Registration Dossier

Administrative data

Description of key information

Based on read-across from Fatty acids, C18-unsaturated, dimers (CAS No. 61788-89-4):
Oral: NOAEL (rat, subchronic) = 741 (males) and 855 (females) mg/kg bw/day (similar to OECD 408, GLP)

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:
30 Nov Nov 1992 - 05 Mar 1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP-compliant, comparable to guideline study with acceptable restrictions. No functional observations performed; most but not all haematological/clinical chemistry, organ weight and histopathological examinations performed.
Reference:
Composition 0
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
no functional observations performed; most but not all haematological/clinical chemistry, organ weight and histopathological examinations performed
GLP compliance:
yes
Limit test:
no
Test material information:
Composition 1
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals 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/cage
- Diet: ESL modified AIN-76A (MODAIN) purified diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 1 week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- 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): experimental diets were initially prepared weekly from 26 Nov to 10 Dec 1992. After obtaining data confirming test material stability in diet for 14 days, diets were prepared every two 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: diets in sealed bags were UV-sterilised for a minimum of 10 h at ambient temperature before entry in the SPF unit, where they were stored at approx. 4 °C prior to use.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
-Stability and homogeneity in diet
The stability of the test substance 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 that the test substance 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 the test substance.
Separation on the HPLC system was based on the interaction of the carboxylic acid of the test substance with free hydroxyl groups at the surface of the diol phase. Thus, interaction increased with the number of carboxylic acid groups. The test substance contains mixtures of mono, di and polyacids. In the assay preparation based on two peaks, di-acid denoted dimer and tri or greater (poly) acid denoted trimer.
The test substance was shown to be stable in diet over 14 days. The results for 0.1% (w/w) test substance 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, the test substance 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% test substance prepared on 26 Nov 1992, 07 Jan 1993 and 18 Feb 1993 were analysed for the achieved concentration of the test substance 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) test substance 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, 7 days/week
Remarks:
Doses / Concentrations:
0.1, 1, 5% (w/w)
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
74.1, 740.9, 3591.2 mg/kg bw/day (males)
Basis:
other: as calculated from the reported mean body weight and food intake data
Remarks:
Doses / Concentrations:
90.5, 854.9, 4085.5 mg/kg bw/day (females)
Basis:
other: as calculated from the reported mean body weight and food intake data
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: twice daily (once on Saturdays and Sundays)
- Cage side observations included: signs of ill-health or reaction to treatment

BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of the study and weekly thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE:
- 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 intake values were determined twice-weekly 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: Yes
- Time schedule for examinations: For each cage of five animals, water consumption values were determined twice-weekly and weekly values were calculated.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: in the week prior to study start and end, respectively
- 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 data
- How many animals: all
- Parameters examined: red blood cell count, platelets, haemoglobin, total white blood cells, differential white cell count (lymphocytes, neutrophils, monocytes, eosinophils, basophils, large unstained cells), mean red cell volume (derived from the red cell volume histogram), haematocrit, mean red cell haemoglobin, mean red cell haemoglobin concentration, reticulocytes, prothrombin time (PT), activated partial thromboplastin time (APTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the test period
- Animals fasted: No data
- How many animals: all
- Parameters examined: sodium, potassium, calcium, magnesium, chloride, inorganic phosphate, creatinine, urea, glucose, triglyceride, total cholesterol, total bilirubin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), hydroxybutyrate dehydrogenase (HBD), alkaline phosphatase (AP), pseudocholinesterase, creatine kinase, 5’-nucleotidase, gamma-glutamyltransferase, total protein, albumin, alpha1-globulin, alpha2-globulin, β-globulin, gamma-globulin, albumin:globulin ratio.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. All animals which survived to the end of the study received a detailed necropsy. All macroscopic abnormalities were recorded and a score was allocated as an assessment of the level of intra-abdominal fat deposition.
Brain, Heart, Liver, Kidneys, Spleen, Testes and Adrenal glands were weighed prior to fixation.
HISTOPATHOLOGY: Yes. The following tissues were taken from each rat and preserved in 10% buffered formalin: Adrenal glands, Jejunum, Prostate, Aorta, Kidneys, Rectum, Bladder, Larynx, Sciatic nerve, Brain, Liver, Spinal cord, Caecum, Lungs, Spleen, Cervix, Lymph nodes (cervical and mesenteric), sternum, Colon, Mammary glands, Stomach, Duodenum, Muscle, Thymus, Femur and stifle joint, Oesophagus, Thyroid and parathyroids, Head, Ovaries and fallopian tubes, Tongue, Heart, Pancreas, Trachea, Ileum, Pituitary, Uterus.
The following tissues were taken from each rat and preserved in Bouin's fixative: Epididymides, Salivary glands, Seminal vesicles, Skin, Testes, Vagina.
The eyes/harderian glands were taken from each rat and fixed in Davidson's fluid.
Bone marrow smears were taken and stained with May-Grunwald Giemsa.
Microscopic examination was carried out on all of the tissues from all animals in the control and hig-dose groups, and on all tissues showing macroscopic abnormalities which were designated as lesions at necropsy. In addition, livers, adrenals, mesenteric lymph nodes, spleens and thyroids (females only) from all animals fed 1.0 and 0.1% test substance were examined microscopically following identification of treatment-related lesions in the high-dose group.
During the examination of tissues, histopathological changes were recorded as present or absent, or graded according to their morphology, using a numerical scale of 0.0 – 5.0 in order to assess degrees of severity or activity. This procedure provided a means of ranking degrees of change which can assist in the interpretation of biological differences.
Statistics:
Statistical analysis was conducted for data on body weight, food and water intake, food conversion efficiency, clinical pathology and organ weights. Initially the data were examined to see if parametric or non-parametric analysis was appropriate.
For parametric data, one way analysis of variance was used to assess differences. A t-test was used to show any significant differences between control and test groups at the 5, 1 and 0.1% probability levels. A multiple T test was used for pairwise comparisons between groups.
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):
effects observed, treatment-related
Description (incidence and severity):
see Details on results.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
see Details on results.
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
see Details on results.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
see Details on results.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
see Details on results.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
see Details on results.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see Details on results.
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No mortality occurred during the study period.
Clinical signs generally involved scabs, alopecia, excoriation, nasal discharge and ocular discharge, and were not considered to be treatment-related as they were observed in all control and treatment groups.

BODY WEIGHT AND WEIGHT GAIN
No treatment-related changes in body weight (gain) were observed during the study period. Statistically significant changes in body weight were few, minor, randomly distributed and of no biological significance. Thus, the mean body weight of females fed 1% test material was slightly (3.7%) increased in Week 0. In the same group, body weight gain was slightly (7.6%) increased in the period comprising Week 0-4.

FOOD CONSUMPTION AND COMPOUND INTAKE
Food consumption was significantly decreased during the first four weeks of the study in males (-5.4%) and females (-8.4%) fed 5.0% test substance in diet. This may reflect an initial reluctance of the animals to eat the diet.

FOOD EFFICIENCY
Food conversion efficiency was significantly increased (9.5%) in females of the 5.0% group during the first four weeks of the study.

WATER CONSUMPTION
Statistically significant changes (increases and decreases) in water consumption were observed but there was no clear treatment-related pattern. The accumulated water consumption was not significantly different between groups.

OPHTHALMOSCOPIC EXAMINATION
At the end of the study, a persistent pupillary membrane was recorded for one male in the 0.1% group and ocular opacity was recorded for one female in the 5.0% group. These findings were not considered to be treatment-related.

HAEMATOLOGY (Table 2)
Mean cell haemoglobin was slightly (2.3%) but statistically significantly increased in females of the 5.0% group. Prothrombin time was likewise slightly but significantly increased in males of the 5.0% group (3%) and in females of the 1.0 and 5.0% groups (2.6 and 4.3%, respectively). The observed changes were slight and unlikely to be toxicologically relevant.
A number of other changes (e.g. decreased neutrophil counts in females fed 1 .0 and 5.0% test substance) were statistically significant by Student's t-test but did not trigger the multiple t-test as significant.

