C18 (branched) fatty acids, esters with mono-, di-, and tri-glycerol

Endpoint summary

Description of key information

To determine the potential of the test substance registered to produce systemic toxicity when administered repeatedly was tested in a 28-dietary study in rats. The study is performed according to OECD 407 in the year 2003. There were no significant effects in rats of either sex after 28 consecutive days of the test substance (target chemical) presented in the diet.
The findings are supported by two carcinogenicity tests performed in rats (2y) and in mice 80 (wk) with the Polyglycerol polyricinoleate (PGPR), i.e. read across substance. The read-across is justifed as both chemicals are of comparable structures. They can be characterized as esters of polyglycerol and fatty acids and undgo the same metabolism (Refer to Read Across Justification presented in Section 13). No carcinogenic effect of PGPR was observed in rats and mice. In addition, dietary PGPR had no adverse effect on growth, food consumption, longevity and haematology. Organ weight analysis revealed an increase in liver and kidney weight in both male and female rats and female mice. Histological analysis of tissues revealed no treatment related adverse effects. Based on the results, it was concluded that the long term treatment of 5%  (i.e. 2500 mg/kg bw/day) in the diet did not induce adverse effects in rats and mice.
Further, no adverse effects were observed in rats after 45 week feeding a diet containing 9 % PGPR (source chemical) (Wilson et al.,1998). 
During 1964 to 1965, PGPR (source chemical) was fed to 19 human volunteers (8 male and 11 female). Subjects received a diet containing constant levels of fat and protein. During the  first week the subjects received a diet without PGPR, then 5g PGPR/day was fed in the second weak and then 10 g PGPR/day was fed in the third week. PGPR was fed in soups, cakes and toffee bars. Fat balance and blood tests showed that digestion and absorption of PGPR took place and no consistent effect of PGPR on various parameters was observed.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

chronic toxicity: oral

Type of information:

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

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Justification for type of information:

READ across justification (See section 13 of IUCLID):
The toxicological profile of C18 (branched) fatty acids, esters with mono-, di-, and tri-glycerol (target chemical) can be derived from the data on Polyglycerol polyrcinoleate (PGPR), source chemical. These two compounds are expected to exhibit comparable toxicity profile, based on the structural chemical similarities and the metabolic considerations.
Based on the above mentioned arguments, the read across approach is justified.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: CARCINOGENIC RISKS IN FOOD ADDITIVES AND PESTICIDES (1960), Ministry of Health, UK

Deviations:

not specified

GLP compliance:

no

Remarks:

Studies were performed prior to implementation of GLP

Limit test:

no

Species:

other: rat and mouse

Strain:

other: rat: Wistar; mouse: C57BL

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Unilever Colworth Laboratory, Sharnbrook, Bedford, UK
- Age at study initiation: 32-46 days (rat); 6-8 weeks (mouse)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

rat study: 2-year
mouse study: 80-week

Frequency of treatment:

feed; continuously

Remarks:

Doses / Concentrations:
0 and 5% (w/w, in diet)
Basis:
nominal in diet

No. of animals per sex per dose:

rat study: 30/sex/group
mouse study: 25/sex/group

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale:
Rat study: The single dose level used for this study was chosen by comparing the likely human intake and the no-effect levels found in earlier chronic studies. Dosage levels for carcinogenicity studies should include a dose that is as high as can be administered without reducing the lifespan of the test animals. Previous studies showed no toxicity in rats fed 9% PGPR in diet for 45 wk. When testing food additives it is recognized that addition to the diet of levels above 5% can disturb the nutritional balance of the diet.
Mouse study: A single dose level was chosen for this study by comparing the likely human intake and the no-effect levels found in earlier subchronic and chronic rat feeding studies. PGPR was added to a semipurified diet at a level of 5% (by weight) by substituting part of the groundnut oil fed to control animals.

- Rationale for animal assignment (if not random): random

Positive control:

no

Observations and examinations performed and frequency:

Rat Study:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- three times weekly and evaluated as a weekly amount.

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 data

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood and how many animals: after 80 wk from four rats of each sex fed PGPR or the control diet, and on all surviving animals at study termination
- Anaesthetic used for blood collection: Yes
- Animals fasted: No data
- Parameters: erythrocyte and leucocyte counts, haemoglobin concentrations and value, red cell fragility and prothrombin time.

CLINICAL CHEMISTRY: No data


URINALYSIS: yes

NEUROBEHAVIOURAL EXAMINATION: No

Mouse Study:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- twice weekly and evaluated as a weekly amount.

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 data

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood and how many animals: all surviving animals at study termination
- Anaesthetic used for blood collection: Yes
- Animals fasted: No data
- Parameters: erythrocyte and leucocyte counts and haemoglobin concentrations

CLINICAL CHEMISTRY: No data


URINALYSIS: yes

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

Rat Study:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Each animal was subjected to gross examination at autopsy. The following organs were weighed and the organ/body weight ratios determined: adrenals, heart, kidney, spleen, liver, testes, thyroid and pituitary. These organs, together with the lung, ovary, uterus, thymus, stomach, intestine, caecum, bladder, lymph nodes, skin, mammary gland, tongue and any macroscopic abnormality were removed, fixed and processed for histological examination.

