Fatty acids, C18-unsatd., dimers, mixed esters with oleic acid and trimethylolpropane

Endpoint summary

Description of key information

Oral: chronic NOAEL (rat, m/f) = 819.8-2312.2 mg/kg bw/day (based on read-across from Fatty acids, C18-unsatd., dimers and after correction for differences in molecular weight)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

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

Adequacy of study:

key study

Study period:

24 Nov 1992 - 05 Mar 1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

there was no satellite group. Some clinical chemistry, gross necropsy and hitopathological data as well as urinalysis are missing.

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 (UK) Ltd.
- Age at study initiation: 4-5 weeks
- Weight at study initiation: 114.5-155.5 g (male); 104.6-142.0 g (female)
- Housing: 5 animals per cage
- Diet (e.g. ad libitum): ESL modified AIN-76A (MODAIN) purified diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 1 week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2°C
- Humidity (%): 55 ± 10%
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 30 Nov 1992 To: 01-05 Mar 1993

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly from 26 Nov 1992 to 10 Dec 1992; every 2 weeks from 17 Dec 1992 to the end of the study
- Mixing appropriate amounts with (Type of food): ESL modified AIN-76A (MODAIN) purified diet
- Storage temperature of food: 4°C

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

-Stability and homogeneity in diet
The stability of Dimer Acid at concentrations of 0.1%, 1% and 5% in diet was determined over periods of 7 and 14 days when stored at 4°C or in an animal room.
The diets were analysed to confirm Dimer Acid was distributed homogeneously in the diet. After mixing the diets, five samples were taken for analysis from the top, the middle and bottom centre, and left and right centre of the mixing bowl.
Diet samples were extracted into propan-2-ol and centrifuged to remove particulate matter. An aliquot was concentrated to dryness, then redissolved and analysed by HPLC on a 5µ Lichrosorb Diol column, detection by a light scattering detector. Quantitation was achieved by comparison of peak areas with external standards of Dimer Acid.
Separation on the HPLC sytem was based on the interaction of the carboxylic acid of Dimer Acid with free hydroxyl groups at the surface of the diol phase. Thus, interaction increased with the number of carboxylic acid groups. Dimer Acid contains mixtures of mono, di and polyacids. In the assay preparation based on two peaks, di-acid denoted dimmer and tri or greater (poly) acid denoted trimer.
Dimer Acid was shown to be stable in diet over 14 days. The results for 0.1% (w/w) Dimer Acid in diet showed more variation that would normally be accepted, but the level was very close to the limits of detection of the analytical method.
Using the methods described, Dimer Acid was shown to be mixed homogeneously in the diet at a concentration of 0.1%, 1% and 5% (w/w).

-Confirmation of achieved concentration
Diets containing 5%, 1% and 0.1% Dimer Acid prepared on 26 Nov 1992, 07 Jan 1993 and 18 Feb 1993 were analysed for the achieved concentration of Dimer Acid after first passing the UV lock. The methodology was the same as that used for the determination of stability.
Analysis of the diets prepared on Week 1 and 13 confirmed the nominal concentration had been achieved within the expected experimental error of the analytical method. The results for 0.1% (w/w) Dimer Acid in diet showed more variation that would normally be accepted, but the level was very close to the limits of detection of the analytical method.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
0, 0.1, 1, 5% (w/w) corresponding to ca. 0, 74.1, 740.9, 3591.2 mg/kg bw/day for males and ca. 0, 90.5, 854.9, 4085.5 mg/kg bw/day for females (average dose weeks 0-13)
Basis:
nominal in diet

No. of animals per sex per dose:

20

Control animals:

yes, plain diet

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were checked at least twice each day (once on Saturdays and Sundays) for signs of ill-health or reaction to treatment.


BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed on the firts day and subsequently at weekly intervals throughout the study.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No. For each cage of five animals, food and water intakes were recorded twice-weekly throughout the study and weekly values were calculated.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No


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 AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water intake was recorded twice-weekly throughout the study and weekly values were calculated.


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: In the week prior to the start of the study all animals were given an ophthalmoscopic examination. In the week prior to the end of the study all animal were re-examined.
- Dose groups that were examined: all


HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the test period
- Anaesthetic used for blood collection: Yes (Halothane)
- Animals fasted: No
- How many animals: 20 per sex per dose
- Parameters checked in table 1 were examined.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the test period
- Animals fasted: No
- How many animals: 20 per sex per dose
- Parameters checked in table 2 were examined.

URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 3)
HISTOPATHOLOGY: Yes (see information on materials and methods)

Statistics:

For body weights, food and water intakes, food conversion efficieny, clinical pathology and organ weights (incuding the ration of organ weight to body weight at terminal kill), a statistical analysis was conducted. Initially the data were examined to see if parametric or non-parametric analysis was appropriate.
If the data were parametric, a one way analysis of variance was used to see if any of the treatments differed. A t-test versus control was used to show any significant differences between control group and any of the other treatment levels at the 5%, 1%, and 0.1% probability levels. A multiple T test was used for pairwise comparisons between groups.

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no decedents during the 13 week treatment period.
The clinical signs observed during the course of the study were not considered to be treatment-related. These generally involved scabs, alopecia, excoriation, nasal discharge and ocular discharge.

BODY WEIGHT AND WEIGHT GAIN
There were no treatment-related changes in body weight during the 13-week dosing period. Statistically significant changes in body weight were few, minor, randomly distributed and of no biological significance.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Food intake was significantly lower during the first four weeks of the study in male and female rats fed 5.0% Dimer Acid in diet, which may refelct an initial reluctance of the rats to eat the diet.

FOOD EFFICIENCY
Food conversion efficiency was statistically significantly higher in females fed 5.0% Dimer Acid during the first four weeks of the study.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
Statistically significant changes in water intake occured but no clear treatment-related pattern in water intake was observed. Accumulated water intake did not differ significantly between groups.

OPHTHALMOSCOPIC EXAMINATION
On ophthalmoscopic examination at the end of the study a persistent pupillary membrane was recorded for one male animal in the 0.1% treatment group and ocular opacity was recorded for one female animal in the 5.0% treatment group. These findings were not considered treatment related.

HAEMATOLOGY
See remarks on results.

CLINICAL CHEMISTRY
See remarks on results.

ORGAN WEIGHTS
See remarks on results.

GROSS PATHOLOGY
Treatment with Dimer Acid has no effects on bodily condition as assessed by estimation of abdominal fat reserves at necropsy.
Mesenteric lymph nodes were slightly or moderately enlarged in a proportion of rats from all groups fed Dimer Acid.
The colour of caecal contents was affected by feeding with Dimer Acid. Caecal contents were predominantly yellow in rats fed 5.0% Dimer Acid and predominantly yellow-green in rats given 1.0% Dimer Acid, compared with green or gray-green in rats fed 0.1% Dimer Acid or the control animals. Dimer Acid is a yellow liquid.
The incidence of uterine fluid distension was slightly increased in rats fed 5.0% Dimer Acid.
All other macroscopic findings were considered to be incidental and within the range expected for this age and strain of rat.

HISTOPATHOLOGY: NON-NEOPLASTIC
On microscopic examination treatment-related findings were observed in mesenteric lymph nodes, in the spleen, the liver, the adrenal glands and thyroidglands (in females). Of these effects, only those in the mesenteric lymph node and spleen extended down to the group fed 0.1% Dimer Acid.

-Mesenteric lymph nodes:
Aggregations of macrophages, some of them containing golden brown pigment, were observed in the paracortex and in the medulary cords in all rats fed 5.0% Dimer Acid, the majority of rats fed 1.0% Dimer Acid and a proportion of rats fed 0.1% Dimer Acid. The incidence and the amount of aggregations were dose-related, with only a few aggregations present in rats fed 0.1% Dimer Acid. The histological findings in the mesenteric lymph nodes correlated with the lymph node enlargement noted at necropsy.

-Spleen:
Macrophages containing golden/dark brown pigment were seen in the red and white pulp in all rats fed 5.0% Dimer Acid, the majority of rats fed 1.0% Dimer Acid and a proportion of female rats fed 0.1% Dimer Acid. The incidence and amount of macrophages showed a dose relationship in both male and female rats. The effect was more pronounced in female rats.

-Liver:
The incidence of bile duct proliferation was increased, as was incidence of sclerosis of the bile ducts, in male rats fed 5.0% Dimer Acid. The sclerosis was associated with a minimal mixed inflammatory cell infiltration.There was a very slight increase in incidence of bile duct proliferation in female rats fed 5.0% Dimer Acid. Periportal cytoplasmic vacuolation was decreased in both male and female rats fed 1.0% or 5.0% Dimer Acid.

