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

Endpoint summary

Description of key information

A repeated dose study via the oral route (OECD 422, GLP) with wistar rats is currently ongoing. Furthermore a Repeated Dose 90-day oral toxicity study in rodents according to OECD 408 is proposed.

A 2 week preliminary oral toxicity dose range finding study has already been performed. Based on the results of this study, it can be concluded that dose levels up to 1000 mg/kg/day can be chosen for a subsequent reproductive toxicity study.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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

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

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

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

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

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

low dose

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.

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

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

Dose descriptor:

NOAEL

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Critical effects observed:

not specified

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:

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.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study planned (based on read-across)

Justification for type of information:

DISCLAIMER

This testing proposal to an analogue substance is based on the knowledge at the time of the dossier update submission. In reference to the testing strategy presented in section 13 of the dossier and in line with the dossier improvement pilot project (CEFIC, ECHA), the registrant upholds the option to reevaluate and change/adapt the strategy, the best suitable test substance, order and extent of studies performed to fulfill the registration requirements, as soon as new knowledge becomes available.

CATEGORY HYPOTHESIS
This read-across justification is based on the category approach. Seven substances are divided in two sub-groups with four substances in sub-group I and three substances in sub-group II. Members of the sub-group I are epoxidized fatty acid esters with free epoxy (EpO) groups (CAS 158318-67-3, CAS 97553-05-4, CAS 8013-07-8 and CAS 95370-96-0). Furthermore, data of CAS 8016-11-3 and CAS 61789-01-3 are used as supporting data. Members of the sub-group II are reaction products of epoxidized fatty acid esters with alcohol and contain reacted EpO groups (CAS 188831-96-1, CAS 211450-54-3 and CAS 85586-35-2). Data of the three substances CAS 96690-51-6, CAS 85586-34-1 and CAS 151661-88-0 are used as supporting data for sub-group II. Substances of both sub-groups are registered separately and are not yet part of a consortium.

This document covers physico-chemical properties, human toxicological parameters, environmental fate and pathways and ecotoxicological data. The read-across hypothesis is based on different compounds with qualitatively and quantitatively similar properties. No relevant variations in properties were observed and effects do not differ in strength among the category members (Read-Across Assessment Framework, RAAF, Scenario 6). Aforementioned chemicals can be included in two sub-groups for reasons presented subsequently.

All members and supporting substances of sub-group I and sub-group II are considered substances of Unknown or Variable composition, Complex reaction product or Biological material (UVCB). Therefore, no clearly definable structures and purities are described in this document. The constituents identified in these UVCBs are summarized in table 2.

Sub-group I:
All substances in this sub-group have a similar chemical structure (Epoxidized fatty acid esters) and have free EpO groups. They exhibit physico-chemical properties in a very comparable range. They are liquid at room temperature with similar melting and boiling points. Their water solubilities are in the same range and they have similar partition coefficients. Next to it, they have a high flash point and are not flammable, not explosive and without oxidizing properties.

Sub-group II:
All substances in this sub-group are reaction products of epoxidized fatty acid esters with alcohol and share a similar chemical structure with reacted EpO groups. Their physico-chemical properties are in a very comparable range and similar to the substances of sub-group I. They are also liquid at room temperature with similar melting and boiling points and have high flash points, are not flammable, not explosive and without oxidizing properties. The only difference is that they are only partially soluble in water and that their partition coefficient (log KOW) is lower.

This document shows that similar structures, physico-chemical properties and toxicokinetic potentials are predicted for all category members. Furthermore, nearly all substances of these two sub-groups do not possess any properties indicating a hazard for human health, environmental fate or ecotoxicity. The only exceptions are a precautionary classification of CAS 211450-54-3 and CAS 158318-67-3 as skin sensitizer Category 1B due to inconsistent data and a classification of CAS 188831-96-1 as reprotoxic Category 2 if the content of trimethylolpropane (TMP) is ≥ 3% because of reprotoxic properties of TMP.

This category approach is considered to be of great significance resulting in a decrease of the use of animal testing due to the redundancy of testing each individual substance for every endpoint. The overall data proved adequate to supporting hazard assessment. In addition, a greater body of data is available in comparison to using the analogue approach. Furthermore, the category approach is strengthened since all chemicals in the two sub-groups have a similar area of application. Detailed information on uses and manufacture can be obtained from corresponding Chemical Safety Report of category members.

Reason / purpose for cross-reference:

other: testing strategy

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Specific details on test material used for the study:

95370-96-0_Fatty acids, C14-22, 2ethylhexylesters, epoxidized

Species:

rat

Route of administration:

oral: unspecified

Endpoint conclusion

Endpoint conclusion:

no study available (further information necessary)

Dose descriptor:

NOAEL

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

No studies on the test substance itself are available at the moment. An OECD 422 study is currently ongoing and a Repeated Dose 90-day oral toxicity study in rodents according to OECD 408 is proposed.

A 2 week oral toxicity dose range finding study has already been performed. 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.

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

Justification for classification or non-classification

Currently no classification is possible due to missing data of the currently ongoing OECD 422 study.