Fatty acids, coco, iso-Bu esters

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The target substance FATTY ACIDS, COCO, ISO-BU ESTERS (CAS 91697-43-7) and the source substance FATTY ACIDS, C16-18, ISOBUTYL ESTERS (CAS 85865-69-6) are both Short Chain Alcohol Esters (SCAE C2-C8) composed by a fatty acid (C16-C18) and a C4 alcohol (isobutanol).
The source and the target substance show therefore the same reactive groups and a similar composition. A read-across to the source is therefore justified.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Both target and source substances are fatty acid esters produced by chemical reaction of an alcohol (isobutanol) with organic acids (e. g. stearic acid) in the presence of an acid catalyst. The esterification reaction is started by a transfer of a proton from the acid catalyst to the acid to form an alkyloxonium ion. The carboxylic acid is protonated on its carbonyl oxygen followed by a nucleophilic addition of a molecule of the alcohol to a carbonyl carbon of acid. An intermediate product is formed. This intermediate product loses a water molecule and proton to give an ester. Monoesters are the final product of esterification.

3. ANALOGUE APPROACH JUSTIFICATION
Since both target and source substances are fatty acid esters produced by chemical reaction of an alcohol (isobutanol) with an organic acid and therefore share similar/overlapping structural features and functional groups, it is justified to use a read across approach. The source substance has been registered already and its oral repeated dose toxicity has been investigated using a grouping of substance and read across approach. All available oral repeated dose toxicity studies within this category resulted in a NOAEL > 1000 mg/kg bw and higher. It has been concluded that no adverse effects are observed for Fatty acids, C16-18, isobutyl esters (CAS No. 85865-69-6).
The same behaviour is predicted for the target substance FATTY ACIDS, COCO, ISO-BU ESTERS (CAS 91697-43-7) .

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions (limited details on test substance purity, lack of urine analysis, neurology and ophthalmoscopy.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

limited details on test substance purity, no urine analysis, no neurology, no ophthalmoscopy performed

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Zentralinstitut für Versuchstierzucht, Hannover, Germany
- Age at study initiation: no data
- Weight at study initiation: mean ca. 150 g (females); ca. 195 g (males)
- Fasting period before study: no data
- Housing: 2-3 males per cage
- Diet: Altromin rat diet No. 1424 DK, ad libitum
- Water: yes, ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): ca. 50
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/23

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Dosing solutions were prepared freshly every day.
Dosing volume was 5 mL/kg bw.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily, 5 days per week

Remarks:

Doses / Concentrations:
100, 500 and 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for selecting satellite groups:
5 additional animals per sex were for analysis of possible post-exposure reversibility of intoxication symptoms

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: No

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at study termination
- Anaesthetic used for blood collection: Yes, ether
- Animals fasted: no data
- How many animals: all
- Parameters checked: Hct, Hb, erythrocyte count, leucocyte count, MCV, thromocyte count, differential leucocyte count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at study termination
- Animals fasted: Yes, ether
- How many animals: all
- Parameters checked: Urea, Sodium, Potassium, Glucose, Calcium, ASAT, ALAT, gamma-Glutamyltransferase, Chloride, total protein, total cholesterol, anorganic phosphorus, AP

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

ORGAN WEIGHTS:
Thyroid gland, thymus, adrenal glands, spleen, heart, kidneys, brain, testes, liver

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, all animals
HISTOPATHOLOGY: Yes, ca. 33 organs of 5 male and 5 female animals of the highest dose group and of the control group were analyzed. Additionally all non-glandulary stomachs from all animals - including those of the recovery group 14 days after the last application - were histopathologically analyzed.
Histologically analyzed organs:
Eye, tongue, salivary gland, trachea, lung, Aorta thoracica, heart, maxillary and mesenterial lymph nodes, thymus, liver, spleen, pancreas, kidney, non-glandular stomach, glandular stomach, small and large intestine, urinary bladder, testes, epididymides, seminal vesicle, prostate, uterus, ovaries, thyroid gland, parathyroid gland, cerebellum, brain stem, cerebrum (hippocampus), sceletal muscle, peripher nerv, skin

Statistics:

t-test, U-test.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

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:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

not treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
No deaths occured and no signs of systemic toxicity were observed in any animals.

