Nonanoic acid

Endpoint summary

Description of key information

Local irritation was seen in the forestomach in this study and in a dermal study using nonanoic acid. Valid dermal and inhalation studies are not available.
Systemic toxicity is low, as evidenced by NOAEL values > 3300 mg nonanoic acid (read across from octanoic and decanoic acid) that were obtained in oral studies using triglycerides instead of the free acid.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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

Remarks:

No macroscopic pathology. No histopathology. No information regarding GLP.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)

Deviations:

yes

Remarks:

No macroscopic pathology, no histopathology

GLP compliance:

not specified

Limit test:

no

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: no data
- Age at study initiation: 1 to 5 years
- Weight at study initiation: no data; estimates: males 6 to 10 kg; females 4-7 kg
- Fasting period before study: no data
- Housing: individually
- Diet: ad libitum, but limited to a feeding period of 3 hours/day
- Water: ad libitum
- Acclimation period: appox 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C
- Humidity (%): 45°C
- Air changes (per hr): no data; according to "Guide for Care and Use of Laboratory Animals", NRC, 1996
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

other: dry food with beef tallow. MCT replaced mass of dry feed tallow, all diets were isocalorically balanced

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): no data
- Mixing appropriate amounts with (Type of food): dry food with beef tallow, without structural lipids. All diets were isocaloricaly balanced
- Storage temperature of food: no data

VEHICLE
- Justification for use and choice of vehicle: dry feed was used for feeding study, MCT replaced mass of dry feed tallow

STABILITY:
Purity,composition and stability for duration of the study of the test article (i.e. diets containg MCT at various levels) was assessed and recorded.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

no data

Duration of treatment / exposure:

91 days

Frequency of treatment:

daily, 7 days/week

Dose / conc.:

0 other: %

Dose / conc.:

5 other: %

Dose / conc.:

10 other: %

Dose / conc.:

15 other: %

No. of animals per sex per dose:

4

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on literature data
- Rationale for selecting satellite groups: no sateliite groups included
- Fasting period before blood sampling for clinical biochemistry:No information
- Post-exposure recovery period in satellite groups: no
- Section schedule rationale (if not random): random

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, 7 days/week

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before release for the study, on Day 1, and during weeks 4, 8, and 12 of the study

BODY WEIGHT: Yes
- Time schedule for examinations: on day of receipt, 2 days prior to study, an dat weakly intervals from study Day 1 (i.e. Days 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, 85, and 91)

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: no data
- Dose groups that were examined: no data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: six days prior to the start of the study and on Days 29, 58, and 91 of the study.
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: all animals
- Parameters examined: blood smear morphology, erythrocytes count, calculated haematocrit, haemoglobin, mean corpuscular haemoglobin, mean corpuscular volume, platelet count, reticulocytes count, total leucocytes count, and the absolute white blood cell differential.
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: six days prior to the start of the study and on Days 29, 58, and 91 of the study.
- Animals fasted: No data
- How many animals: all
- Parameters examined: albumin, alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, calcium, chloride, cholesterol, creatinine kinase, creatinine, direct bilirubin, gamma-glutamyl transferase, globulin, glucose, Lactate dehydrogenase, phosphorus, potassium, sodium, total bilirubin, total protein, triglycerides and urea nitrogen.

URINALYSIS: Yes
- Time schedule for collection of urine: six days prior to the start of the study and on Days 29, 58, and 91 of the study.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data
- Parameters examined: appearance, colour, bilirubin, ketone levels, occult blood, pH, urine glucose and red blood cells, casts, epithelial cells, mucus, sperm, bacteria, yeast, amorphous sediment, and crystal formation

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

Animals were not sacrificed at termination. At the conclusion of the study, all animals were returned to stock. No tissue samples were taken, nor were any animals terminated.

