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Administrative data

Description of key information

Repeated dose toxicity (oral): Based on the experimental results on analogue substances, the weight of evidence approach was applied and the 90d-NOAEL for the substance Fatty acids, C16-18 (even numbered), ammonium salts was determined to be 2500 mg/kg bw/day in rats.

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:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
Groups of 20 male and 20 female six week old rats were fed diets containing 5, 10 or 20 % magnesium stearate. The diets were semi synthetic in which sodium caseinate replaced casein. The animals were weighed once weekly and food utilization and weight gain was calculated for each sex of all groups of rats. Blood samples were taken from 8 males and 8 females from each group prior to dosing and at 8 and 12 weeks. At the termination of the study, the rats were sacrificed and organs were weighed. Microscopic examination of tissues were performed on high dose and control animals.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 6 weeks old.
- Diet (e.g. ad libitum): See below "Details on oral exposure".
- Water (e.g. ad libitum): Acidified water (pH 3.5), ad libitum
Route of administration:
oral: feed
Vehicle:
not specified
Details on oral exposure:
The diets were semi synthetic in which sodium caseinate replaced casein. The carbohydrates of the diet were substituted by magnesium stearate as follows: 0 (control), 5, 10, 20 % in diet (magnesium stearate); 67.3, 62.3, 57.3 and 47.8 % in diet (carbohydrate)
The diets fed were considered isocaloric, as stearate has a calorific value of about 9, and a pilot study demonstrated that 35-40% of the stearate is absorbed at a 10% level in the diet.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
3 months
Frequency of treatment:
Daily
Dose / conc.:
5 other: %
Remarks:
nominal in diet
Dose / conc.:
10 other: %
Remarks:
nominal in diet
Dose / conc.:
20 other: %
Remarks:
nominal in diet
No. of animals per sex per dose:
20 animals per sex and per dose.
Control animals:
yes, concurrent no treatment
Observations and examinations performed and frequency:
BODY WEIGHT: Yes
- Time schedule for examinations: Once weekly.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food utilization was calculated for each sex of all groups of rats.

HAEMATOLOGY: Yes
- Blood samples were taken from 8 males and 8 females from each group prior to dosing and at 8 and 12 weeks.
- Parameters checked: Hemoglobin, packed cell volume (PCV), red cell count, total white cell count reticulocyte count, differential white cell count.

CLINICAL CHEMISTRY: Yes
- Blood samples were taken from 8 males and 8 females from each group prior to dosing and at 8 and 12 weeks.
- Parameters checked: Glucose, urea, aspartate amino transferase, alkaline phosphatase
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
At the termination of the study, the rats were sacrificed and the following organs were weighed: thymus, liver, kidneys, adrenals, testes/ovaries, heart, lungs, brain and pituitary.

HISTOPATHOLOGY: Yes
Samples of the organs listed above and the following tissues were taken for light microscopy: urinary bladder, stomach, duodenum, pancreas, jejunum, cecum, colon, thyroid, parathyroid, triceps, brachial muscle, ischiadic nerve, axillar lymph node, uterus, sternum, eye, Harderian gland, skin and submandibular gland. Microscopic examination was undertaken on the high dose and control animals only .
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Males at 20% group were quiet with slow and unsteady movements. Four males at 20% group died within the first 2 months and all had stone formation in the lower urinary pathways and the deaths were considered to be related to this finding. One other male in this group was incontinent. In the remaining males, the symptoms receded during the following 4 weeks.
There were no clinical effects in females in any group.
Mortality:
mortality observed, treatment-related
Description (incidence):
Four males at 20% group died within the first 2 months and the deaths were considered to be related to this finding.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The weight gains of the 20% males were significantly less than the corresponding controls during the first 8 weeks of the study.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No effects were reported.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
A reduction in PCV (P<0.01) was found in the 20% males compared to controls. No other hematological differences were reported.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No effects were reported.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The relative liver and kidney weights were lower (p<0.001) (see below Table 1).
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
In addition to the findings reported in the males that died in the 20% group, changes were also found in the renal pelvis and in the lower urinary pathways (due to stone formation) at autopsy in 4 males and one female in the 20 % group.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Nephrocalcinosis was seen in all females and in 12/20 males in the control group. In 18 of the females nephrocalcinosis was regarded as severe. Slight to moderate nephrocalcinosis was observed in 19/20 of the females in the 20% group and 7/20 of the males were affected only slightly.
Deposition of iron was found in various amounts in kidney and in liver, the amount was increased in the liver of both sexes in the 20% group.
Liver glycogen showed a marked decrease in males in the 20% group and no difference was found in the females.