BODY WEIGHT AND WEIGHT GAIN
Control and test animals showed similar gain in body weight.

HAEMATOLOGY
No significant treatment-related changes observed.

CLINICAL CHEMISTRY
No significant treatment-related changes observed.

GROSS PATHOLOGY
No treatment related abnormalities observed.

HISTOPATHOLOGY: NON-NEOPLASTIC
Hyperplastic changes were found in the non-glandular stomach of treated and control animals. Manifest mucosal hyperplasia was found in animals of the highest dose group. In few cases the mucosal hyperplasia was found combined with a submucosal inflammatory oedema. In the recovery group these findings were not present in the non-glandular stomach, indicating the reversibility of the changes. The changes were judged to be induced by the olive oil used as vehicle and not by the test substance itself.
Therefore, no treatment-related changes were observed.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects on: clinical signs; mortality; body weight; haematology; clinical chemistry; gross pathology; histopathology.

Critical effects observed:

not specified

Conclusions:

In a 28 day oral gavage study a NOAEL of 1000 mg/kg bw/day was found for male and female Wistar rats.

Cross-reference 2

Reason / purpose for cross-reference:

read-across: supporting information

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study with acceptable restrictions (analytical purity of test substance not specified; no urine and neurobihavioural examinations).

Qualifier:

according to guideline

Guideline:

other: 87/302/EWG, Annex, Part B

Deviations:

yes

Remarks:

analytical purity of test substance not specified; no urine and neurobihavioural examinations

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

analytical purity of test substance not specified; no urine and neurobihavioural examinations

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga, Sulzfeld, Germany.
- Age at study initiation: ca. 40 days
- Weight at study initiation: 143 - 168 g (153.4 g mean body weight)
- Housing: 2-3 animals per Makrolon Type III cage on softwood bedding (ARWI-Center, Essen, Germany).
- Diet: pelleted Altromin 1324, Altromin, Lage, Germany, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 48-60
- Air changes (per hr): nodata, 80-490 Lux (the cages were rotated weekly in the rack)
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: March 28th, 1989 - April 28th, 1989

Route of administration:

oral: gavage

Vehicle:

peanut oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The application volume was 5 mL/kg bw.
The dosing solutions were prepared daily immediately before application.
The following concentrations were prepared: 100 mg/ kg bw /day: 2%; 500 mg/kg bw/day: 10%, 1000 mg/kg bw/day: 20%
The total number of applications was 23 or 24 (depending on the day of necropsy).

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily, 5 days/week

Remarks:

Doses / Concentrations:
100, 500 and 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for selecting satellite groups: Additionally 5 male and 5 female animals per dose were dosed with 0 and 1000 mg/kg/day to determine the reversibility of possible compound-related findings (recovery group).
- Post-exposure recovery period in satellite groups: 27 days

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day, 5 days a week for mortality and clinical signs

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly

WATER CONSUMPTION: Yes
- Time schedule for examinations: weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at study termination
- Dose groups that were examined: control group and highest dose group

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at study termination
- Anaesthetic used for blood collection: not reported
- Animals fasted: No data
- How many animals: all animals
- Parameters checked: RBC, HCT, MCV, HGB, WBC, PLT, differential blood count (banded neutrophils, segmented neutrophils, lymphocytes, eosinophils, monocytes, basophils, myelocytes).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at study termination
- Animals fasted: No data
- How many animals: all animals
- Parameters checked: Urea, Creatinine, Sodium, Potassium, Glucose, Calcium, ASAT, ALAT, AP, gamma-GT, Bilirubin, Chloride, total protein, total Cholesterol.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

ORGAN WEIGHTS:
Absolute and relative organ weights were determined for the following:
Brain, testes, heart, liver, spleen, adrenals, kidneys, thymus.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, all animals
HISTOPATHOLOGY: Yes, animals of the control and highest dose group.
Following organs were examined microscopically:
Aorta thoracica, eyes, large and small intestinum, glandular stomach, cerebrum, urinary bladder, skin, heart, testes, pituitary, cerebellum, liver, trachea, lung, maxillary lymph nodes, mesenterial lymph nodes, spleen, epididymides, adrenal, peripheral nerv, kidney, ovaries, pancreas, prostate, vesicular gland, thymus, salivary gland, oesophagus, sceletal muscle, thymus, uterus, stomach, tongue.