CLINICAL CHEMISTRY (Table 3)
The following statistically significant changes were observed but not considered to be toxicologically relevant, since they were minor (< 10%) and/or no dose-relationship was noted: decrease in plasma calcium (males at 5.0% and all female groups), glucose (females at 1.0%), total protein (males and females at 5.0%) and increase in albumin/globulin ratio (males at 1.0 and 5.0%).
Aspartate aminotransferase was increased in females of the 0.1 and 5.0% groups and serum albumin was increased in males and females of the 5.0% group, but without dose-relationship.
Similarly, no clear evidence for a dose dependency was seen for the decrease in 5’-nucleotidase (males at 0.1 and 5.0% and females at 5.0%) and the increase in alanine aminotransferase (males and females at 5.0%).
Bilirubin was statistically significantly increased in males of the 1.0 and 5.0% groups. However, the levels measured were below the sensitivity of the method used and must therefore be viewed with caution.
Total cholesterol and triglycerides were significantly and dose-dependently decreased in males and females of the 1.0 and 5.0% groups. A decrease in beta-globulin was observed in males of the 5% group.
The statistically significant and dose-dependent increase in alkaline phosphatase observed in males and females of the 1.0 and 5.0% groups was marked, the level being more than double that of the control group in animals fed 5.0% test substance.

ORGAN WEIGHTS (Table 4)
Absolute and relative spleen weights were statistically significantly reduced in males of the 1.0 and 5.0% groups. Kidney weights were slightly but significantly reduced in females of the 1.0% (only relative weight) and 5.0% (absolute and relative weights) groups. Absolute liver weight was significantly reduced in females of the 0.1% group, while the relative weights were significantly reduced in all female groups, but without any dose-relationship. Absolute and relative liver weights were slightly but significantly reduced in males of the 1.0 and 5.0% groups.
The observed effects had no relation to any effect which might have been expected on the basis of the histopathology findings.

GROSS PATHOLOGY (Table 5)
Treatment with the test substance had no effects on bodily condition as assessed by estimation of abdominal fat reserves at necropsy.
Mesenteric lymph nodes were slightly enlarged in 1/20 male and 2/20 females of the 0.1% groups, in 13/20 males and 14/20 females of the 1.0% groups and in 13/20 males and 16/20 females of the 5.0% groups. Moderate enlargement was seen in 1/20 males at 1.0% and in 7/20 males and 2/20 females at 5.0%.
The colour of caecal contents was affected by feeding with the test substance. Caecal contents were predominantly yellow in rats fed 5.0% test substance and predominantly yellow-green in rats given 1.0% test substance, compared with green or gray-green in rats fed 0.1% test substance or the control animals. It should be note that the test substance was a yellow liquid.
The incidence of uterine fluid distension was slightly increased in rats fed 5.0% test substance (15/20 animals) compared with the control (8/20 animals).
All other macroscopic findings were considered to be incidental and within the range expected for this age and strain of rat.

HISTOPATHOLOGY: NON-NEOPLASTIC (Table 6)
Microscopic examination revealed treatment-related findings in mesenteric lymph nodes, spleen, liver, adrenal glands and thyroid glands (in females). Only those effects seen in the mesenteric lymph nodes and spleen extended down to the group fed 0.1% test substance.

-Mesenteric lymph nodes:
Macrophage aggregation(s) (some containing a golden brown pigment) were noted in the paracortex and in the medullary cords in all rats fed 5.0% test substance, in 11/19 male and 17/19 female rats fed 1.0% test substance and in 3/18 male and 8/19 female rats fed 0.1% test substance. Incidence and number of aggregations were dose-related, with only a few aggregations being present in rats given 0.1% test substance. There was a correlation between histological findings in the mesenteric lymph nodes and lymph node enlargement seen at necropsy.

-Spleen:
Golden/dark brown pigmented macrophages were observed in the red and white pulp of all animals fed 5.0% test substance, 16/20 males and 19/20 females fed 1.0% test substance and 5/20 females fed 0.1% test substance. Incidence and amount of pigmented macrophages was dose-related in male and female rats, the effect being more pronounced in females.