Mouse Study:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Each animal was subjected to gross examination at autopsy (at test termination and for those animals dying during the test). The following organs were weighed: heart, kidney, liver and testes. These organs, together with lung, spleen, adrenals, skin, stomach, intestine, thyroid, thymus, mammary gland and lymph nodes, together with any macroscopic abnormality were removed, fixed and processed for histological examination.

Other examinations:

In rat study, Liver function was examined after 84 and 103 week using the bromosulfothalein excretion test. Kidneyfunction was assessed at the same times by measuring urine concentration.

Statistics:

All records were examined separately for each sex by analysis of variance to assess the significance of any inter-group differences. Organ weightswere also analysed if justified by an analysis of covariance on final body weight.
In the analysis of variance, the F-ratio test was used to test whether the treatments differed. Student's t-tests were then used to compare every treatment mean individually against the control.

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:

no effects observed

Clinical biochemistry findings:

not specified

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

ORGAN WEIGHTS
Rat study:
Organ weight measurements showed that kidneys from male and female rats and livers from female (but not male) rats fed PGPR were heavier than those fed the control diet.

Mouse study:
Organ weight measurements revealed that livers and kidneys from female mice fed PGPR were heavier than those from mice fed the control purified diet.

Key result

Critical effects observed:

not specified

Rats in both treatment and control groups occasionally showed clinical signs of respiratory disease, which in some cases was accompanied by loss of body weight and reduced food consumption. The incidence of respiratory disease was most evident from wk 50 onwards. Bronchiectasis and emphysema were the main cause of death among animals in both test and control groups. The incidence of respiratory disease was comparable between treatment and control groups.

Mean weights of liver and kidney of rats and mice.

Species	Organ	Mean organ weights (g)
		Males		Females
		PGPR diet	Purified diet (control)	PGPR diet	Purified diet (control)
Rats	Liver	12.18	11.45	9.15 *	8.67
	Kidney	3.59 *	3.19	2.52 *	2.37
Mice	Liver	2.81	2.54	3.55 *	2.26
	Kidney	0.70	0.68	0.66 *	0.53

 * significantly different from control (P=0.05)

Conclusions:

The treatment of 5% target chemical in the diet (approx. 2500 mg/kg bw/day) is not considered likely to induce adverse effects in rats and mice.

Executive summary:

The repeated dose toxicity of the target substance was assessed based on the analogue approach using PGRP as a read-across supporting substance.

The long-term toxicity potential of PGPR was evaluated in rats and mice. Groups of 60 male and 60 female rats were given purified diets containing 5% of either PGPR or groundnut oil for 2 years. Groups of 25 male and 25 female mice were given purified diets containing 5% of either PGPR or groundnut oil for 80 weeks. No carcinogenic effect of PGPR was observed. In addition, dietary PGPR had no adverse effect on growth, food consumption, longevity and haematology. Organ weight analysis revealed an increase in liver and kidney weight in both male and female rats and female mice. Histological analysis of tissues revealed no treatment related adverse effects. Based on the results, it was concluded that treatment of 5% test item in the diet (approx. 2500 mg/kg bw/day) did not induce adverse effects in rats and mice. Likewise, treatment with the target chemical of the same level is not considered likely to induce adverse effects.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

2 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

To determine the potential of the test substance to produce systemic toxicity when administered repeatedly was tested in a 28 -dietary study in rats with the target substance. The study is performed according to OECD 407. The findings are supported by two carcinogenicity tests performed in rats (2y) and in mice 80 wk) with the source substance Polyglycerol polyricinoleate (PGPR), i.e. the read across substance. The read-across is justifed as both chemicals are of comparable structures. They can be characterized as esters of polyglycerol and fatty acids and undgo the same metabolism (Refer to "Read Across Justification" presented in Section 13).

Additional information

To determine the potential of the test substance to produce systemic toxicity when administered repeatedly was tested in a 28-dietary study in rats with the test substance registered. The study is performed according to OECD 407 in the year 2003. Eight groups of five rats (male or female only) each were presented with a diet containing 0% (Groups 1 and 2), and approximately 0.012% (Groups 3 and 4), 0.12% (Groups 5 and 6) or 1.2% (Groups 7 and 8) of the test substance. Targeted dietary intake levels of the test substance were 0, 10, 100, and 1,000 mg/kg/day. There were no mortalities and no significant effects in rats of either sex after 28 consecutive days of the test substance (target chemical) presented in the diet. Thus the No-Observed-Adverse-Effect Level (NOAEL) for this study is considered to be 845 mg/kg/day in males and 922 mg/kg/day in females, i.e. the highest dose tested.

A read across approach is used to fill the data gap for succhronic toxicity.The read-across to PGPR is justifed as both chemicals are of comparable structures. They can be characterized as esters of polyglycerol and fatty acids and undgo the same metabolism (Refer to "Read Across Justification" presented in Section 13).