Statistics:

T-tests according to Sachs and Dunnett; steel-test

Clinical signs:

no effects observed

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):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
All doses applied were tolerated without lethality. No symptoms were noted as compound-related effects.

BODY WEIGHT AND WEIGHT GAIN
The total body weight gain was comparable to control in all male and female test groups.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
The mean food consumption of all groups was comparable to the control.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
The mean water intake of the male groups 2 and 4 showed a slight increase which was not considered to be compound-related.

OPHTHALMOSCOPIC EXAMINATION
The examination of the eyes by slit lamp microscope showed no compound-related effects.

HAEMATOLOGY
The haematological examinations showed no compound-related effects.

CLINICAL CHEMISTRY
The clinical chemistry showed no compound-related effects.

ORGAN WEIGHTS
The absolute and relative organ weights showed no compound-related effects.

GROSS PATHOLOGY
The macroscopical examination of the organs showed no compound-related effects. Some observations like hydrometra, hydronephrosis,
discolouration of the thymus and one suspicion of hydrocephalus internus were considered to be spontaneous.

HISTOPATHOLOGY: NON-NEOPLASTIC
The histological examination of the organs of all groups displayed no compound-related effects.
The histological examination of the organs of the recovery group was not performed, because in the main groups no target organ was evaluated.

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

Conclusions:

According to the described study, a daily administration of 2-Ethylhexylstearate up to 1000 mg/kg bodyweight/day for 28 days caused no cumulative-systemic toxicity to rats. The NOAEL was found to be 1000 mg/kg bw/day.

Executive summary:

2-Ethylhexylstearate was tested for systemic toxicity at repeated doses of 0 (group 1), 100 (group 2), 500 (group 3) and 1000 (group 4) mg/kg body weight/day. The compound was administered daily by gavage over a period of 28 days. 10 male and 10 female rats were used for each dose. In addition to the groups 1 and 4, 5 male and 5 female animals were used to determine the reversibility of possible compound-related findings (recovery group).

 

All doses applied were tolerated without lethality. No symptoms were noted as compound-related effects. The mean food consumption of all groups was comparable to the control. The mean water intake of the male groups 2 and 4 showed a slight increase which was not considered to be compound-related. The total body weight gain was comparable to control in all male and female test groups. The haematological examinations showed no compound-related effects. The clinical chemistry showed no compound-related effects. The examination of the eyes by slit lamp microscope showed no compound-related effects. The absolute and relative organ weights showed no compound-related effects. The macroscopical examination of the organs displayed no compound-related effects. Some observations like hydrometra, hydronephrosis, discolouration of the thymus and one suspicion of hydrocephalus internus (animal No. 67) were considered to be spontaneous. The histological examination of the organs of all groups displayed no compound-related effects.The histological examination of the organs of the recovery group was not performed, because in the main groups no target organ was evaluated.

Cross-reference 3

Reason / purpose for cross-reference:

read-across: supporting information

Reference

Endpoint:

chronic toxicity: oral

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Study period:

1953

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable published study, basic data given (analytical purity of test substance not specified, only limited parameters evaluated (no clinical chemistry, urinalysis, behavioral examinations), limited documentation).

Qualifier:

no guideline followed

Principles of method if other than guideline:

Groups of male Sprague-Dawley rats were treated with the test item offered via feed during 2 years. 16 animals per dose level were used. The tested dose levels were 0.01, 0.05, 0.25, 1.25 and 6.25%, but only data of the 2 highest dose levels were considered for reporting.

GLP compliance:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 5-6 weeks
- Weight at study initiation: 65-66 g

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

2 years

Frequency of treatment:

continously in diet

Remarks:

Doses / Concentrations:
1.25 and 6.25%
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
2500 and 6000 mg/kg bw/day
Basis:
other: (corresponding daily doses)

No. of animals per sex per dose:

16

Control animals:

yes, plain diet

Observations and examinations performed and frequency:

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were measured twice weekly during the first month, thereafter at weekly intervals.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food intakes were measured twice weekly during the first month, thereafter at weekly intervals.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: In the interval between 1 and 2 years, animals were killed whenever they approached a state of debility which suggested that death might be imminent. In such cases smears of peripheral blood and bone marrow were examined.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, in the interval between 1 and 2 years, animals were killed whenever they approached a state of debility which suggested that death might be imminent. In such cases the following organs were subjected to gross pathological examination: portions of the lungs, heart, liver, spleen, adrenals, kidneys, stomach, small intestine, thyroid, and brain.