-Liver:
In male rats fed 5.0% test substance, the incidence of bile duct proliferation was increased (18/20 animals compared to 5/20 animals in the control group), as was incidence of sclerotic bile ducts (13/20 animals compared to 3/20 animals in the control group). Sclerosis was associated with minimal infiltration of mixed inflammatory cells. There was a very slight increase in the incidence of bile duct proliferation in female rats given 5.0% test substance (5/20 treated compared to 2/20 control animals). Periportal cytoplasmic vacuolation was decreased in males and females given 1.0% and 5.0% test substance.

-Adrenals:
In female rats fed 5.0% or 1.0% test substance, cortical vacuolation was noted in the adrenal glands (13/20 and 20/20 rats, respectively). Trace levels of vacuolation were seen in one female fed 0.1% test substance. This finding was not considered toxicologically important as vacuolation may occasionally be seen in control female animals. Cytoplasmic rarefaction was decreased in females given 5.0% test substance (0/20 treated rats compared to 19/20 control rats).
Cortical extramedullary haemopoiesis was not present in females fed 5.0% test substance. The incidence of extramedullary haemopoiesis was slightly reduced in female rats fed 1.0% or 0.1% test substance (6/20 and 5/20 animals respectively compared to 10/20 control animals). However, this was not considered of toxicological importance given that the incidence of this finding generally varies considerably among groups of untreated animals.

-Thyroids:
A slight increase in follicular epithelial hypertrophy was observed in female rats fed 5.0% test substance (15/20 animals) when compared with the control (5/20 animals).

-Spontaneous pathology:
Microscopic examination of the uteri from rats showing macroscopic fluid distension revealed that this was due to a variety of different reasons: luminal dilatation or dilated/cystic endometrial glands. This finding is not thought to be of any toxicological importance, in view of this, and in view of the variation in uterine size with different phases of the estrous cycle.
The incidence of retinal folding/atrophy was higher in animals of the test groups. However, since the overall incidence was very low and no dose relationship was observed, these lesions were not considered toxicologically relevant.
There was a slight increase in the incidence of lesions in the nasal passage in rats of the 5.0% group compared with the control groups. 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 observed in rats from all test groups without evidence of a treatment-related distribution. Findings were within the spectrum of spontaneous lesions commonly seen in laboratory rats of this age and strain and were thus considered no to be related to the test substance.
Dose descriptor:
NOAEL
Effect level:
1 other: % in diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: adaptive effects on clinical chemistry (increase in alkaline phosphatase, slight decrease in total cholesterol and triglycerides) and histopathology (pigmented macrophage aggregation in lymph nodes and spleen)
Dose descriptor:
NOAEL
Effect level:
ca. 741 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
average dose calculated on the basis of the reported body weight and food intake data
Sex:
male
Basis for effect level:
other: corresponding to 1% in diet based on reported mean body weight and food intake data
Dose descriptor:
NOAEL
Effect level:
ca. 855 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
average dose calculated on the basis of the reported body weight and food intake data
Sex:
female
Basis for effect level:
other: corresponding to 1% in diet based on reported mean body weight and food intake data
Critical effects observed:
not specified

Table 1. Achieved dose levels.

Treatment

Body weight (g)

Mean body weight (g)

Food intake (g)

Mean food intake (g/day)

Mean compound intake (g/day)

Mean dose level (mg/kg bw/day)

Week 0

Week 13

Week 0-13

Week 0-13

Week 0-13

Week 0-13

Week 0-13

Males

5.0%

133.7

541.5

337.6

2182.3

24.2

1.21

3591.2

1.0%

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

Control

133.3

545.5

339.4

2215.3

24.6

0.00

0.0

Females

5.0%

120.2

317.5

218.85

1609.4

17.9

0.89

4085.5

1.0%

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

Control

119.2

317.5

218.35

1693.8

18.8

0.00

0.0

 

Table 2. Haematology

Treatment

Mean cell haemoglobin (pg)

Prothrombin time (s)

Males

5.0%

18.16*

11.90**

1.0%

17.74

11.65

0.1%

17.58

11.54

Control

17.75

11.55

Females

5.0%

18.45

11.92***

1.0%

18.57

11.73*

0.1%

18.51

11.58

Control

18.41

11.43

 

* Statistically significantly different versus control (P = 0.5)

** Statistically significantly different versus control (P = 0.1)

*** Statistically significantly different versus control (P = 0.01)

 

Table 3. Clinical chemistry.