-Adrenals:
Cortical vacuolation was observed in the adrenal gland of female rats fed 5.0% or 1.0% Dimer Acid. One female rats fed 0.1% Dimer Acid had trace levels of vacuolation, which is not considered toxicologically important as vacuolation may occasionally be seen in control females. Cytoplasmic rarefaction was decreased in female rats fed 5.0% Dimer Acid.
Cortical extramedullary haemopoiesis was absent in female rats fed 5.0% Dimer Acid. In rats fed 1.0% or 0.1% Dimer Acid extramedullary haemopoiesis was slightly reduced in incidence in the females. However, since the incidence of this finding generally varies considerably among groups of untreated rats this was not considered of toxicological importance.

-Thyroids:
Follicular epithelial hypertrophy was slightly increased in female rats fed 5.0% Dimer Acid.

-Spontaneous pathology:
Microscopic examination of uteri with macroscopic fluid distension revealed this to be due to a variety of different reasons - luminal dilatation or dilated/cystic endometrial glands. In view of this, and in view of the variation in uterine size with different phases of the oestrous cycle, this finding is not thought to be of any toxicological importance.
The incidence of retinal folding/atrophy was higher in rats fed Dimer Acid. However, as the overall incidence was very low and there was no dose relationship, these lesions were not considered to be of any toxicological importance.
There were slightly more lesions in the nasal passage in rats fed 5.0% Dimer Acid compared with controls.These lesions were minor in nature and included focal epithelial hypertrophy or hyperplasia associated with mucosal inflammatory cells or a luminal inflammatory exudate. Lesions of this nature are a common finding in control rats, and, while it is possible that they could be exacerbated by inhalation of diet containing irritant test material, the incidence in treated rats was still within the normal range.
A variety of spontaneous changes was recorded in animals from all dose groups with no evidence of a treatment-related distribution. These findings were within the spectrum of spontaneous lesions commonly encountered in laboratory rats of this age and strain and were considered to be unrelated to the feeding of Dimer Acid.

Dose descriptor:

NOAEL

Effect level:

ca. 855 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No adverse effcts were observed at this dose level

Remarks on result:

other: 1% in the diet

Dose descriptor:

NOAEL

Effect level:

ca. 741 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No adverse effcts were observed at this dose level

Remarks on result:

other: 1% in the diet

Dose descriptor:

LOAEL

Effect level:

4 085.5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical biochemistry

histopathology: non-neoplastic

Remarks on result:

other: 5% in the diet

Dose descriptor:

LOAEL

Effect level:

3 591.2 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical biochemistry

histopathology: non-neoplastic

Remarks on result:

other: 5% in the diet

Critical effects observed:

not specified

Doses

Based on compund concentrations in diet as well as data on body weight and food intake over the 13 -weeks study period, averege doses in mg/kg bw/day were calculated as presented in the table below.

	body weight		mean body weight (g)	food intake (g)	food intake (g/day)	compund intake(g/day)	dosis (mg/kg/day)
	week 0	week 13	week 0-13	week 0-13	week 0-13	week 0-13	week 0-13
Male
5%	133,7	541,5	337,6	2182,3	24,2	1,21	3591,2
1%	135,1	533,5	334,3	2229,1	24,8	0,25	740,9
0,1%	135,8	560,8	348,3	2321,6	25,8	0,03	74,1
0%	133,3	545,5	339,4	2215,3	24,6	0,00	0,0
Female
5%	120,2	317,5	218,85	1609,4	17,9	0,89	4085,5
1%	123,6	335,9	229,75	1767,7	19,6	0,20	854,9
0,1%	120,2	317,7	218,95	1782,7	19,8	0,02	90,5
0%	119,2	317,5	218,35	1693,8	18,8	0,00	0,0

Haematology

Statistically significant changes by Student's t-test and by multiple t-test in haematology parameters are summarised in the table below. In this table the symbols M and F are used to indicate a significant change in a particular parameter and whether this is seen in male (M) or female (F) rats.