HISTORICAL CONTROL DATA:
According to the authors the occurrence of nephrocalcinosis is a common finding in animals fed semi-synthetic diets. The increased magnesium content of the diet could explain the reduction of nephrocalcinosis in the 20% animals. A high magnesium content of the diet has also been previously associated with a greater incidence of stone formation in the lower part of the urinary tract.
Histopathological findings: neoplastic:
not specified
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
5 other: % in diet (~2500 mg/kg bw)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
Key result
Critical effects observed:
no

Table 1: The relative liver and kidney weights:

Dietary (%)

Sex

Liver (g/100 g bw)

Kidney (g/100 g bw)

0 (control)

Male

3.25±0.21

633±48.6

5

Male

3.13±0.21*

614±51.5

10

Male

2.99±0.23***

599±40.6*

20

Male

2.82±0.18***

640±80.7

0 (control)

Female

3.30±0.24

768±103

5

Female

3.33±0.18

661±86.5***

10

Female

3.31±0.31

667±54.0***

20

Female

3.16±0.23*

646±55.8***

 

* P< 0.05

*** P<0.001

 

Conclusions:
The NOAEL-90 days for repeated dose toxicity by feed of Magnesium stearate was determined to be 5% in diet (~2500 mg/kg bw)(basis for effect: liver weight).
Executive summary:

A repeated dose toxicity study was performed on Magnesium stearate. Groups of 20 male and 20 female six week old rats were fed diets containing 5, 10 or 20 % magnesium stearate. The diets were semi synthetic in which sodium caseinate replaced casein. The animals were weighed once weekly and food utilization and weight gain was calculated for each sex of all groups of rats. Blood samples were taken from 8 males and 8 females from each group prior to dosing and at 8 and 12 weeks. At the termination of the study, the rats were sacrificed and organs were weighed. Microscopic examinations of tissues were performed on high dose and control animals. The weight gains of the 20% males were significantly less than he corresponding controls during the first 8 weeks of the study. Moreover, four males in this group died within the first 2 months and all had stone formation in the lower urinary pathways. Nephrocalcinosis was seen in both control and 20% dose groups in males and females. Deposition of iron was found in various amounts in kidney and in liver in both sexes in the 20% dose groups and liver glycogen showed a marked decrease but only in males. According to the authors the occurrence of nephrocalcinosis is a common finding in animals fed semi-synthetic diets. The increased magnesium content of the diet could explain the reduction of nephrocalcinosis in the 20% animals. A high magnesium content of the diet has also been previously associated with a greater incidence of stone formation in the lower part of the urinary tract. The authors concluded that when liver weight was used as a measure of adverse effect, the no effect level was estimated to be 5% magnesium stearate in the diet, corresponding to 2500 mg/kg body weight.

Endpoint:
repeated dose toxicity: oral, other
Remarks:
(combined repeated dose and reproduction/developmental screening test)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan, Hino, Tokyo
- Age at study initiation: 8 weeks
- Weight at study initiation: 312.1 - 363.7 g males; 205.3 - 230.8 g females
- Housing: metal wire floor cages
- Diet (ad libitum): CE-2, CLEA Japan
- Water (ad libitum): tap water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 ± 1 ºC
- Humidity (%): 50 - 65%
- Air changes (per hr): 15 per hour
- Photoperiod (hrs dark / hrs light): 12 hours dart/ 12 hours light
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Solutions prepared more frequently than once a week. Aliquots kept refrigerated in airtight conditions.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Insoluble in water
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no. (if required): V6H2050
Duration of treatment / exposure:
Exposure peirod:
Males, 42 days.
Females, from 14 days prior to mating to day 3 of lactation.
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
vehicle
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
13 animals per sex and per dose.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on preliminary result in a 14 day-repeated dose toxicity study, where no signs of toxicity were found.
Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once a day

BODY WEIGHT: Yes
- Time schedule for examinations: Once a week (body weight and body weight gain).

FOOD CONSUMPTION:
- Time schedule: Once a week.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 42 days, prior to sacrifice
- Anaesthetic used for blood collection: Yes (identity): pentobarbital sodium
- Animals fasted: 18 - 24 hours before sacrifice
- How many animals: all surviving animals
- Parameters checked: Red blood cell count (RBC), white blood cell count (WBC), platlet count, hemoglobin (Hb), hematocrit (Ht), mean corpuscular volume (MCV),mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), differentiation of leukocytes (band neutrophil, segmented neutrophil, eosinophil, basophil, monocyte, lymphocyte).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 42 days, prior to sacrifice
- Animals fasted: 18 - 24 hours before sacrifice
- How many animals: all surviving animals
- Parameters checked: total protein, albumin, A/G, blood urea nitrogen (BUN), creatinine, glucose, total cholesterol, total bilirubin, triglyceride, sodium (Na), potassium (K), chloride (Cl), calcium (Ca), inorganic phosphorus (IP), alkaline phosphatase (ALP), GPT, GOT, γ-GTP.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Animals examined: all animals
Organs examined: Males: tyroid gland, thymus, lung, liver, spleen. Females: thymus, spleen, adreanl gland, kidney.
Organs weights: Males: heart, liver, kidneys, thymus, testes, epididymides. Females: heart, liver, kideny and thymus.