HISTOPATHOLOGY: Yes, sections of various organs were prepared from the sacrificed animals mentioned above, for microscopical examination. Therefore, heart, spleen, adrenals, stomach, small intestine, thyroid, and brain were fixed in formalin, sectioned from paraffin, and stained with hematoxylin and eosin.

Clinical signs:

no effects observed

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, treatment-related

Description (incidence and severity):

slight changes in ratio of types of leucocytes in both, control and treated animals (non adverse)

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

non adverse

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

non adverse

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
No significant differences were observed between treated and control groups.

BODY WEIGHT AND WEIGHT GAIN
No substance related effects were observed.

HAEMATOLOGY
As the animals increased in age, slight changes in distribution of leucocytes were found, but these trends occurred in both the control and the treatment groups.

GROSS PATHOLOGY
Following gross pathology lesions were observed in animals killed at various time points during the experiment: acute and chronic inflammatory changes in the lungs, nephrosis in which the kidneys were enlarged and tan in colour, and fatty changes in the liver. Further pathological changes, such as tumors, infections of the ear and preputial glands, and an arteritis similar to periarteritis nodosa, occurred less frequently. These changes were found primarily in older animals.

HISTOPATHOLOGY: NON-NEOPLASTIC
Heart muscle, stomach, spleen, small intestine, bladder, thyroid, and adrenal gland exhibited only minor and insignificant changes. In lungs, liver, and kidneys, pathologic changes were observed frequently. The lesions of the lungs were for the most part typical of the chronic rat pneumonia described by Saxton et al. (1941) and others in old rats. The principal finding in the liver was fatty infiltration, usually diffuse in distribution; occasionally necrosis of the hepatic cells surrounding a central vein was seen. Chronic nephrosis were found in some rats. These lesions also were as described by Saxton et al. (1941) in old rats.

Dose descriptor:

NOAEL

Effect level:

6 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no treatment-related effects were observed.

Critical effects observed:

not specified

Table 1: Hematologic findings

% test substance in diet	Erythrocytes x 10^6/Cu. Mm.	Hemoglobin g/100 mL	Leucocytes x 10^6/Cu. Mm.
0.00	7.4 ± 0.5	14.8 ± 0.8	7.8 ± 1.2
1.25	7.2 ± 0.5	14.2 ± 0.9	8.1 ± 1.9
6.25	7.6 ± 0.7	14.2 ± 1.4	7.9 ± 1.3

% test substance in diet	Leucocytes (%)
	Neutrophiles			Lymphocytes	Monocytes	Eosinophiles
	Non-segmented	Segmented	Total
0.00	1.0	23.6	24.6	71.0	3.1	1.4
1.25	1.2	24.6	25.8	70.2	2.5	1.5
6.25	1.3	23.1	24.4	72.5	2.4	0.8

Table 2: Incidences of pathologic lesions at necropsy

% test substance in diet	Number of rats subjected to necropsy	Number of rats affected
		Gross pathologic lesions
		None	Pneumonitis	Renal Disease	Preputial gland infection	Ear infection	Arteritis	Tumors, all types	Other
0.00	16	3	9	6	3	1	1	2	3
1.25	15	2	7	4	3	1	1	0	1
6.25	16	3	6	7	5	3	3	2	2

Cross-reference 4

Reason / purpose for cross-reference:

read-across: supporting information

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Study period:

23 Sep 2003 - 07 Apr 2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP-guideline study with acceptable restrictions (lack of details on test substance, no urine analysis performed).

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

lack of details on test substance; no urine analysis performed.