	Dimer Acid (% w/w)
Haematological change	Control	0.1	1.0	5.0
Increase in mean cell haemoglobin				M
Increase in prothrombin time			F	M/F

The changes observed were slight and unlikely to be of any toxicological significance. A number of other changes, for instance a decrease in neutrophil count in females fed 5.0% or 1.0% Dimer Acid, were statistically significant by Student's t-test but did not trigger the multiple t-test as significant.

In the absence of any treatment-related changes in either the red or white cell count it was considered unnecessary to examine the bone marrow smears.

Clinical chemistry

Statistically significant changes by Student's t-test and by multiple t-test in clinical chemistry parameters are summarised in the table below. In this table the symbols M and F are used to indicate a significant change in a particular parameter and whether this is seen in male (M) or female (F) rats.

	Dimer Acid (% w/w)
Clinical chemistry change	Control	0.1	1.0	5.0
Decrease in plasma calcium		F	F	M/F
Increase in alkaline phosphatase			M/F	M/F
Decrease in 5'-nucleotidase		M		M/F
Increase in alanine aminotransferase				M/F
Increase in aspartate aminotransferase		F		F
Increase in bilirubin			M	M
Decrease in total cholesterol			M/F	M/F
Decrease in triglycerides			M	M/F
Decrease in glucose			F
Decrease in total serum protein				M/F
Decrease in serum albumin				M/F
Decrease in beta globulin fraction			M	M
Increase in albumin/globulin ratio			M	M

The changes in plasma electrolyte levels were slight. The increase in plasma alkaline phosphatase were marked, the level being more than double that of the control group in rats fed 5.0% Dimer Acid. Other fluctuations in plasma enzyme levels were not so marked.

Serum triglyceride and cholesterol levels were decreased in rats fed 5.0% or 1.0% Dimer Acid in diet. Serum protein levels were also decreased in rats fed 5.0% Dimer Acid.

It should be noted that while a very small increase in plasma bilirubin was observed in male rats fed Dimer Acid, the levels measured were below the sensitivity of the method and must be viewed with caution.

Statistically significant changes in plasma glucose appear to be random and not part of a treatment-related pattern. Plasma levels of pseudocholinesterase were statistically significantly increased in males fed 5.0% Dimer Acid but decreased in females.

Organ weights

Statistically significant changes by Student's t-test and by multiple t-test in organ weights are summarised in the table below. In this table the symbols M and F are used to indicate a significant change in a particular parameter and whether this is seen in male (M) or female (F) rats.

	Dimer Acid (% w/w)
Organ weight change	Control	0 .1	1.0	5.0
Decrease in spleen weight			M	M
Decrease in relative spleen weight			M	M
Decrease in kidney weight				F
Decrease in liver weight		F	M	M
Decrease in relative liver weight		F	M/F	M/F

Relative kidney weight was significantly lower in rats fed 5.0% or 1.0% Dimer Acid by Student's t-test though not by multiple t-test. A dose-related trend in this parameter was evident.

Conclusions:

Based on clinical chemistry parameters and histopathological findings, 1.0 % (w/w) test material in diet can be considered a no-observed-adverse-effect-level (NOAEL), corresponding to a dose of 741 and 855 mg/kg bw/day for males and females, respectively.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

The available information comprises adequate, reliable (Klimisch score 2) and consistent studies from reference substances with similar structure and intrinsic properties. Read-across is justified based on the structural similarity between the source and target substances, as the source substances comprise hydrolysis products (CAS 77-99-6 and CAS 61788-89-4) of the target substance. Refer to endpoint discussion for further details. The selected study is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006.

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

Justification for grouping of substances and read-across

There are no data available for the assessment of repeated dose toxicity of Fatty acids, C18-unsatd., dimers, mixed esters with oleic acid and trimethylolpropane (CAS 147256-33-5). In order to fulfil the standard information requirements set out in Annex IX, 8.6, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006, read-across from structurally related substances was conducted.

In accordance with Article 13 (1) of Regulation (EC) No 1907/2006, "information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI are met.” In particular for human toxicity, information shall be generated whenever possible by means other than vertebrate animal tests, which includes the use of information from structurally related substances (grouping or read-across).

Having regard to the general rules for grouping of substances and read-across approach laid down in Annex XI, Item 1.5, of Regulation (EC) No 1907/2006 whereby substances may be predicted as similar provided that their physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity.

