Registration Dossier

Administrative data

Description of key information

In a 36 week feeding study in young male F344 rats (Scimeca et al., 1998), a NOAEL of above 467 mg/kg bw/d was determined.

In an 90-d feeding study in young Wistar rats (O´Hagan, 2003), a NOAEL of 2433 mg/kg bw/d was determined.

In a 18 month feeding study in young male F344 rats (Park et al., 2005), a NOAEL between 400 and 800 mg/kg bw/d can be estimated.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1997
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
- Principle of test: 36 week feeding study in male rats.
- Short description of test conditions: 40 male Fischer 344 rats were given either a basal diet (control) or the same diet supplemented with 1.5 % CLA for 36 weeks.
- Parameters analysed / observed: food consumption, body weight, cageside observation, histopathological evaluation of 15 organs, haematological analysis
GLP compliance:
not specified
Limit test:
yes
Species:
rat
Strain:
Fischer 344
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague Dawley (Madison, WI, USA)
- Age at study initiation: 5 - 6 weeks
- Housing: individually in stainless-steel cages
- Diet: Purina Rat Chow 5012, ad libitum
- Water: ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 5
- Humidity (%): 60 ± 20

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): monthly
- Mixing appropriate amounts with (Type of food): semi-purified AIN-76A diet
- Storage temperature of food: refrigerated

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The CLA was analysed by GC. Ca. 90 % of the linoleic acid was isomerized to CLA.
Three isomers accounted for 85 % of the total isomers: c9, t11, t9. c11, and t10, c12-octadecadienoic acid
Duration of treatment / exposure:
36 weeks
Frequency of treatment:
daily
Dose / conc.:
0 other: %
Remarks:
in diet
Dose / conc.:
1.5 other: %
Remarks:
in diet
No. of animals per sex per dose:
20 male/dose group
Control animals:
yes, concurrent no treatment
Positive control:
not examined
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily except for weekends

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the day of sacrifice
- Anaesthetic used for blood collection: Yes (Ether)
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table 1 were examined.




Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes (see table 2)
Statistics:
Body weight gain, food consumption, organ weights, relative organ weights, and haematological parameters were compared by Students t-test.
Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One animal in the control group had to be sacrificed pre-scheduled due to signs of distress. A histopathological evaluation revealed a transmural ulcer of the small intestine.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean body weight gain in the control animals was 270.5 ± 31.8 g. The mean body weight gain in the treatment group (258.3 ± 46.5 g) was not significantly different from control.
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
Description (incidence and severity):
One control rat revealed markedly elevated platelet and granulocyte count. Furthermore, this animal showed depressed values for red blood cells, haematocrit and haemoglobin, indicators of anaemia. However, histopathological evaluation did not confirm this.
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
The average daily intake of the test substance in the treatment group ranged from 1970 ± mg/kg bw/d in week 1 to 467 ± 52 mg/kg bw/d in week 36.
Key result
Dose descriptor:
NOAEL
Effect level:
> 467 - < 1 970 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Key result
Critical effects observed:
no
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2002
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
The 9cis,11trans and 10trans,12cis CLA isomers are present in approximately equal proportions in the test item, and these two isomers account for approximately 75% of the total fatty acid content of the product. Other CLA isomers are also present and account for approximately 4.4% of the total fatty acid content. The remaining fatty acids are mainly mono-unsaturated e.g. oleic acid, and saturated e.g. palmitic and stearic acid. The poly- and saturated e.g. palmitic and stearic acid. The poly-unsaturated fatty acids, other than CLA, consist mainly of linoleic acid. The typical fatty acid composition of the test item is given in table 1.
Species:
rat
Strain:
Wistar
Remarks:
Crl:(WI)WU BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 7 weeks
- Weight at study initiation: ca 140 - 180 g
- Housing: individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 2 - 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET
Low fat control (LF control) : AIN-93G diet, standard fat content of 7 % w/w (safflower oil)
High fat control (HF control): AIN-93G diet containing 15 % w/w fat (safflower oil)
Treatment groups: modified AIN-93G diets containing 15% w/w fat made up from different levels of saffiower oil and ClarinolTM G-80.
High fat (HF) - low dose
High fat (HF) - mid dose
High fat (HF) - high dose




Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The diet was homogenous over the 0 - 15 % dose range for test and control oil.
Safflower oil became unstable after a storage period of 8 days in the refrigerator or 4 days at room temperature, respectively.
Analyses of the total fat content and the ratio of CLA to linoleic acid of the dose groups were within the determined ranges.
Duration of treatment / exposure:
90 d
Frequency of treatment:
daily
Dose / conc.:
0 other: %
Remarks:
Low fat control
Dose / conc.:
0 other: %
Remarks:
High fat control
Dose / conc.:
1 other: %
Remarks:
High fat - low dose
Dose / conc.:
5 other: %
Remarks:
High fat - mid dose
Dose / conc.:
15 other: %
Remarks:
High fat - high dose
No. of animals per sex per dose:
Main group: 20
Recovery group: 10 (LF control, HF Control, HF - high dose)
Control animals:
yes, concurrent no treatment
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily; daily on weekends

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to treatment and on a weekly basis during the treatment period.