GLP compliance:

yes (incl. QA statement)

Remarks:

the Department of Health of the Government of the United Kingdom

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent.
- Age at study initiation: 5-6 weeks
- Weight at study initiation: males weighed 189 to 235 g, and females weighed 142 to 203 g
- Housing: groups of 5 animals in polypropylene grid-floor cages suspended over trays lined with absorbent paper
- Diet: pelleted diet (Rodent 5LF2 (Certified) Diet, lnternational Product Supplied Ltd., Northants, UK), ad libitum
- Water: mains drinking water, ad libitum
- Acclimation period: 14 days
Environmental enrichment was provided in the form of wooden chew blocks (B & K Universal Ltd., Hull, UK) and cardboard fun tunnels (Datesand Ltd., Cheshire, UK).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Air changes (per hr): 55 ± 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Dose volume: 4 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples were taken once weekly of each test material formulation and analysed for concentration at Safepharm Analytical Laboratory.
The results indicate that the prepared formulations were within ± 10 % of nominal.

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
5, 50 and 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing and one and five hours after dosing during the working week.
Animals were observed immediately before dosing and one hour after dosing at weekends and on public holidays.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE:
Food consumption was recorded for each cage group (5 animals) at weekly intervals throughout the study

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: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily, for each cage group by visual inspection of the water bottles for overt changes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: The eyes of all control and high dose animals were examined pre-treatment, and all treatment
groups were examined prior to termination of treatment (during Week 12).

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at study termination
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: all surviving animals
- Parameters checked: Hb, RBC, Hct, MCH, MCV, MCHC, total leucocyte count, differential leucocyte count, platelets, reticulocytes, Prothrombine time, Activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at study termination
- Animals fasted: No data
- How many animals: all surviving animals
- Parameters checked: Urea, Glucose, total protein, albumin, sodium, potassium, chloride, calcium, inorganic phosphorus, ASAT, ALAT, AP, Creatinine, total cholesterol, total bilirubin

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: weekly
- Dose groups that were examined: all surviving animals
- Battery of functions tested: sensory activity / grip strength / motor activity:
- - Behavioural Assessment

ORGAN WEIGHTS:
Adrenals, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Spleen, Testes, Thymus, Uterus

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
On completion of the dosing period all surviving animals were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination. All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

HISTOPATHOLOGY: Yes
Samples of the following tissues were removed from all animals and preserved:
Adrenals, Aorta (thoracic), Bone & bone marrow (femur including stifle joint), Bone & bone marrow (sternum), Brain (including cerebrum, cerebellum and pons), Caecum, Colon, Duodenum, Epididymides, Eyes, Gross lesions, Heart, Ileum (including Peyer's patches), Jejunum, Kidneys, Liver, Lungs (with bronchi), Lymph nodes (cervical and mesenteric), Mammary glands, Muscle (skeletal), Oesophagus, Ovaries, Pancreas, Pituitary, Prostate, Rectum, Salivary glands (submaxillary), Sciatic nerve, Seminal vesicles, Skin (hind limb), Spinal cord (cervical, mid-thoracic and lumbar), Spleen, Stomach, Testes, Thymus, Thyroid/parathyroid, Tongue, Trachea, Urinary bladder, Uterus
All tissues were despatched to Propath UK Ltd., Willow Court, Netherwood Road,, Rotherwas, Hereford, UK for processing (Principal Investigator: H Young).

Initially, tissues from all control and high dose groups were prepared as paraffin blocks, sectioned at nominal thickness of 5 μm and stained with haematoxylin and eosin for subsequent microscopic examination. Since there were indications of treatment-related hepatic changes, examination was subsequently extended to include similarly prepared sections of liver tissue from all animals in the other treatment groups.
Microscopic examination was conducted by the Study Pathologist. All findings were entered into the ROELEE Pathology computerisation system for tabulation and report production.

Statistics:

Haematological, blood chemical, organ weight (absolute and relative to terminal bodyweight), weekly bodyweight gain and quantitative functional performance and sensory reactivity data were assessed for dose response relationships by linear regression analysis, followed by one way analysis of variance (ANOVA) incorporating Levene's test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnett's test. Where Levene's test showed unequal variances the data were analysed using non-parametric methods: Kruskat-Wallis ANOVA and Mann-Whitney 'U' test.

Clinical signs:

no effects observed

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:

no effects observed

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

considered non-adverse

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

considered non-adverse

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

considered non-adverse

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no deaths during the study. No clinical signs of systemic toxicity were observed.