Overview of repeated dose toxicity:

CAS	147256-33-5 (a)	61788-89-4 (b)	77-99-6
Chemical Name	Fatty acids,C18-unsatd., dimers, mixed esters with oleic acid and trimethylolpropane	Fatty acids, C18-unsatd., dimers	Propylidynetrimethanol
MW	624.97; 1115.78; 1606.58; 677.05; 1219.92; 1762.80	556.87; 564.93	134.18
Repeated dose toxicity oral	RA: CAS 61788-89-4 RA: CAS 77-99-6	Experimental result: NOAEL female = 855 mg/kg bw/day (female) NOAEL = 741 mg/kg bw/day (male)	Experimental result: NOAEL ≥ 800 mg/kg bw/day (male/female)

(a) Substances subject to the REACh Phase-in registration deadline of 31 May 2013 are indicated in bold font.

(b) Substances that are either already registered under REACh or not subject to the REACh Phase-in registration deadline of 31 May 2013 are indicated in normal font. Lack of data for a given endpoint is indicated by “--“.

The above mentioned substances are considered to be similar on the basis of the structural similar properties and/or activities. The available endpoint information is used to predict the same endpoints for Fatty acids, C18-unsatd., dimers, mixed esters with oleic acid and trimethylolpropane (CAS 147256-33-5).

A detailed analogue approach justification is provided in the technical dossier (see IUCLID Section 13).

 

Discussion

Repeated dose toxicity

Oral

CAS 61788-89-4

A GLP-compliant subchronic oral toxicity study with Fatty acids, C18-unsaturated, dimers was performed similar to OECD guideline 408 in male and female Sprague-Dawley rats (Spurgeon and Hepburn, 1993). Twenty animals per sex and group received the test substance ad libitum at dietary levels of 0, 0.1, 1, 5% (w/w) for a period of 13 weeks. Based on the reported average body weight and food intake data during Week 1-13, the approximate doses were determined to be 0, 74.1, 740.9, 3591.2 mg/kg bw/day for males and ca. 0, 90.5, 854.9, 4085.5 mg/kg bw/day for females, respectively.

No mortalities and no treatment-related clinical signs were observed in any treatment group during the entire study period. The lower food intake during the first four weeks of the study in rats fed the test material at 5% in the diet may reflect an initial reluctance of the rats to eat the diet. However, no treatment-related changes in body weight were observed in treated animals of any dose group compared to control during the entire study period.

Ophthalmoscopic examination at study termination reveal a persistent pupillary membrane in one male animal of the 0.1% treatment group and ocular opacity in one female animal of the 5.0% treatment group. However, these findings were not considered to be treatment-related.

At necropsy, mesenteric lymph nodes were slightly or moderately enlarged in individual rats of all dose levels compared to controls. Caecal contents appeared yellow-green to yellow in rats given 1.0 and 5.0% in the diet, which was attributed to the yellow colour of the test substance. Furthermore, the incidence of uterine fluid distension was slightly increased in rats fed 5.0% of the test substance in the diet. All other macroscopic findings were considered to be incidental and within the range expected for this age and strain of rat.

Statistically significant decreases in absolute and/or relative organ weight of spleen, kidney and liver were mainly observed in males treated with 1.0% and 5.0% in the diet. In females, a statistically significant decrease in relative organ weights of the liver was observed at all dose levels, but absolute liver weight was only decreased in females treated with 0.1% in the diet. However, these changes were not accompanied by any corresponding histopathological findings, and were thus not considered to be of adverse nature.

Plasma alkaline phosphatase activity (ALP) was increased in males and females fed the test material at 1.0% or 5.0%. In addition, alanine aminotransferase (ALT) was increased in male and female rats fed at 5.0%, which correlated with the increase in biliary hyperplasia and sclerosis in animals of the 5.0% treatment group at histopathological examination. A small increase in plasma bilirubin was observed in male rats fed the test material at 5.0% or 1.0%. However, the levels measured were below the sensitivity of the method and were thus considered with caution. A small reduction in plasma calcium was observed in male and female rats fed the test material at 5.0%. Small reductions in both total serum protein and albumin were also observed in male and female rats of the 5.0% treatment group. However, in the absence of any correlating effect, these changes were not considered to be toxicologically relevant.

