

Health condition control: The health condition will be controlled daily during the check-in, acclimatisation period, during the administration and during the recovery period in groups. Pre-experimental observation of all rats will be performed to ensure that only the animals exhibiting normal behavioural activity would be entered into the study. In administration period this observation will be performed before, during and immediately after application.

General clinical observation: All rats will be observed daily during the administration period.
This observation will be made in order to record possible clinical effects after application and all changes in behaviour of animals. So it will be done after application at the same time every day – at the time of expectation of maximal effect of the test item. Animals will be observed in natural conditions in their cages.

Detailed clinical observations – clinical observational battery
Detailed clinical observations will be made:
•the first part (in the cage) - posture, position of eyelids, tonic or clonic movements, piloerection, stereotypic or bizarre behaviour
•the second part (during the removal from cage) – reaction to handling, elasticity of skin, colour of mucous membranes, salivation, lacrimation, cleanliness of fur around foramina

Functional observation: Detailed clinical observations: sensory reactivity on auditory, visual and proprioceptive stimuli, grip strength, motor activity, pupillary reflex will be evaluated.

Ophthalmological examination: Both eyes will be examined by the means of an ophthalmoscope before the start of administration and the last week of study.
The ophthalmological examination will be performed in animals of control and high groups, if findings in the high group will be detected, animals of low and middle doses and recovery groups will be examined.

Urinalysis: The rats will be kept in the metabolic cages for the collection of urine for one hour. Immediately before entering metabolic cages the animals will be administered by 2 mL drinking water for 100g of body weight by gavage to the stomach.
The following parameters will be determined by analyser PocketChem PU-4210 (Arkray, Inc., Japan): volume, cloudiness, colour, odour, glucose, protein, bilirubin, urobilinogen, pH, specific weight, ketones, blood, nitrites and leucocytes.

Haematological examination
The blood samples for this examination will be taken from orbital plexus by glass micropipette under the light ether narcosis into the PVC test tubes (containing anticoagulation system), which will be prepared for individual examinations.
The following parameters will be determined: total erythrocyte count, total leucocyte count, total platelet count, haematocrit, haemoglobin concentration and average volume of erythrocyte by haematology analyzer URIT-5160Vet. The values of blood coagulation will be detected by Coagulometer ACL Coagulation System (Instrumentation Laboratory).

Clinical Biochemistry
The blood samples for this examination will be taken during the sampling for haematology. The animals will be starved for 18 hours before the blood collection, but they will be supported with drinking water ad libitum.
The blood serum samples will be prepared by spontaneous coagulation and they will be centrifuged 10 minutes in centrifuge.
Total protein, total bilirubin, urea, creatinine, glucose, transaminases (Aspartate Aminotransferase-AST, Alanine transaminase-ALT), total cholesterol, HDL, LDL, albumin, alkaline phosphatase (ALP), bile acids, triglycerides, cholinesterase, phosphorus and calcium will be measured by automatic clinical chemistry analyser Accent-200 (Cormay, Poland). Sodium, potassium and chloride ions will be measured by automatic electrolyte analyser with ion-selective electrodes SPOTCHEMTM EL SE-1520 (Arkray, Inc.).
Blood samples from males and females will be assessed for serum levels of thyroid hormones thyroxine (T4, T3 and TSH) by ELISA kit. The examination of thyroid hormones will be conducted in animals of control and high group of main and recovery groups, if findings in the high group are detected, samples of low and middle doses and recovery groups will be analysed.

Biometry of organs
Terminal body weight and absolute weights of liver, kidneys, adrenal glands, brain, testes, epididymis, prostate gland, seminal vesicles with coagulating glands, ovaries, spleen, thymus, uterus, pituitary gland, thyroid gland and heart will be recorded from all animals.

Sacrifice and pathology:

Pathological examination
In the study all animal of each group will be necropsied and all macroscopic abnormalities will be recorded. Careful examination of the external surface of the body, all orifices and the cranial, thoracis and abdominal cavities and their contents will be recorded. At necropsy the oestrus cycle of all females will be determined ty taking vaginal smears. Samples of the following tissues and organs will be collected from all animals at necropsy and fixed in buffered 4% formaldehyde solution (v/v) for eventual further histopathology evaluation:
Adrenal glands, aorta, bone marrow (femur), brain, male and female mammary gland area, heart, small and large intestines (including Peyer´s patches), kidneys, liver, lungs (infused with formalin), lymph nodes – paraaortal and mesenteric, oesophagus, ovaries, pancreas, pituitary gland, prostate gland, rectum, salivary glands, seminal vesicle, sciatic nerve, spleen, spinal cord (cervical, mid-thoracis, lumbar), skeletal muscle, skin, stomach, thymus, thyroid gland, parathyroid gland, trachea, urinary bladder, uterus with cervix, vagina and all gross lesions.
Testes and epididymis will be fixed in modified Davidson’s fixative for further histopathology evaluation.

Histopathological examination
•all tissues collected at the scheduled sacrifice from all animals of the control and the high dose group
•if treatment-related changes will be found the target organs will be investigated in medium and low dose group too
•all tissues collected at the scheduled sacrifice from all animals of the control satellite and the high dose satellite group
For histopathological processing the routine histological paraffin technique with synoptic haematoxylin-eosin staining will be used. Other special staining will be used in indicated cases.

Statistics:

Statistical treatment of main study results
For statistical evaluation the software Statgraphic ® Centurion (version XVII, USA) will be used.
Males/females from control group will be compared with males/females from three treated groups. Satellite males/females from control group will be compared with satellite males/females from treated group. The results statistically significant on probability level 0.05 will be indicated

Results and discussion

Effect levels

Dose descriptor:

NOAEL

Remarks on result:

other: to be determined

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Executive summary:

The test item is orally administered daily in graduates doses to several groups of experimental animals, one dose per group for a period of at least 90 days. During the period of administration, the animals are observed closely for signs of toxicity of the test item. In the end of administration period the blood collection for haematological and biochemical examination are performed. Animals are humanely killed and necropsied. The organs for biometry of organs and histopathological examination are collected.