Treatment

Calcium

(mmol/L)

5’-Nucleotidase

(U/L)

Alkaline phosphatase

(U/L)

Alanine aminotransferase

(U/L)

Aspartate aminotransferase

(U/L)

Triglycerides

(mmol/L)

Total cholestertol

(mmol/L)

Males

5.0%

2.502***

28.4***

810.6***

65.1***

104.5

0.618***

1.901***

1.0%

2.559

32.7

606.3***

52.8

107.3

1.247***

2.380*

0.1%

2.598

32.2*

424.7

40.4

95.1

1.701

2.517

Control

2.604

36.3

389.5

41.0

93.8

1.820

2.803

Females

5.0%

2.567***

47.0**

512.6***

43.1***

93.1***

0.475***

1.766***

1.0%

2.626**

63.4

393.3***

30.5

75.9

0.876

2.181*

0.1%

2.612***

74.3

250.4

29.1

81.5**

0.965

2.362

Control

2.694

76.3

216.4

29.6

71.1

1.120

2.441

 

Treatment

Glucose

(mmol/L)

Bilirubin

(µmol/L)

Total protein

(g/L)

Albumin

(g/L)

Albumin/globulin ratio

beta-globulin

(g/L)

Males

5.0%

9.353

3.1***

62.07***

29.23*

0.905**

10.64***

1.0%

9.815

2.7*

64.09*

30.44

0.905**

11.89

0.1%

10.258

2.5

65.44

30.16

0.860

12.29

Control

10.053

2.3

66.58

30.52

0.845

12.36

Females

5.0%

9.420

2.7

67.29**

33.76***

1.015

10.65

1.0%

9.373*

2.8

70.59

36.24

1.055

10.70

0.1%

9.993

2.6

71.10

35.97

1.025

11.15

Control

9.852

2.6

72.35

37.12

1.050

11.43

 

* Statistically significantly different versus control (P = 0.5)

** Statistically significantly different versus control (P = 0.1)

*** Statistically significantly different versus control (P = 0.01)

 

Table 4. Organ weights.

Treatment

Spleen

(g)

Spleen

(g/100 g bw)

Kidney

(g)

Kidney

(g/100 g bw)

Liver

(g)

Liver

(g/100 bw)

Males

 

 

 

 

 

 

5.0%

0.7520***

0.1381***

3.429

0.631

18.169*

3.343***

1.0%

0.8436*

0.1566*

3.441

0.641

18.739*

3.472**

0.1%

0.9571

0.1691

3.565

0.633

20.756

3.660

Control

0.9454

0.1723

3.635

0.663

20.610

3.737

Females

 

 

 

 

 

 

5.0%

0.5573

0.1765

2.028*

0.640**

10.781

3.395**

1.0%

0.5904

0.1764

2.178

0.650*

10.804

3.210***

0.1%

0.5808

0.1851

2.064

0.657

9.862***

3.125***

Control

0.6079

0.1924

2.167

0.688

11.520

3.640

 

* Statistically significantly different versus control (P = 0.5)

** Statistically significantly different versus control (P = 0.1)

*** Statistically significantly different versus control (P = 0.01)

 

 

Table 5. Incidence of macroscopic findings.

Macroscopic observation

Treatment

Control

0.1%

1.0%

5.0%

Males

No. of animals/group

20

20

20

20

Caecum

Contents green

5

6

4

0

Contents gray-green

15

13

0

0

Contents yellow

0

0

0

19

Contents yellow-green

0

1

16

1

Mesenteric lymph node

Enlarged (moderate)

0

0

1

7

Enlarged (slight)

0

1

13

13

Females

No. of animals/group

20

20

20

20

Caecum

Contents green

7

8

3

0

Contents gray-green

13

12

1

0

Contents yellow

0

0

0

20

Contents yellow-green

0

0

16

0

Mesenteric lymph node

Enlarged (moderate)

0

0

0

2

Enlarged (slight)

0

2

14

16

Uterus

Moderate fluid distention

1

4

6

3

Severe fluid distention

1

0

0

0

Slight distention

0

0

1

0

Slight fluid distention

8

7

6

15

 

Table 6. Incidence of microscopic findings.