The plasma lipids cholesterol and triglyceride were reduced in male and female rats in the 5.0% and 1.0% treatment groups, respectively, which may be a result of interference of the test substance with the absorption of lipid and other nutrients from the gut. The reduction in periportal hepatocyte vacuolation seen on histological examination of the liver could correlate with the reduced plasma lipids, indicating some alteration in lipid metabolism, another possible explanation for the plasma lipid, serum protein and calcium changes.

At microscopic examination, a dose-related increase in the incidence of macrophage aggregation in the mesenteric lymph nodes was observed, which correlated with the mesenteric lymph node enlargement noted at necropsy. The incidence and amount of macrophages was also dose-dependently increased in the spleens of both male and female rats. The macrophages of the mesenteric lymph nodes and spleen revealed a golden/dark brown pigmentation in animals of all dose groups. However, there was no evidence of any degenerative effect associated with these histopathological findings.

Increased cortical vacuolation in the adrenals, associated with decreased cytoplasmic rarefaction was probably due to altered steroidogenesis. However, this finding was not accompanied by any evidence of degenerative change in the adrenal. The significance of the reduced extramedullar haemopoiesis in the adrenals was uncertain, but may possibly correlate with the reduction in neutrophil count in females fed the test material at 5.0% and 1.0%. However, all of the changes observed in the adrenal were minor in nature and thus of no toxicological relevance.

In thyroids, an increase in follicular epithelial hypertrophy was observed in female rats fed 5% of the substance in the diet. Since this effect was only slight and not correlated with any other effects, it was not considered to be toxicologically relevant.

A variety of spontaneous changes was also recorded in animals from all dose groups, which were not considered to be a treatment-related, as they commonly occur in laboratory rats of this age and strain.

Based on the results of this study, a NOAEL of 1% (w/w) was derived for Fatty acids, C18-unsaturated, dimers, corresponding to doses of 741 and 855 mg/kg bw/day for male and female Sprague-Dawley rats, respectively.

CAS 77-99-6

In a GLP-compliant subacute oral toxicity study performed according to OECD guideline 422, male and female Slc:SD rats were exposed to Propylidynetrimethanol (TMP) at dose levels of 12.5, 50, 200 and 800 mg/kg bw/day (MHLW, 1994). The test substance dissolved in distilled water was orally administered to 12 animals per sex and dose via gavage. Males were treated for a period of 45 days, whereas females were exposed to the test substance 14 days prior to mating, during gestation and until Day 3 of lactation. A similar constituted group of animals received the vehicle and served as control. No mortality and no clinical signs were observed during the study period. During the 14-day pre-mating period, the absolute body weights in males and the body weight gain in females was statistically significantly decreased at 800 mg/kg bw/day compared to the controls. However, no effects on the body weights were observed in treated animals during the remainder of the study. No effect on food consumption was observed in male and females of any treatment groups compared to controls. Clinical chemistry analysis in males revealed a slight, but statistically significant increase in total bilirubin and chloride at 800 mg/kg bw/day compared to controls. At 800 and 200 mg/kg bw/day, a slight increase (stat. significant) in total protein, albumin, and albumin/globulin ratio was observed in males compared to controls. The values of blood urea nitrogen were statistically significantly increased in males of the 50, 200 and 800 mg/kg bw/day dose groups. At haematological examination, a slight, statistically significant decrease in the values of mean corpuscular haemoglobin (MCH) and the mean corpuscular haemoglobin concentration (MCHC) at all dose levels in males was observed compared to controls. In addition, haemoglobin concentration was statistically significantly reduced at 800 mg/kg bw/day. Due to the absence of any correlating effects, changes in clinical chemistry and haematological parameters were considered to be of non-adverse nature. All other significant changes in clinical chemistry and haematology parameters were not dose-related and thus of no toxicological relevance. A significant increase in absolute and relative liver weight in males was observed at 800 mg/kg bw/day. In females of the same dose group, only a slight and not statistically significant increase in absolute and relative liver weight was noted. At necropsy, enlargement of the liver was found in 3/10 males, which correlated with the observed increase in liver weight in this gender. In females, the increase in the incidence of macroscopic findings in the lungs (red patch/zone) at all dose levels was not considered to be of toxicological relevance, since they were not unequivocally attributable to the test substance administration due to a lacking dose-response relationship. At histopathological examination, a slight basophilic alteration of the renal tubular epithelial cells was observed in 1/11 females receiving 50 mg/kg bw/day, in 2/10 females receiving 200 mg/kg bw/day and in 5/10 females (stat. significant) receiving 800 mg/kg bw/day. However, these changes were not unequivocally attributable to the test substance administration, because of their limited distribution and limited degree, and because similar lesions were observed in male rats of all groups including the controls. Although necropsy revealed liver enlargement in 3/10 male rats receiving 800 mg/kg bw/day, histopathological examination did not show any definite morphological lesions in the liver. All other histopathological alterations in treated animals were also observed to a similar degree in the corresponding control animals, and were thus of no toxicological significance.