HISTOPATHOLOGY: Yes
Animals examined: all animals in control and 1000 mg/kg.
Organs examined: Males: brain, heart, liver, spleen, thymus, kidney, urinary bladder, testis, epididymis. Females: brain, heart, liver, thymus, kidney, urinary bladder, adrenal gland, ovary.
Statistics:
Dunnett’s or Scheffe’s test for continuous numerical data, Chi square test for copulated index and fertility index, and Mann-Whitney U test or Fisher’s test for histopathological examination data.
Clinical signs:
no effects observed
Description (incidence and severity):
No abnormalities in general condition were observed in any of the treated groups.
Mortality:
no mortality observed
Description (incidence):
No deaths were observed in any of the treated groups.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no changes related to the dosing of compound in body weight gain.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no changes related to the dosing of compound in food consumption.
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):
At autopsy following treatment for 42 days in males and on postpartum day 4 in females, no adverse effects were found in the hematological examinations.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
At autopsy following treatment for 42 days in males and on postpartum day 4 in females, no adverse effects were found in the biochemical examinations.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
At autopsy following treatment for 42 days in males and on postpartum day 4 in females, no adverse effects were found for internal organs.
Gross pathological findings:
no effects observed
Description (incidence and severity):
At autopsy following treatment for 42 days in males and on postpartum day 4 in females, no adverse effects were found for internal organs.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
At autopsy following treatment for 42 days in males and on postpartum day 4 in females, no adverse effects were found in the histopathological examinations.
Histopathological findings: neoplastic:
not specified
Other effects:
no effects observed
Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects observed at the highest dose.
Key result
Critical effects observed:
no
Conclusions:
The NOEL of docosanoic acid for repeated oral dose toxicity in rats was 1000 mg/kg bw/day for males and females.
Executive summary:

A combined repeated dose and reproduction/developmental screening was performed on docosanoic acid according to OECD Guideline 422. 13 rats per sex and per dose were exposed to 0 (vehicle), 100, 300 and 1000 mg/kg bw/day, males for 42 days and females from 14 days prior to mating to day 3 of lactation. No deaths or abnormalities in general condition were observed in any of the treated groups. Also, there were no changes related to the dosing of compound in body weight gain and food consumption. At autopsy, no adverse effects were found for internal organs and findings of histopathological, hematological and biochemical examinations. Based on these results, the NOEL for repeated dose toxicity was determined to be 1000 mg/kg bw/day for both males and females.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
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)
GLP compliance:
not specified
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 Laboratories
- Age at study initiation: 3 weeks old
- Weight at study initiation: 71-98 g males, 68-88 g females
- Fasting period before study: No.
- Housing: All rats were housed individually in suspended wire-bottomed cages.
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 5 days. Diet and water: Ad libitum access to water and corn oil control diet. Housing: 2 per cage.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 ºC
- Humidity (%): 50±20%
- Photoperiod (hrs dark / hrs light): 12 hrs dart/ 12 hrs light
Route of administration:
oral: feed
Vehicle:
not specified
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Each group was provided access ad libitum to one of five diets that contained 0 (control, corn oil), 0 (control, MCT corn oil), 5.25%, 10.25% and 15.00% (w/w of the diet) caprenin. Corn oil served as a typical dietary fat control and was added to all diets, at a level no lower than 3%, to provide essential fatty acids. MCT oil served as an additional control owing to its high medium chain fatty acid (MCFA) content.

DIET PREPARATION
- Rate of preparation of diet (frequency): Fresh diets were prepared weekly and place in glass jars twice weekly. All diets were prepared to provide 4000 kcal/kg based on the assumption that the caloric densities (or physiological fuel values) of corn oil, MCT oil and caprenin were 9, 7 and 5 kcal/g,
respectively.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The fatty acid compositions of caprenin, Dukes corn oil and medium chain triglyceride (MCT) oil were determined. For all three triglycerides, levels of free fatty acids were measured by titration and expressed as lauric acid equivalents. Peroxide values were measured by a modified iodometric titration procedure (see details in Table 1.
Duration of treatment / exposure:
91 days
Frequency of treatment:
Continously (by feed, ad libitum).
Dose / conc.:
5.23 other: % (w/w)
Remarks:
nominal in diet
Dose / conc.:
10.23 other: % w/w
Remarks:
nominal in diet
Dose / conc.:
15 other: % w/w
Remarks:
nominal in diet
No. of animals per sex per dose:
25 animals per sex and per dose.
Control animals:
yes
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice per day
- Cage side observations checked: mortality, moribundity and obvious indications of toxic effects.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed physical examinations were performed at each weekly interval.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly
- Body weight gains were calculated.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal was determined (g food consumption).
- Compound intake was calculated as time-weighted averages from the consumption and body weight gain data (g caprenin/kg bw/day)
- Other: Spillage was measured and used to correct food consumption values

FOOD EFFICIENCY:
- Body weight gain in g/food consumption in g per unit time X 100 was calculated as time-weighted averages from the consumption and body weight gain data (% fedd consumption efficiency).