Microscopic observation

Treatment

Control

0.1%

1.0%

5.0%

Males

Adrenals

No. examined

20

20

20

20

 

Without abnormalities

3

0

0

1

Adrenal cortex

Vacuolation

17

20

20

19

 

Extramedullary haemopoiesis

0

2

0

0

Thyroids

No. examined

20

0

0

20

 

Without abnormalities

10

0

0

8

 

Follicular epithelium hypertrophy

10

0

0

12

Mesenteric lymph node

No tissue available

0

2

1

0

 

No. examined

20

18

19

20

 

Without abnormalities

12

12

1

0

 

Pigmented macrophage aggregation(s)

0

1

11

20

 

Macrophage aggregation(s)

1

3

10

20

Spleen

No. examined

20

20

20

20

 

Without abnormalities

20

19

4

0

 

Pigmented macrophages

0

0

16

20

 

Extramedullary haemopoiesis

0

1

2

0

Liver

No. examined

20

20

20

20

 

Without abnormalities

0

0

1

0

 

Cytoplasmic vacuolation (periportal)

7

6

1

0

 

Bile duct proliferation

5

4

6

18

 

Sclerotic bile duct(s)

3

4

7

12

 

Mixed inflammatory cell infiltration (periportal)

1

1

1

11

Females

Adrenal cortex

No. examined

20

20

20

20

 

Without abnormalities

0

0

1

0

 

Vacuolation

0

1

13

20

 

Extramedullary haemopoiesis

10

6

5

0

 

Cytoplasmic rarefaction

19

16

14

0

Thyroids

Follicular epithelium hypertrophy

5

5

6

15

Mesenteric lymph node

No tissue available

0

1

1

0

 

No. examined

20

19

19

20

 

Without abnormalities

17

5

1

0

 

Pigmented macrophage aggregation(s)

0

3

9

19

 

Macrophage aggregation(s)

0

8

17

20

 

Focus(i) foamy macrophages

0

1

0

0

Spleen

No. examined

20

20

20

20

 

Without abnormalities

20

15

1

0

 

Pigmented macrophages

0

5

19

20

Liver

No. examined

20

20

20

20

 

Without abnormalities

0

1

5

5

 

Cytoplasmic vacuolation (periportal)

10

13

6

2

 

Bile duct proliferation

2

0

0

5

 

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 an adequate and reliable study (Klimisch score 2) from a reference substance with similar structure and intrinsic properties. Read-across is justified based on structural similarity as a result of common origin and production process line (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, 8.6.2, 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

Mode of Action Analysis / Human Relevance Framework

Additional information

Justification for grouping of substances and read-across

There are no data available on the repeated dose toxicity of Fatty acids, C18-unsaturated, trimers, hydrogenated. In order to fulfil the standard information requirements set out in Annex IX, Section 8.6.2, in accordance with Annex XI, Section 1.5 of Regulation (EC) No 1907/2006, read-across from a structurally similar substance is 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 physicochemical, toxicological and ecotoxicological properties may be predicted from data for reference substance(s) by interpolation to other substances on the basis of structural similarity, Fatty acids, C18-unsatd., dimers (CAS 61788-89-4) is selected as reference substances for assessment of repeated dose toxicity.

The read-across is based on structural similarity as a result of common origin and production process line. Shortly, the source and target substances are derived from catalytically di- and trimerised long-chain fatty acids; dimers and trimers are separated by distillation and unsaturated alkyl chains are hydrogenated as specifically required. A detailed analogue approach justification is provided in the technical dossier (see IUCLID Section 13).

Overview of repeated dose toxicity

 

Target substance

Source substance

CAS No.

