Fatty acids, C16-18 (even numbered, C18 unsaturated), 2-ethylhexyl esters, epoxidized

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

other information

Study period:

February 2021

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

dose finding test study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

14-day dose-range-finding study with daily intake concentrations of 0, 300, 1000 mg/kg bw of the test substance. The test substance was administered orally by gavage at a dose volume of 4 mL/kg bw. Control animals were dosed with the vehicle alone (corn oil).

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, C16-18 (even numbered, C18 unsaturated), 2-ethylhexyl esters, epoxidized

Molecular formula:

Unspecified

IUPAC Name:

Fatty acids, C16-18 (even numbered, C18 unsaturated), 2-ethylhexyl esters, epoxidized

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch number of test material: 0022865630
- BASF compound no.: 12/0076-2
- Retest date: 27 May 2021
- The determination of the identity, strength, purity, composition and stability of the test item was the responsibility of the Sponsor.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient temperature
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: The analytical method was qualified in the present study in the range from 75 to 250mg/mL. Samples of the preparations prepared in Week 1 were analysed to check the homogeneity
and concentration. The results of the analysis were within the acceptance limit for concentration and homogeneity.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- the test item was dissolved/suspended in the vehicle to reach required concentrations
- preparations were made daily
- concentrations were calculated and expressed in terms of test item as supplied

FORM AS APPLIED IN THE TEST (if different from that of starting material)
- in the vehicle: corn oil

OTHER SPECIFICS
- Appearance: Clear yellowish liquid

Test animals

Species:

rat

Strain:

other: Wistar Hannover

Details on species / strain selection:

The Wistar Hannover rat was the species and strain of choice because it is accepted by many regulatory authorities and there are ample experience and background data on this species and strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS srl, San Pietro al Natisone (UD), Italy.
- Females (if applicable) nulliparous and non-pregnant: no data
- Age at study initiation: 9-10 weeks
- Weight at study initiation: 200-225 g for males and 175-200 g for females
- Fasting period before study: no data
- Housing: 5 animals of one sex per cage, in clear polysulfone solid bottomed cages
- Diet (e.g. ad libitum): laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019, SettimoMilanese (MI), Italy), ad libitum
- Water (e.g. ad libitum): water bottles, ad libitum
- Acclimation period: 6 weeks

DETAILS OF FOOD AND WATER QUALITY:
There was no information available to indicate that any non-nutrient substance likely to influence the effect of the test item was present in the drinking water or the diet.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C±2 °C
- Humidity (%): 55%±15 %
- Air changes (per hr): 15-20
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 09 February 2021 To: 26 February 2021

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The oral route was selected as it is a possible route of exposure of the test item in man and has been specifically requested by the Regulatory Authorities.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): Daily

VEHICLE
- The vehicle was corn oil.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis was performed in order to validate the analytical method, the preparation procedure and to verify the stability of the preparations. Samples of the preparations prepared in week 1 were analysed to check the homogeneity (in the case of suspension) and concentration.
Results were within the limit of the acceptance.

Duration of treatment / exposure:

14 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

low dose

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

high dose

No. of animals per sex per dose:

4

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels have been selected in consultation with the Sponsor (dose range finding study)
- The rats were allocated to the 3 groups by computerised stratified randomisation to give approximately equal initial group mean body weights.

Examinations

Observations and examinations performed and frequency:

MORTALITY: Yes
- Time schedule: Throughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and public holidays, a similar procedure were followed except that the final check was carried out at approximately mid-day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All clinical signs were recorded for individual animals. Once before commencement of treatment and daily during the study, each animal was observed and any clinical signs was recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions.

BODY WEIGHT: Yes
- Time schedule for examinations: Each animal was weighed on the day of allocation, twice weekly thereafter and on the day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- The weight of food consumed by each cage of rats was recorded at the same intervals as per body weight following allocation, where possible. The group mean daily intake per rat was calculated.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: as part of sacrificial procedure
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all viable animals
- Following parameters were examined: Haematocrit, Haemoglobin, Red blood cell count, Reticulocyte count, mean red blood cell volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, white blood cell count, differential leucocyte count (Neutrophils, Lymphocytes, Eosinophils, Basophils, Monocytes, large unstained cells), Platelets

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: as part of sacrificial procedure
- Animals fasted: Yes
- How many animals: all viable animals
- Following parameters were examined: Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Urea, Creatinine, Glucose, total bilirubin, total cholesterol, total protein, Sodium, Potassium, Calcium, Chloride, Bile Acids

PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (Adrenal glands, Liver, Kidneys, Spleen)

HISTOPATHOLOGY: No

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Sign of hairloss, from slight to moderate severity, was recorded in 3 out of 4 females at 1000 mg/kg/day (see table 1). No clinical signs were observed in males at both dose levels or in females at 300 mg/kg/day.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

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):

No treatment related changes were recorded. Males dosed at 1000mg/kg/day showed mean corpuscular haemoglobin concentration statistically significantly higher than controls (4%) and eosinophils statistically significantly lower than controls (46%). Changes were not consistent between sexes and were within the range of expected biological variation, therefore they were considered to be incidental.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment related changes were recorded. The statistically significant increase of calcium recorded in females dosed at 300 mg/kg/day (13%) was not dose related, therefore it was considered to be incidental.

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:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At post mortem evaluation treated males did not show treatment-related changes. Three out of 4 females receiving test item at 1000 mg/kg/day showed moderate hairloss mainly in the forelimbs, hindlimbs and thoracic region or in scapular region in females nos. Such findings could be considered treatment-related. The macroscopic changes noted in single females dosed at 300 mg/kg/day as single dark, depressed area - 2x1mm - in the glandular region of the stomach or unilateral (right) moderate pelvic dilatation in kidney were considered most likely stress related or incidental, respectively. Any other macroscopic observations were within the range of observed and expected incidental or spontaneous pathological changes in rats of the same age and considered unrelated to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Effect levels

Dose descriptor:

NOAEL

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1: 2 WEEK PRELIMINARY ORAL TOXICITY STUDY IN RATS: Appearance of hairloss (Females)

Females	Control		300 mg/kg/day		1000 mg/kg/day
	a	b	a	b	a	b
Hairloss, Suprascapular, Bilateral, Moderate	0	0	0	0	1	1.0
Hairloss, Suprascapular, Bilateral, Slight	0	0	0	0	1	1.0
Hairloss, Thoracic, ventral, Moderate	0	0	0	0	1	10.0
Hairloss, Upper forelimb, Slight	0	0	0	0	2	2.0
Hairloss, Ventrum, Moderate	0	0	0	0	1	1.0

a = Number of animals affected
b = Number of days with clinical sign/animal

 

Table 2:

Applicant's summary and conclusion

Conclusions:

Based on the results, it can be concluded that dose levels up to 1000 mg/kg/day can be chosen for a subsequent reproductive toxicity study.

Executive summary:

The aim of this preliminary study was to investigate toxic effects of the test substance when given at 300 and 1000 mg/kg/day for 2 consecutive weeks. The animals received the test item, or vehicle for the control rats, by oral gavage at the dose volume of 4 mL/kg. At completion of 2 week treatment period, no changes were seen in the body weight and food consumption throughout the study. No clinical signs were recorded with the exception of hairloss observed in females at 1000 mg/kg/day. No treatment-related changes were seen at clinical pathology  examination. The necropsy confirmed the presence of hairloss area in females at 1000 mg/kg/day. No differences were found in the absolute or relative organ weights.