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before initiation of the study and at week 13.
- Dose groups that were examined:

HAEMATOLOGY: Yes (blood samples were taken through the orbital sinus)
- Time schedule for collection of blood: At the end of the study
- Anaesthetic used for blood collection: Yes, with a C02/02 mix
- Animals fasted: Yes, fasted overnight with water available
- How many animals: 20 rats/sex/group
- Parameters checked: leucocyte count, corrected leucocyte count, leucocyte differential, erythrocyte count, haemoglobin, haematocrit, platelet count, cell morphology, absolute reticulocyte count, reticuiocyte count, mean cell volume, mean cell haemoglobin, and mean cell haemoglobin concentration.
- Other: Immediately after orbital sinus bleeding under CO2/O 2 anaesthesia rats were anaesthetized with sodium pentobarbital and samples for coagulation (prothrombin time, and activated partial thromboplastin time) were collected from the abdominal aorta.

CLINICAL CHEMISTRY: Yes (blood samples were taken through the orbital sinus)
- Time schedule for collection of blood: At the end of the study
- Anaesthetic used for blood collection: Yes, with a C02/02 mix
- Animals fasted: Yes, fasted overnight with water available
- How many animals: 20 rats/sex/group
- Parameters checked: sodium, potassium, chloride, calcium, inorganic phosphorus, glucose, total bilirubin, blood urea nitrogen (BUN), total protein, albumin, globulin, creatinine, total cholesterol, lipoproteins, triglycerides, γ-glutamyltransferase (GGT), alkaline phosphatase, aspartate aminotransferase (AST) and alanine aminotransferase (ALT).
Sacrifice and pathology:
20 rats/sex/group were sacrified at test termination:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
Tissues from the corn oil, MCT oil and high-dose caprenin groups were preserved in 10% neutral buffered formalin, embedded in paraffin, sectioned, stained with haematoxylin and eosin, and examined microscopically. Additional sections from the liver and heart were stained with Oil Red O and graded under microscopic evaluation for fat content. Extra sections of kidney were stained with alizarin red and graded for incidence and severity of nephrocalcinosis.
Weights of brain, spleen, liver, kidneys, heart, gonads, adrenals, caecum (after rinsing with saline) and colon (after rinsing with saline) were determined and expressed as absolute and relative (organ-to-body weight and organ-to-brain weight) values.
Other examinations:
The remaining five rats/sex/group were anaesthetized with sodium pentobarbital and exsanguinated. The livers and hearts from the animals were trimmed of excess fat, homogenized, and analysed for total fat and behenic acid content.
Statistics:
All results were analysed statistically by analysis of variance (ANOVA). Comparisons were made using the protected least significant difference when Bartlett's test of homogeneity of variance was not significant. If Bartlett's test was significant, comparisons were made by the t-test technique that allows for unequal variances. In this latter case, Wilcoxon's rank sum test also was applied. In addition, the Fisher-Irwin exact test was used to compare the incidence of autopsy observations in control and caprenin-fed groups. All statistical tests were conducted at a 5% two-sided risk level.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There was a low frequency of clinical findings in all dose groups during this study. The signs were common to rats of this strain and age and considered unrelated to the consumption of caprenin.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One corn oil control male rat was found dead at week 6 of the study (cause of death undetermined) and one high-dose female rat was found dead at week 8 of the study (cause of death attributed to urinary calculus with associated renal inflammation). Nevertheless, no treatment-related deaths were observed.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no statistically significant differences in body weight gain at study termination.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Male rats on the high-dose caprenin diet consumed significantly more food, which resulted in significantly lower feed efficiency values, relative to the corn oil or MCT oil controls. These results demonstrate that the caprenin diets were not isocaloric relative to the corn oil or MCT control diets.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Male rats on the high-dose caprenin diet consumed significantly more food, which resulted in significantly lower feed efficiency values, relative to the corn oil or MCT oil controls. These results demonstrate that the caprenin diets were not isocaloric relative to the corn oil or MCT control diets.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Ophthalmoscopic examinations did not reveal any treatment-related ocular abnormalities.
Haematological findings:
no effects observed
Description (incidence and severity):
According to the authors, the haematological parameter differences were considered to be minor and unrelated to treatment since they fell within the normal historical range for rats of this sex and strain.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were a number of statistically significant changes in clinical chemistry parameters for rats maintained on caprenin diets relative to both the corn and MCT oil control groups. Nevertheless and according to the authors, most of these changes were considered to be unrelated to caprenin intake, since they were generally mild, showed no dose-response relationship, and they occurred inconsistently between sexes. There were no other findings, including histopathological correlates, to suggest that the changes were toxicological or related to treatment. Furthermore, the changes were within, or closely approximated to, the normal historical range for rats of this sex and strain.
Changes in alkaline phosphatase (males), ALT (males and females) and total protein, albumin and globulin (females) appear to be dose related, thus attributable to caprenin intake. Nevertheless, all of these latter individual differences also were mild and within the historical range of control rats of this sex and strain. Furthermore, they were not accompanied by any changes in histopathology. According to the authors, these changes appear to represent a normal physiological response to the intestine to diets that differed in both type and amount of absorbable fat and/or protein.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
The only significant differences noted in absolute or relative organ weights were confined to the liver, colon, kidneys, heart and spleen. These three latter changes were mild, restricted to organ-to-body weight ratios only, not dose related and thus considered unrelated to treatment.
There was a general tendency for rats administered caprenin to have lower liver weights, but this was not accompanied by consistent changes in other measures of liver weight, nor were there any microscopical indications that the livers were affected by caprenin.
According to the authors, the higher colon weights, colon-to-body-weight ratios, and colon-to-brain weight ratios observed in rats fed caprenin, relative to those in the corn oil controls, were determined to be not a toxicological response, but appear to reflect a hypertrophic adaptation of this organ to a diet that provided greater faecal bulk.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There was a statistically significant increase in the number of kidneys that were described as 'granular/pitted/rough' (15/19) among the high-dose females compared with the controls fed corn oil (9/20). According to the authors, these changes may reflect an exacerbation of the spontaneously occurring nephrocalcinosis that was observed in all control and treated rats.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no histopathological alterations associated with the administration of caprenin.
Histopathological findings: neoplastic:
not specified
Other effects:
no effects observed
Description (incidence and severity):
Tissue fat content and levels of behenic acid at selected depot sites:
Liver fat content was significantly lower in high-dose females than in the corn oil controls; however, the fat content of hearts from either male or female rats was unaffected by caprenin. Behenic acid was poorly retained by male and female rats. The concentration of this VLCFA never exceeded 1.75% in any depot site, for either sex, at any level of caprenin intake.
Key result
Dose descriptor:
NOAEL
Effect level:
> 15 other: % (w/w) caprenin in diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no treatment-related adverse effects were observed
Remarks on result:
other: (NOAEL > 13200 mg/kg bw/day males, 14600 mg/kg bw/day females)
Key result
Critical effects observed:
no