Since no adverse effects were observed up to and including the highest dose level, a NOAEL of 800 mg/kg bw/day was derived for propylidynetrimethanol (TMP) in male and female Slc:SD rats.

Inhalation

There are no data available. Due to their physicochemical properties (low vapour pressure), exposure to Fatty acids, C18 unsatd., dimers, mixed esters with oleic acid and trimethylolpropane via inhalation is unlikely.

Dermal

There are no studies available in which potential toxic effects after long-term dermal exposure have been studied. However, the whole body of evidence on the toxicokinetic behaviour and toxicological activity of Fatty acids, C18 unsatd., dimers, mixed esters with oleic acid and trimethylolpropane indicate that both systemic bioavailability and toxic effects are unlikely to occur upon dermal exposure.

Conclusion for repeated dose toxicity

No studies investigating repeated dose toxicity are available for Fatty acids, C18-unsatd., dimers, mixed esters with oleic acid and trimethylolpropane. Oral, dermal or inhalative absorption of the registered substance is deemed unlikely due to its very high MW and physico-chemical properties. As hydrolysis is possible to a limited extend, it will most likely be the cleavage products that are absorbed, thus a worst case approach considering the repeated dose toxicity of the potential hydrolysis products was performed. None of the potential hydrolysis products (Propylidynetrimethanol, Fatty acids, C18-unsaturated, dimers or Oleic acid) poses a risk for human health effects. Oleic acid is an integral part of a standard western diet; the daily intake is far above any exposure levels that are considered here, thus the other two potential cleavage products were considered first. Since no adverse effects were observed up to and including the highest dose level, a NOAEL of ≥ 800 mg/kg bw/day was derived for propylidynetrimethanol (TMP) in male and female Slc:SD rats.. For Fatty acids, C18-unsaturated, dimers, the third potential cleavage product, a NOAEL of 741 and 855 mg/kg bw/day for male and female Sprague-Dawley rats, respectively, was derived.

The lowest long-term NOAEL of 741 mg/kg bw/day for Fatty acids, C18-unsaturated, dimers (molecular weight = 556.87-564.93g/mol) is selected for hazard assessment of Fatty acids,C18-unsatd., dimers, mixed esters with oleic acid and trimethylolpropane (molecular weight = 624.97-1762.80 g/mol). In order to account for differences in molecular weight, full enzymatic hydrolysis of Fatty acids, C18-unsatd., dimers, mixed esters with oleic acid and trimethylolpropane into Fatty acids, C18-unsaturated, dimers, Propylidynetrimethanol and oleic acid is assumed in a worst case approach.Thus, a resulting estimated long-term NOAEL ranging from 819.8-2312.2 mg/kg bw/day was established for Fatty acids, C18-unsatd., dimers, mixed esters with oleic acid and trimethylolpropane.

However, even the lowest long term NOAEL used for hazard assessment represents an overestimation, as the registered substance is not anticipated to be absorbed and hydrolysed in any significant amounts. Having regard to the fact that the NOAEL was near to the conventional limit dose of 1000 mg/kg bw/day, no adverse effects for humans are expected and thus no DNEL was derived.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Hazard assessment is conducted by means of read-across from structural analogues and surrogates. The selected study is the most adequate and reliable study based on the identified similarities in structure and intrinsic properties between source and target substance and overall assessment of quality, duration and dose descriptor level (refer to the endpoint discussion for further details).

Justification for classification or non-classification

Based on read-across from the surrogate substances, the available data on repeated dose toxicity do not meet the classification criteria according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.

There are no data available on repeated dose toxicity by the inhalation and dermal routes.