1373883-45-4

61788-89-4

Chemical name

Fatty acids, C18-unsaturated, trimers, hydrogenated

Fatty acids, C18-unsatd., dimers

Repeated dose toxicity: oral (subacute)

RA: CAS 61788-89-4

Experimental result:

NOAEL (rat, m) ≥ 1450 mg/kg bw/day

NOAEL (rat, f) ≥ 1692 mg/kg bw/day

(OECD 421)

Repeated dose toxicity: oral (subchronic)

RA: CAS 61788-89-4

Experimental result:

NOAEL (rat, m) = 741 mg/kg bw/day

NOAEL (rat, f) = 855 mg/kg bw/day

(similar to OECD 408)

 

Repeated dose toxicity: oral

Subacute

CAS 61788-89-4

A Reproduction/Developmental Toxicity Screening Test was conducted with Fatty acids, C18-unsatd., dimers following OECD guideline 421 and under GLP conditions (Clubb and Sutherland, 2004). This study is discussed in detail under Toxicity to reproduction.

Based on the lack of toxicologically relevant effects up to the highest dose level tested, the systemic toxicity as well as the reproductive toxicity/fertility NOAEL values were ≥ 1450 mg/kg bw/day and ≥ 1692 mg/kg bw/day for males and females, respectively.

Subchronic

CAS 61788-89-4

A subchronic oral toxicity study is available for Fatty acids, C18-unsaturated, dimers (Spurgeon and Hepburn, 1993). The 13-week feeding study was conducted in rats following a protocol equivalent to OECD Guideline 408 and in compliance with GLP. Three groups of 20 Sprague-Dawley rats per sex and dose were fed a plain diet or the test substance at 0.1, 1.0 and 5.0% (w/w) in purified diet ad libitum for 90 days. Based on the reported body weight and food intake data, the approximate doses were 74.1, 740.9, 3591.2 mg/kg bw/day for males and ca. 90.5, 854.9, 4085.5 mg/kg bw/day for females (average dose weeks 0-13).

No mortality occurred and no treatment-related changes in clinical signs, body weight or at ophthalmoscopic examination were observed during the study period. Food intake was lower during the first 4 weeks of the study in males and females of the 5.0% group. The lower food consumption was probably due to an initial reluctance of the rats to eat the diet. There were no treatment-related changes in water intake. Haematological examinations showed no toxicologically relevant changes.

Plasma alkaline phosphatase activity (AP) was increased in males and females fed the test material at 1.0% or 5.0%. Alanine aminotransferase (ALT) was also increased in male and female rats fed at 5.0%. An increase in plasma AP activity may reflect induction (increased synthesis) in liver cells rather than increased release from damaged cells.

A small reduction in plasma calcium, total serum protein and albumin was observed in males and females fed the test substance at 5.0%. It is possible the plasma calcium and serum protein changes may be connected. However, reduction in plasma calcium was not always accompanied by a simultaneous reduction in plasma albumin. Changes in plasma calcium and serum proteins were small, probably representing a physiological rather than a pathological response to treatment with the test substance.

Total cholesterol and triglycerides in plasma were reduced in male and female rats in the 5.0% and 1.0% treatment groups. The effect was very small for the 1.0% group. It is possible that the test substance blocks the absorption of lipid and other nutrients from the gut. This could also explain changes in plasma electrolytes and intermediate metabolites.

Decreases in absolute and/or organ weights of spleen (males), kidney (females) and liver (males and females) were observed, mainly after feeding the test material at 5.0% and less at 1.0%, but there was no relation to any effect which might have been expected on the basis of the findings at histopathological examination.

In addition to increases in plasma AP level, treatment-related effects were observed on microscopic examination of the liver. Histopathological examination of livers from animals in the 5.0% group showed an increase in biliary hyperplasia. Interference in bile flow could be related to the observed increase in AP, which is a sensitive indicator of cholestasis. Changes in AP develop before any detectable increases in plasma bilirubin levels. It should be noted that while a very small increase in plasma bilirubin was observed in male rats fed the test material at 5.0% and 1.0%, the bilirubin levels measured were below the sensitivity of the analytical method and must therefore be regarded with caution. Although the bile duct proliferation and sclerosis recorded in the liver may correlate with the increase in AP, it should be noted that only very minor biliary changes were observed in a few number of female rats. The reduction in periportal hepatocyte vacuolation noted at histopathological examination of the liver could correlate with the reduced plasma lipids, as described above, indicating some alteration in lipid metabolism, another possible explanation for the plasma lipid, serum protein and calcium changes.