Table 1. Fatty acid composition of caprenin and control trigycerides:

Fatty acid

Normalized fatty acid concentration (% w/w)

Caprenin

Corn oil

MCT oil

C6:0

-

-

4.5

C8:0

23.2

-

70.6

C10:0

26.6

-

25.9

C12:0

0.3

-

-

C16:0

0.2

11.3

-

C18:0

0.9

2.1

-

C18:1

-

25.4

-

C18:2

-

60.7

-

C18:3

-

0.5

-

C20:0

2.7

-

-

C22:0

45.0

-

-

C24:0

1.1

-

-
Conclusions:
The NOAEL for 90 days feeding repeated dose toxicity of caprenin in rats was determined to be >15% w/w caprenin in diet (13200 mg/kg bw/day for males and 14600 mg/kg bw/day for females).
Executive summary:

A 90 days repeated dose toxicity test was performed on Caprenin, a randomized triglyceride primarily comprising caprylic (C8:0, 23.2%), capric (C 10:0, 26.6%), and behenic (C22:0, 45.0%) acids. Test item was administered in a semi-purified diet to weanling Sprague-Dawley rats (25/sex/group) at dose levels of 5.23, 10.23 or 15.00% (w/w) for 91 days. Corn oil was added at 8.96, 5.91 and 3.00%, respectively, to provide essential fatty acids and digestible fat calories. Corn oil alone (12.14%) and a blend of medium-chain triglyceride (MCT) oil plus corn oil (11.21 and 3.13%, respectively) served as controls. Mortality, clinical signs, body weight, feed consumption, feed efficiency, organ weights, haematological and clinical chemistry parameters were evaluated in all groups. Histopathology of a full complement of tissues was evaluated in the corn oil and MCT oil control groups as well as the high-dose caprenin group. Additional rats (n = 5/sex/group) were included in the study to determine whether there was marked storage of C22:0 in heart, liver or perirenal fat at the end of the 91-day feeding period. No significant differences in body weight gain were measured with the balanced caloric diets, although feed conversion efficiency was reduced in the high-dose caprenin group. No adverse effects from the ingestion of caprenin were detected, nor were significant amounts of C22: 0 present in the fat extracted from the selected fat depot sites. According to these results, the 90 days-NOAEL for caprenin was determined to be > 15% (w/w) caprenin in the diet (or more than 83% of total dietary fat), which is equal to a mean exposure level of >13200 mg/kg/day for male rats and more than 14600 mg/kg/day for female rats.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: No specific test guideline was reported; a scientifically defensible approach was used but limited number of animals and examinations were reported.
Principles of method if other than guideline:
-Principle of test: Feeding study, where lauric acid was given at a concentration of 10% in the diet to 5 male rats for 18 weeks
GLP compliance:
no
Limit test:
yes
Species:
rat
Strain:
Osborne-Mendel
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 40-50 g
- Diet (e.g. ad libitum): basal diet of ground commercial biscuit (ad libitum)
- Water (e.g. ad libitum): water (ad libitum)
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): every two weeks
- Mixing appropriate amounts with (Type of food): by blending the basal diet of ground commercial biscuit with lauric acid
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
18 weeks
Frequency of treatment:
Feeding ad libitum
Dose / conc.:
10 other: % (~5000 mg/kg bw/day)
Remarks:
nominal in diet
No. of animals per sex per dose:
5 males per dose.
Control animals:
yes, concurrent no treatment
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the study
- How many animals: All animals (5 males per dose)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: No.
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs were observed.
Mortality:
no mortality observed
Description (incidence):
No mortality was observed.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related effects were observed.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not specified
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
Description (incidence and severity):
There was no significant differences between the controls and test animals.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There was no significant differences between the controls and test animals.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
10 other: % in diet (~5000 mg/kg bw/day)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: (no treatment-related effects observed)
Critical effects observed:
not specified
Conclusions:
The NOAEL for repeated dose toxicity by feed of lauric acid was determined to be >10% in diet (~5000 mg/kg bw/day).
Executive summary:

Lauric acid was fed to five male rats at the 10% level of their diet for 18 weeks. A control group of 5 males was fed concurrently. There were no observable clinical effects, no adverse effects on weight gain, nor was there any mortality. Gross organ pathology and comparison of individual organ weights showed no significant differences between the controls and test animals. The NOAEL (18 weeks) for repeated dose toxicity by feeding route of lauric acid in male rats was determined to be >10% in diet (~5000 mg/kg bw/day).

Endpoint:
repeated dose toxicity: oral, other
Remarks:
(subacute + subchronic)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: No specific test guideline was reported. The scientific approach was defensible but the examinations were limited to clinical signs and blood analysis.
Principles of method if other than guideline:
- Principle of test: The effect of a recent hyperammonemic model, consisting of a high ammonia diet for 3, 7, 15, 45, and 90 days, to female Wistar rats has been studied.
- Short description of test conditions: The high ammonia diet was prepared by mixing a standard diet with ammonium acetate (20% wt/wt); in addition, 5 mM of ammonium acetate was added to the water supply. Fifty female rats were divided into ten groups (five control and five experimental groups), each one including five animals.
GLP compliance:
not specified
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 200-250 g
- Diet and Water (e.g. ad libitum): Control rats were fed ad libitum with a standard diet (UAR A04, Panlab, Spain) (17% protein); the experimental animals were fed ad libitum with a high ammonia diet and received ammonium acetate in the water supply (5mM).
Route of administration:
other: feed + drinking water
Vehicle:
not specified
Details on oral exposure:
DIET PREPARATION
The high ammonia diet was prepared by mixing standard diet with ammonium acetate (20% m/wt) as described by Azorin et al.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The ammonia concentrations in the standard and high ammonia diets were determined by means of the Lucke and Giedel's method and were 0.024% and 3.377% respectively (g NH4/100 g food).
Duration of treatment / exposure:
3, 7, 15, 45, or 90 days.
Frequency of treatment:
Daily
Dose / conc.:
20 other: % (ammonium acetate in the diet)
Remarks:
nominal
Dose / conc.:
5 other: mM (ammonium acetate in the drinking water)
Remarks:
nominal
No. of animals per sex per dose:
Fifty female rats were divided into ten groups (five control and five experimental groups), each one including five animals.
Control animals:
yes, concurrent vehicle
Positive control:
No data.
Observations and examinations performed and frequency:
Blood samples for ammonia determination were collected at the moment of decapitation; plasma was separated immediately and stored at - 70 ºC.
Blood ammonia level was determined with an Ammonia Kit.
Sacrifice and pathology:
Each group was sacrificed at different times: 3, 7, 15, 45, or 90 days.
Rats were killed by decapitation and the spinal cord was immediately removed as described by Meikle and Martin and frozen at - 80 ºC in sodium dodecyl sutfate (SDS) extraction buffer (0.15% SDS, 0.05 M phosphate buffer, 0.02% EDTA and 0.9% NaCl; pH:8).
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Significant differences in weight were observed after the third day. At dose of 3150.4 mg/kg bw/day: 10 % reduction in final body weight at 3 or 7 days, 15 % reduction in final body weight at 90 days.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No observable differences were noted in the water consumption between control and high ammonia diet rats.
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Description (incidence and severity):
Blood ammonia analysis demonstrated a significant difference between high ammonia diet and control animals at 7th day. No significant differences between experimental and control animals were observed at the other times studied.
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No observable differences were noted in the behaviour between control and high ammonia diet rats.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
no effects observed
Key result
Dose descriptor:
LOAEL
Effect level:
3 150.4 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
Critical effects observed:
not specified

The NOAEL was 22 mg/kg bw/day and the LOAEL was 3150.4 mg/kg bw/day.