BODY WEIGHT: Yes
- Time schedule for examinations: day 0, once weekly thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE
- Food consumption for each animal determined and mean daily diet consumption (periods of 7 days) calculated as g food/rat/day: Yes

FOOD EFFICIENCY:
- Body weight gain in g weight gain/g food consumed.: Yes

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: during 4-day periods in week 1, 6 and 12

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to the start of treatment in all animals and towards the end of the study (day 87)
- Dose groups that were examined: both control groups and HF - high dose group and in the control and high-dose groups towards the end of the recovery period.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On days 22 (males) and 24 (females) in week 4 and on days 51 (females) and 52 (males) in week 8 10 rats/sex/group of the main study and at necropsy (day 90).
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes, over night
- How many animals: all animals of the respective groups

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: see HAEMATOLOGY
- Animals fasted: Yes, over night
- How many animals: see HAEMATOLOGY
- Parameters examined: Aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), and alkaline phosphatase (AlkP)

URINALYSIS: Yes
- Time schedule for collection of urine: week 13, 10 rats/sex/group in the main study groups and all aninials of the recovery groups. For all male animals of die recovery groups this procedure was repeated in week 17.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, water 24 h, food at least 16 h


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during the last week of treatment
- Dose groups that were examined: 10 sex/group of the main study
- Battery of functions tested: sensorimotor reflex testing, grip strength, measurement and motor activity assessment
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
Statistics:
Analysis of covariance (covariate: body weight on day 0) followed by Dunnett's multiple comparison tests: Body weights
One-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison tests: Food and water consumption, food efficiency, red blood cell and clotting potential variables, total white blood cell counts, absolute differential white blood cell counts, clinical chemistry values and organ weights.
Kruskal-Wallis non-parametric ANOVA followed by Mann-Whitney U-tests: Other blood cell counts and urinary parameters.
Fisher's exact probability test: Histopathology changes
Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One control animal of the recovery group was sacrificed in moribund state on day 107.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
According to the reduced food intake, the animals of the HF - high dose showed a delayed growth and body weight gain. The reduced growth resulted in a significantly reduced body weight in the HF - high dose animals, with a more severe impact on female animals.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption was significantly lower in the HF - high dose group. The reason was the reduced palatability of the 15 % test item diet.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), and alkaline phosphatase (AlkP) were significantly increased in the HF - high dose group. In male animals these values normalized during the recovery period. In female animals ALAT and ASAT remained elevated compared to the LF - control group. This indicates, that the observed changes in liver were adaptive effects to the HF diet.
The test item increased plasma insulin levels in males and females of the HF - high dose group. In male rats, this increase was transient. At the end of the treatment period, no significant differences between HF - high dose and control values were observed.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
In male and female rats in the HF- high dose group, liver enlargement was observed. Females developed hepatocellular hypertrophy. These effects were completely reversible during the recovery period. This indicates, that the observed changes in liver were adaptive effects to the HF diet.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Liver enlargement was observed in male and female rats in the high - dose group. Femals showed additionally hepatocellular hypertrophy. During recovery period, a complete reversal of the effects was observed in males. In females, the reversal was not complete. The observed changes indicate an adaptive effect in response to feeding high levels of the test item in the diet. There was no evidence of fat accumulation in the liver.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
2 433 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Key result
Dose descriptor:
NOAEL
Effect level:
2 728 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Key result
Critical effects observed:
no
Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2002 - 2004
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
- Principle of test: oral feeding of the test item to rats.
- Short description of test conditions: Male Fischer 344 rats received a diet with or without 1 % conjugated linoleic acid (CLA) for a period of 18 months.
- Parameters analysed / observed: survival rate, weight gain, food consumption, clinical chemistry, hematology, necropsy and histopathology
GLP compliance:
not specified
Limit test:
yes
Specific details on test material used for the study:
Test item composition:
88.7% CLA( cis-9,trans-11, 41.9%; trans-10,cis-12, 43.5%; trans-9,trans-11/trans-10,trans-12, 1.5%; others, 1.8%) with the remainder as oleic acid, 5.6%; palmitic acid, 1.4%; linoleic acid, 0.5%; and other fatty acids, 3.8%.
Species:
rat
Strain:
Fischer 344
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague-Dawley, Madison, WI, USA
- Age at study initiation: Weanling
- Weight at study initiation: 110 - 120 g
- Housing: individually in wire-bottomed cages
- Diet: TD94060, 99% basal mix, ad libitum
- Water: ad libitum
- Acclimation period: 5 days

- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Duration of treatment / exposure:
18 month
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
600 mg/kg bw/day (nominal)
Remarks:
based on the addition of 1 % CLA to the feeding, an individual dose between 400 - 800 mg/kg bw/d was determined
No. of animals per sex per dose:
untreated control: 10
Treatment group: 11
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The 1 % diet supplement was chosen to approximate the dose of a previous study in mice (850 mg/kg bw).
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: "closely" observed

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: At week 70

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at three different days at week 69 and 70.
- Anaesthetic used for blood collection: No
- Animals fasted: Yes, overnight

CLINICAL CHEMISTRY: Yes

URINALYSIS: Yes
- Time schedule for collection of urine: At week 70
- Metabolism cages used for collection of urine: Yes


Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
Statistics:
Data were analysed with Student t-test. Fasted glucose, survival, necropsy, and histopathology data were analysed with Fisher´s exact test. Fasted plasma samples for glucose analysis were collected in three different days, therefore data were analysed with two-way ANOVA.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Three rats from each group were sacrificed before the completion of the study. All of these animals had severe chronic renal disease. Three animals of each group also had pituitary or testicular tumors.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
In total, four animals in each group had to be sacrificed before the end of the study, due to clinical implications.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Feeding of CLA reduced blood glucose concentration in both fasted and fed animals significantly.
Clinical biochemistry findings:
effects observed, non-treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no differences in tissue weights of treated and untreated animals.
Gross pathological findings:
effects observed, non-treatment-related
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
All animals showed signs of chronic renal disease (chronic interstitial nephritis, nephrosis and/or glomerulosclerosis). This may have been due to the high protein content of the diet compared to regular chow (14 – 16 %).
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Pituitary tumors, testicular tumors/masses, and/or prostatitis were observed in both treatment groups. One of the CLA-fed animals had an enlarged spleen, which was diagnosed as granular cell lymphoma. Also, two animals from each treatment had early stage granular cell lymphoma. Other observations included gastritis, cardiomyopathy, focal hepatopathy, and interstitial pneumonitis, which were observed in one animal from each treatment group.
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
>= 400 - <= 800 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Key result
Critical effects observed:
no

Table 2: Histopathology

 

Treatment

Control

CLA

Total number of animals

7

8

Chronic renal diseases

7

8

Prostatitis

4

6

Testicular tumor or mass

3

4

Pituitary tumor

3

3

Lymphoma

2

2

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
476 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Klimisch code 2

Additional information

In a subchronic 36 week oral toxicity study, the test item was administered to 20 young male F344 rats at a dose level of 1.5 % in the daily diet (Scimeca, 1998). The mean substance intake ranged from 1970 mg/kg bw/d in week 1 to 467 mg/kg bw/d in week 36, based on the data of food consumption. There were no compound related effects in mortality, clinical signs, body weight, food consumption, hematology, organ weights, or gross and histologic pathology.

Based on the results of the study a NOAEL of above 467 mg/kg bw was determined.

In a subchronic oral toxicity study according to OECD guideline 408 (O´Hagan, 2003), the test item was administered to 20 young male or female Wistar rats per group at a dose level of 1, 5, or 15 % in the daily diet. Two control groups were run in parallel (Low fat - control and High fat - control). The test substance had no effect on mortality, clinical signs, bodyweight, food consumption or food conversion efficiency. Investigations of the clinical biochemistry parameters indicated a transient elevation of alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT) and alkaline phosphatase (AlkP) as well as blood insulin levels. The gross pathological investigation revealed a test substance related transient hepatocellular hypertrophy but no accumulation of fat in the liver. However, since the observed effects had a transient nature, they were not considered as adverse.

Based on the results of the study a NOAEL of above 2433 mg/kg bw/d in male rats was determined.

In a chronic oral toxicity study (18 months) (Park, 2005), the test item was administered to 11 young male Fisher 344 rats at 1 % in the daily diet. A control group was run in parallel (plain diet). The test substance had no effect on mortality, clinical signs, bodyweight or food consumption. Investigations of the clinical biochemistry parameters indicated a reduced blood glucose level in the treatment group. However, this effect was not considered as adverse. At necropsy, no changes in the organ weights were observed. All animals of both treatments showed signs of chronic renal disease. It was proposed, that this effect is due to the elevated protein content of the diet.

In conclusion, no adverse effects were observed in the available studies.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is not considered to be classified for repeated dose oral toxicity, as amended for the tenth time in Regulation (EU) No 2017/776.