BODY WEIGHT AND WEIGHT GAIN
No adverse effect on bodyweight gain was detected during the study.

FOOD CONSUMPTION AND FOOD EFFICIENCY
No adverse effect on dietary intake or food efficiency (the ratio of bodyweight gain to dietary intake) was detected for test animals in comparison to controls during the study.

WATER CONSUMPTION AND COMPOUND INTAKE
Daily visual inspections of water bottles revealed no overt intergroup differences.

OPHTHALMOSCOPIC EXAMINATION
No treatment-related ocular effects were detected.

HAEMATOLOGY
No treatment-related effects were detected.

CLINICAL CHEMISTRY
Males treated with 1000 mg/kg/day showed statistically significant increases in cholesterol and creatinine levels (p < 0.05).

NEUROBEHAVIOUR
There were no treatment-related changes in the functional performance parameters measured.
There were no treatment-related changes detected in the sensory reactivity parameters measured.

ORGAN WEIGHTS
Elevated liver weights, both absolute and relative to terminal bodyweight were detected for animals of either sex treated with 1000 mg/kg/day (p < 0.001). Elevated adrenal weight was also observed for females treated with 1000 mg/kg/day (absolute p < 0.01, relative p < 0.05). An increase in absolute spleen weight (p < 0.05) in 1000 mg/kg/day males and an increase in relative kidney weight (p < 0.01) was also observed for 1000 mg/kg/day females, however, in the absence of the supporting histopathological evidence to suggest an effect of treatment, were considered to be unrelated to treatment.

GROSS PATHOLOGY
No treatment-related macroscopic abnormalities were detected.

HISTOPATHOLOGY: NON-NEOPLASTIC
Treatment-related observed changes:
A marginal effect on hepatocyte size was observed in relation to treatment for female animals treated with 1000 mg/kg/day with a few animals from this group demonstrating centrilobular hepatocyte enlargement (p < 0.05). A similar effect was not convincingly seen for 1000 mg/kg/day males, or for animals of either sex treated with 50 or 5 mg/kg/day.
No other treatment-related changes were observed.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Dose descriptor:

NOEL

Effect level:

50 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: clinical sings, body weight, food consumption, food efficiency, water consumption, ophthalmoscopic examination, hematology, clinical chemistry, neurobehavioural examination, organ weights, gross necropsy and histopathological examinations.

Critical effects observed:

not specified

Materials and methods

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, coco, iso-Bu esters

EC Number:

294-304-2

EC Name:

Fatty acids, coco, iso-Bu esters

Cas Number:

91697-43-7

Molecular formula:

Not available for UVCB substances

IUPAC Name:

Fatty acids, coco, iso-Butyl esters

Test material form:

liquid

Results and discussion

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

other:

Basis for effect level:

other: no effects on: clinical signs; mortality; body weight; haematology; clinical chemistry; gross pathology; histopathology.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The source substance has been registered already and its oral repeated dose toxicity has been investigated using a grouping of substance and read across approach. All available oral repeated dose toxicity studies within this category resulted in a NOAEL > 1000 mg/kg bw. It has been concluded that no adverse effects are observed for Fatty acids, C16-18, isobutyl esters (CAS No. 85865-69-6).
The same behaviour is predicted for the target substance FATTY ACIDS, COCO, ISO-BU ESTERS (CAS 91697-43-7) .

Executive summary:

The target substance FATTY ACIDS, COCO, ISO-BU ESTERS (CAS 91697-43-7) and the source substance FATTY ACIDS, C16-18, ISOBUTYL ESTERS (CAS 85865-69-6) are both Short Chain Alcohol Esters (SCAE C2-C8) composed by a fatty acid (C16-C18) and a C4 alcohol (isobutanol).

The source substance has been registered already and its oral repeated dose toxicity has been investigated using a grouping of substance and read across approach. All available oral repeated dose toxicity studies within this category resulted in a NOAEL > 1000 mg/kg bw. It has been concluded that no adverse effects are observed for Fatty acids, C16-18, isobutyl esters (CAS No. 85865-69-6).

The same behaviour is predicted for the target substance FATTY ACIDS, COCO, ISO-BU ESTERS (CAS 91697-43-7) .