Conclusions:
The NOAEL was 22 mg/kg bw/day and the LOAEL was 3150.4 mg/kg bw/day.
Executive summary:

The effect of a recent hyperammonemic model, consisting of a high ammonia diet for 3, 7, 15, 45, and 90 days, to female Wistar rats has been studied. The high ammonia diet was prepared by mixing a standard diet with ammonium acetate (20% wt/wt); in addition, 5 mM of ammonium acetate was added to the water supply. Fifty female rats were divided into ten groups (five control and five experimental groups), each one including five animals. Blood ammonia analysis demonstrated a significant difference between high ammonia diet and control animals at 7th day. No significant differences between experimental and control animals were observed at the other times studied. No observable differences were noted in either the behaviour or the water consumption between control and high ammonia diet rats; however, significant differences in weight were observed after the third day. The LOAEL were determined to be 3150.4 mg/kg bw/day.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: No specific test guideline was reported. The scientific approach was defensible but the examinations were limited to clinical signs and blood analysis.
Principles of method if other than guideline:
-Principle of test: Female Wistar rats were fed a high ammonia diet prepared by mixing a standard diet with ammonium acetate (20% w/w); in addition, 5 mM of ammonium acetate was added to their water supply. Control rats were fed with a standard diet. The animals were sacrificed at 3, 7 and 15 days of ammonia ingestion. Thirty female rats were divided into six groups (three control and three experimental groups), each one including five animals.
GLP compliance:
not specified
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 220-250 g
- Diet and Water (e.g. ad libitum): Control rats were fed ad libitum with a standard diet (UAR A04, Panlab, Spain) (17% protein); the experimental animals were fed ad libitum with a high ammonia diet and received ammonium acetate in the water supply (5mM).
Route of administration:
other: feed + drinking water
Vehicle:
not specified
Details on oral exposure:
DIET PREPARATION
The high ammonia diet was prepared by mixing standard diet with ammonium acetate (20% m/wt) as described by Azorin et al.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
3, 7, or 15 days
Frequency of treatment:
Daily
Dose / conc.:
20 other: % (ammonium acetate in the diet)
Remarks:
nominal
Dose / conc.:
5 other: mN (ammonium acetate in the drinking water)
Remarks:
nominal
No. of animals per sex per dose:
Thirty female rats were divided into six groups (three control and three experimental groups), each one including five animals.
Control animals:
yes, concurrent no treatment
Positive control:
No data.
Observations and examinations performed and frequency:
Blood samples for ammonia determinations were collected at the moment of decapitation; plasma was separated immediately and stored at - 70 ºC.
Blood ammonia level was determined with an Ammonia Kit.
Sacrifice and pathology:
The animals were killed by decapitation at 3, 7 and 15 days after starting treatment.
The brains were removed and the frontoparietal cortex and hippocampus were rapidly dissected.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Significant differences in weight were observed by the third day on the ammonia diet and were maintained for up to 15 days.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Ammonia levels in blood increased from 0.13 mM in control rats to 0.4 mM in rats fed the high ammonia diet at 7 days of starting this diet.
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Rats had a decreased number of available somatostatin receptors in the frontoparietal cortex and hippocampus and lower weigh increase.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Key result
Dose descriptor:
NOAEL
Effect level:
3 102.2 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
gross pathology
haematology
Critical effects observed:
not specified

The NOAEL was 3102.2 mg/kg bw/day.

Conclusions:
The NOAEL for Ammonium acetate was 3102.2 mg/kg bw/day
Executive summary:

Female Wistar rats were fed a high ammonia diet prepared by mixing a standard diet with ammonium acetate (20% w/w); in addition, 5 mM of ammonium acetate was added to their water supply. Control rats were fed with a standard diet. The animals were sacrificed at 3, 7 and 15 days of ammonia ingestion. Thirty female rats were divided into six groups (three control and three experimental groups), each one including five animals. Significant differences in weight were observed by the third day on the ammonia diet and were maintained for up to 15 days. Ammonia levels in blood had increased ca. 3-fold at 7 days of ammonia ingestion. Rats with high NH4+ intake from administration of 20% ammonium acetate in the diet and 5 mM ammonium acetate in the water for up to 15 days had a decreased number of available somatostatin receptors in the frontoparietal cortex and hippocampus. The NOAEL was 3102.2 mg/kg bw/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
2 500 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Several studies were available on rats. The klimisch scores were between K1 and K3. A weight of evidence approach was applied and the overall quality of the database was determined as appropriate for assessment.