Aggregation of macrophages (some pigmented) was observed in mesenteric lymph nodes of all treated rats. In the spleen, macrophages containing pigment were seen mainly in males and females fed 1.0 and 5.0% test substance. The incidence and amount of macrophages increased in a dose-related manner. The pigment present in the macrophages in the mesenteric lymph nodes and spleen did not stain with Perls' stain for haemosiderin or aldehyde fuchsin for lipofuscin, but did stain with Schmorl's stain for lipofuscin. Lipofuscin is derived from oxidation of unsaturated lipids or lipoproteins. Since the test substance is composed of a mixture of monomers, dimers and trimers of various fatty acids, it is likely that the pigment represents either lipofuscin produced by oxidation of some component of the test substance, or that the test substance itself stains positively.

There was no evidence of any degenerative effect associated with pigmented macrophages. It is probable that they represent a physiological response to dietary administration of lipid materials such as the test substance. Accumulations of macrophages in the mesenteric lymph nodes commonly occur as a result of ageing, or following administration of pigmented or lipid substances in the diet (Gopinath et al., 1987; Mohr et al., 1992). Although the pigment appeared darker in the spleen than in the mesenteric lymph node, this may have reflected a difference in density. The pigment was present alongside normal levels of haemosiderin which appeared tinctorially distinct. The coloured nature of the test substance is also likely to have been responsible for the alteration in the colour of the caecal contents that was noted at necropsy.

Increased cortical vacuolation in the adrenals of females fed 1.0 and 5.0% test substance, coupled with decreased cytoplasmic rarefaction, was observed. This probably indicated altered steroidogenesis. However, this finding was not accompanied by any evidence of degenerative change. The relevance of the reduced extramedullary haemopoiesis seen in the adrenals of the 5.0% females is uncertain, but may possibly correlate with the reduction in neutrophil count in females fed the test material at 5.0% and 1.0%. All of the changes in the adrenal glands were minor in nature and of limited importance.

An increase in thyroid follicular epithelial hypertrophy was observed in female rats fed 5.0% test substance. This finding was unusual and not dose-related. The hypertrophy comprised of a slight increase in height of the follicular epithelium combined with a reduction in the size of the follicular lumen and in the amount of colloid therein.

Based on clinical chemistry parameters and histopathological findings regarded to be of adaptive nature, 1% (w/w) test material in diet is 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.

Conclusions for repeated dose toxicity

There are no data available on the repeated dose toxicity of Fatty acids, C18-unsaturated, trimers, hydrogenated. Therefore, in order to fulfil the standard information requirements set out in Annex IX, Section 8.6.2, in accordance with Annex XI, Section 1.5 of Regulation (EC) No 1907/2006, read-across from a structurally similar substance is conducted.

A subchronic oral toxicity study is available for Fatty acids, C18-unsatd., dimers (CAS 61788-89-4), in which NOAEL values of 741 and 855 mg/kg bw/day were identified for males and female rats (corresponding to 1% in diet), based on adaptive changes in clinical chemistry parameters and histopathological findings.

Furthermore, in a reproduction/developmental toxicity screening study with Fatty acids, C18-unsatd., dimers, no toxicologically relevant effects were observed in rats treated with the test item up to and including the highest dose level, thus resulting in NOAEL values of at least 1450 (males) and 1692 (females) mg/kg bw/day.

Based on read-across, the substance is considered to be of low toxicity after long-term oral exposure, and no hazard was identified since in a subchronic study toxicologically relevant effects were observed only at dose levels above the currently applicable limit dose level of 1000 mg/kg bw/day.

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

References

Gopinath, C. et al. (1987). In : Atlas of Experimental Toxicological Pathology, pp 112, 113 and 127. MTP Press Ltd., Falcon House, Queen Sq., Lancaster, England.

Mohr, U. et al. (Eds.) (1992). In: Pathobiology of the Ageing Rat, pp 54, 55. ILSI Press, 1126 Sixteenth St., N.W., Washington, D.C.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Hazard assessment is conducted by means of read-across from a structural analogue. 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 a structurally similar substance following an analogue approach, the available data on the repeated dose oral toxicity of the substance 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.