No adverse effects were observed in rats after 45 week feeding a diet containing 9 % PGPR (source substance).

Haematological parameters were not affected. No effects on organ weight (kidneys, spleen, testes, adrenals and pituitary) were observed. The livers of rats fed 45 weeks were heavier but not the livers from animals fed 30 weeks (Wilson et al., 1998). In a separate experiment (Wilson et al., 1998) DNA of livers was determined to distinguish between hyperplasia and hypertrophy. The total DNA content of enlarged livers was not increased indicating that enlargement was not due to an increase in the number of parenchymal cells (hyperplasia). Therefore, the findings of increased liver weight are considered to be an physiological effect of adaptive metabolic response.

The findings are supported by two carcinogenicity tests (Smith et al., 1998) performed in rats (2 y) and in mice (80 wk) with the read across analogue substance Polyglycerol polyricinoleate (PGPR) (i.e. source substance). Rats and mice received purified diets containing 5 % of PGPR. No carcinogenic effect of PGPR was observed in rats and mice. In addition, dietary PGPR had no adverse effect on growth, food consumption, longevity and haematology. Organ weight analysis revealed an increase in liver and kidney weight in both male and female rats and female mice. Histological analysis of tissues revealed no treatment related adverse effects. The effect on liver weight might be interpreted as adaptive physiological response. Based on the results, it was concluded that treatment of 5% (approx. 2500 mg/kg bw/day) in the diet did not induce adverse effects in rats and mice.

During 1964 to 1965, PGPR (source chemical) was fed to 19 human volunteers (8 male and 11 female). Subjects received a diet containing constant levels of fat and protein. During the first week the subjects received a diet without PGPR, then 5g PGPR/day was fed in the second week and 10 g PGPR/day was fed in the third week. PGPR was fed in soups, cakes and toffee bars. Fat balance and blood tests showed that digestion, absorption and excretion of PGPR took place and no consistent effect of PGPR on various parameters was observed (Wilson R. and Smith M., 1998).The quantities consumed, up to 10 g/day, was equivalent to approximately 63 times theestimated maximum per capita mean daily intake by man of 2.64 mg/kg body weight/day [FAC (1992) Food Advisory Committee Report on the Review of the Emulsifiers and Stabilisers in Food Regulations FdAC/REP/11. pp. 1-71. HMSO, London]. It is therefore concluded from this study that the consumption of ADMUL WOL, a brand of PGPR, has no adverse effects in man.

Furthermore, the source chemical PGPR is well assessed as food additive. Polyglycerol esters of fatty acids have been evaluated for acceptable daily intake by the Joint FAO/WHO Expert Committee on Food Additives in 1966. In its evaluation of PGPR in 1974, the Joint FAO/WHO Expert Committee on Food Additives (JECFA 17th report) considered that the rat reproduction study (Wilson R. and Smith M., 1998), with a dietary level of PGPR of 1.5%, should be used to estimate an acceptable daily intake (ADI) of PGPR for man since the study showed a no-effect level for liver enlargement. Accordingly, a rat intake equivalent to 750 mg PGPR/kg body weight was used to set the ADI for PGPR of 7.5 mg/kg body weight. This intake is more than maximum likely from the use of PGPR in tin-greasing emulsions or in block chocolate and chocolate couverture.

No study is provided for the systemic inhalation toxicity. Inhalation of the registered test substance is unlikely due to the following reason: The vapour pressure is very low with 0.000418 Pa (20°C). During manufacturing and processing inhalation exposure is unlikely due to efficient control measures in place. There is no spray application.

No study is provided for the systemic dermal toxicity. Systemic exposure via repeated dermal application of the registered test substance is considered to be of no concern due to the following reason: The manufacture is performed in closed systems. The test substance is shown to be of low acute and systemic toxicity. Due to the size of the molecule (MW is about 700 g/mol) it is unlikely that the substance will be absorbed through the skin in large quantities.Even when absorbed through the skin, due to the demonstrated low systemic toxicity of the test item it would be of no concern. Further, the test substance is not a skin irritant or skin sensitizer, neither in rats nor in human (patch tests). This assumption is well supported by the finding that the analogue source chemical PGPR is well evaluated and used as food additive.

Based on the similar structure and the same metabolism of both the target and source chemical (refer to Section 13: “Justification for Read Across”) and taken all the available data on repeated dose toxicity with both the target and the source chemical together, it is considered that the target substance is of low toxicity. Therefore, the NOAEL for repeated dosing is determined to be 2000 mg/kg bw/day. This value will be used as NOAEL for the repeated oral toxicity of the target chemical.

Justification for classification or non-classification

Based on the results from the available repeated dose toxicity data, it is concluded that the registered substance is not subject to classification and labelling according to the criteria of the EU Dangerous Substances Directive (67/548/EEC) (DSD) and of the EU Classification, Labelling and Packaging Regulation (1972/2008/EC) (CLP).