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

REPEATED DOSE TOXICITY (ORAL):

Weight of Evidence Approach (see rationale attached in IUCLID5 Section 13):

A combined repeated dose and reproduction/developmental screening was performed by the Ministry of Health, Labour and Welfare (Japan, 1998) on docosanoic acid according to OECD Guideline 422. Rats were exposed up to 1000 mg/kg bw/day, males for 42 days and females from 14 days prior to mating to day 3 of lactation. No deaths or abnormalities were observed in any of the treated groups. At autopsy, no adverse effects were found for internal organs and findings of histopathological, hematological and biochemical examinations. The NOEL for repeated dose toxicity was determined to be 1000 mg/kg bw/day for both males and females.

In accordance with Webb DR et al. (1993), a 90 days repeated dose toxicity test was performed on Caprenin, a randomized triglyceride primarily comprising caprylic (C8:0, 23.2%), capric (C 10:0, 26.6%), and behenic (C22:0, 45.0%) acids. Test item was administered in a semi-purified diet to rats at dose levels up to 15.00% (w/w) for 91 days. Additional rats (n = 5/sex/group) were included in the study to determine whether there was marked storage of C22:0 in heart, liver or perirenal fat at the end of the 91-day feeding period. No adverse effects from the ingestion of caprenin were detected, nor were significant amounts of C22: 0 present in the fat extracted from the selected fat depot sites. The 90 days-NOAEL was determined to be > 15% (w/w) caprenin in diet, which is equal to a mean exposure level of >13200 mg/kg/day for male rats and > 14600 mg/kg/day for female rats.

A repeated dose toxicity study was performed on Magnesium stearate by Sondergaard D et al. (1980). Rats were fed diets containing up to 20 % magnesium stearate for 3 months. The diets were semi synthetic in which sodium caseinate replaced casein. four males in this group died within the first 2 months and all had stone formation in the lower urinary pathway. Nephrocalcinosis was seen in both control and 20% dose groups and deposition of iron was found in various amounts in kidney and in liver in the 20% dose groups. According to the authors the occurrence of nephrocalcinosis is a common finding in animals fed semi-synthetic diets. The increased magnesium content of the diet could explain the reduction of nephrocalcinosis in the 20% animals. A high magnesium content of the diet has also been previously associated with a greater incidence of stone formation in the lower part of the urinary tract. The authors concluded that when liver weight was used as a measure of adverse effect, the no effect level was estimated to be 5% magnesium stearate in the diet, corresponding to 2500 mg/kg body weight.

In a study performed by Fitzhuhg OG (1960), lauric acid was fed to five male rats at the 10% level of their diet for 18 weeks. There were no observable clinical effects, no adverse effects on weight gain, nor was there any mortality. Gross organ pathology and comparison of individual organ weights showed no significant differences between the controls and test animals. The NOAEL (18 weeks) was determined to be >10% in diet (~5000 mg/kg bw/day) in male rats.

The effect of a recent hyperammonemic model, consisting of a high ammonia diet for up to 90 days, to female rats has been studied by Bodega G et al (1993). The high ammonia diet was prepared by mixing a standard diet with ammonium acetate (20% wt/wt); in addition, 5 mM of ammonium acetate was added to the water supply. Blood ammonia analysis demonstrated a significant difference between high ammonia diet and control animals at 7th day. No significant differences between experimental and control animals were observed at the other times studied. No observable differences were noted in either the behaviour or the water consumption between control and high ammonia diet rats; however, significant differences in weight were observed after the third day. The LOAEL were determined to be 3150.4 mg/kg bw/day.

In a study by Boyano-Adánez MC et al. (1996), female rats were fed a high ammonia diet prepared by mixing a standard diet with ammonium acetate (20% w/w) up to 15 days. In addition, 5 mM of ammonium acetate was added to their water supply15 days of ammonia ingestion. Significant differences in weight were observed by the third day on the ammonia diet and were maintained for up to 15 days. Ammonia levels in blood had increased ca. 3-fold at 7 days of ammonia ingestion. Rats with high NH4+ intake from administration of 20% ammonium acetate in the diet and 5 mM ammonium acetate in the water for up to 15 days had a decreased number of available somatostatin receptors in the frontoparietal cortex and hippocampus. The NOAEL was determined to be 3102.2 mg/kg bw/day.

Taking into account the available experimental results, the weight of evidence approach was applied and the 90 day-NOAEL for repeated dose toxicity by oral exposition for the substance Fatty acids, C16 -18 (even numbered), ammonium salts was determined to be 2500 mg/kg bw/day in rats (based on subchronic study, worst case assumption).

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

The study with the longest duration (subchronic) and lowest NOAEL  (worst case assumption).

Justification for classification or non-classification

Based on the available information, the test substance Fatty acids, C16 -18 (even numbered), ammonium salts is not classified for repeated exposure according to CLP Regulation (EC) No 1272/2008.