Statistics:

Data variances were determined for homogeneity across test groups at the 0.05 level by Bartlett's test. Tests for differences between comparison groups were made using Dunnetts test. For non-homogenous data (from Bartlett's test), test for pair-wise differences between each of the comparison groups were made using Cochran and Cox's modified two-sample t-test. Statistical significance for each comparison was reported at the 0.05 level

Clinical signs:

no effects observed

Description (incidence and severity):

no mortality, no clinical observation considered to be related to treatment

Mortality:

no mortality observed

Description (incidence):

no mortality, no clinical observation considered to be related to treatment

Body weight and weight changes:

no effects observed

Description (incidence and severity):

no significant group mean body weight changes noted

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

no significant differences across test groups; reduced food intake an several consecutive days in MCT groups was noted and attributed to palatibility of MCT

Food efficiency:

not examined

Description (incidence and severity):

no effect, based on comparable body weights

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

significant increase of blood urea nitrogen in both sexes in the 10% and 15% fed groups. nonsignificant increase of cholesterol in both sexes of the 10% and 15% group.
At the 15% level, an increase in serum potassium and a decrease in globulin were observed, but did not follow a dose-dependent response.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

urinary volume decreased in the 10% and 15% groups, concomitant with increased specific gravity.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

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:

ca. 15 other: % in diet

Based on:

other: MCT

Remarks:

corresponds to octanoic acid/kg and day: males 2295 (1720-2870) mg, females 3385 (2470-4300) mg; corresponds to decanoic acid/kg and day: males 984 (738-1230) mg , females 1451 (1058-1845) mg

Sex:

male/female

Basis for effect level:

other: no adverse effect noted

Remarks on result:

other: Highest dose tested, which was above limit dose, did not induce effects

Critical effects observed:

not specified

Conclusions:

Based on the findings of this 91-day MCT feeding study, there are no indications of toxicological effects in dogs fed up to 15% MCT in the diet.

Executive summary:

In this 91 day study, 4 male and 4 female beagle dogs were treated with 0, 5, 10, or 15% MCT via the diet. The study followed a protocol similar to OECD TG 409. No statement regarding GLP is given.

A reduced food intake an several consecutive days in the groups with MCT exposure was noted and attributed to palatibility of MCT. Bodyweight developed normal.

There was a singificant increase of blood urea nitrogen in the 10% and 15% groups of both sexes and a non-significant increase of cholesterol in both sexes of the 10% and 15% group.

The changes were not considered to be adverse. The same was concluded for the observed reduction of urine volumes in the mid- and high-dose groups.

In this study animals were not sacrificed at termination. No tissue samples were taken, nor were any animals terminated.

No other effects were observed. The NOAEL was therefore 15% MCT in the diet in the beagle dog.

The estimated NOAEL-values in the beagle dog are as follows, based on calculations described in the attached document (Hillesheim, 2014):

octanoic acid:      

males           2295 (1700 – 2900) mg/kg bw and day                            

females        3385 (2500 – 4300) mg/kg bw and day

decanoic acid:       

males            984 (740 – 1200) mg/kg bw and day

females        1451 (1060 – 1850) mg/kg bw and day

Reference 2

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

Remarks:

Histopathology performed only for some organs. No information regarding GLP.

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 Inc., Raleigh, NC, USA
- Age at study initiation: approx 4weeks
- Weight at study initiation: males 85 (71-98) g; females 78 (68-88) g
- Fasting period before study: no
- Housing: two per cage
- Diet: ad libitum;


- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 50 +/- 20
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: receipt of animals 5 days prior to study To: Day 91 of study

Route of administration:

oral: feed

Vehicle:

other: All diets contained corn oil. For treatment, corn oil was substituted by caprenin

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

Control diets: corn oil (12.1%); MCT (11.21%)
Test article: Caprenin 5.23%, 10.23%, and 15.0%
Al diets prepared to provide about 4000 kcal/kg

DIET PREPARATION
- Rate of preparation of diet (frequency): weeks
- Storage temperature of food: refrigerated. Freshly diet was placed in glass jars twice weekly.
- Stability: less than 0.03% free fatty acid and 1.4 mEq peroxide was initially contained in caprenin, MCT and corn oil. No increase was not during 91 days of bulk refrigerated storage, nor in diets held for up to 7 days at room temperature.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

91 days

Frequency of treatment:

7 days/week

Dose / conc.:

5.23 other: %

Dose / conc.:

10.23 other: %

Dose / conc.:

15 other: %

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

other: MCT 11.21%

Details on study design:

- Dose selection rationale: 0, 5, 10, and 15% caprenin was used in a preceeding 28-day rat study (both sexes) where the NOAEL was 15% in the diet.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to study and at week 13
- Dose groups that were examined: no data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at end of study
- Anaesthetic used for blood collection: Yes (CO2/O2 mix)
- Animals fasted: Yes
- How many animals: 20 per sex/group
- Parameters examined: leucocyte count, corrected leucocyte count, leucocyte differential, erythrocyte count, haemoglobin, haematocrit, platelet count, cell morphology, absolute reticulocyte count, mean cell volume, mean cell haemoglobin, and mean cell haemoglobin concentration, prothrombin time.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination
- Animals fasted: No data
- How many animals: 20 per sex/group
- Parameters examined: Sodium potassium, chloride, calcium, inorganic phosphorus, glucose, total bilirubin, blood urea nitrogen (BUN), total protein, albumin, globulin, creatinine, total cholesterol, lipoproteins, triglycerides, gamma-glutamyltransferase (GGT), alkaline phosphatase (AP), aspartate aminotransferase (AST) and alanine aminotransferase (ALT)

URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: No


OTHER: behenic acid (C22:0) content in heart, liver, perirenal fat tissue. 5 rats per sex/group

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. Organ weights: liver, kidneys, brain, adrenals, spleen, heart, colon, caecum, testes, ovaries

HISTOPATHOLOGY: Yes; no exhaustive list provided. Histopathology was at least performed for: liver, heart, kidneys, bone marrow. possibly more organs.

Other examinations:

content of C22:0 acid in fat from heart, liver, and perirenal fat

Statistics:

ANOVA, Bartlett's test, t-.test, Wilcoxon rank sum test. Fisher-Irwin exact test.

All results were analysed statistically by 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 allowing for unequal variances. In this case, Wilcoxons rank sum test was also applied. In addition, The Fisher-Irwin exact test was used to compare the incidence of autopsy observations in control and treatment froups. All statistical tests were conducted at a 5% two-sided risk level.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Description (incidence):

No clinical signs noted. One corn oil control male and one high-dose female died in week 6 and week 8 respectively. Deaths were not related to treatment.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Male rats in the high dose group consumed significantly more food, which resulted in significantly lower feed efficiency values compared to corn oil or MCT controls (see below)

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Efficiency decreased in both sexes at the top caprenin dose, compared with corn oil and MCT controls. This was considered to reflect that the caprenin diets was not isocaloric with the corn oil and MCT control diets, due to an incomplete absorption of behenic acid. Further, poor absorption also considered to cause increased colon weights in caprenin groups (see below).

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Description (incidence and severity):

Few small isolated changes were noted. Serum haemoglobin of mid (+4%) and high-dose males were significantly higher than that of corn oil controls. Relative to MCT oil, mean cell volume was significantly greater in low (+2%) and mid-dose (+2%) males, mean cell haemoglobin was greater in all treated males (range of +2 to +3%), and activated partial thromboplastin time was greater in high-dose males (+9%). Platelet count was higher (+10%) with MCT oil relative to corn oil.
All these 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, treatment-related

Description (incidence and severity):

several parameters were statistically significantly changed compared to corn oil or MCT controls. However, most of these changes were generally mild, showed no dose-relationship, and occurred inconsistently between sexes. As there were no other findings including histopathology, and because the changes were close to or within the historical range, the changes were not considered to be of toxicological relevance. Examples of such observations included glucose, total cholesterol, LDL, HDL, triglycerides, potassium calcium, chloride, AST.

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):

Statistically significant changes in absolute or relative organ weights compared to corn oil controls or MCT controls were only seen in the liver, colon, kidneys, heart, and spleen. The three latter changes were mild, restricted to relative organ weights only, were unrelated to treatment and were, therefore, not considered to be related to treatment. There was a tendency of lower liver weights with increasing caprenin dose. The effect was small and did not gain statistical significance. Significantly higher relative colon weights were seen in males and females whereas absolute weight was significantly higher only in mid- and high-dose males. These changes were all small, lacked a dose-response relationship, were inconsistent to sex, and lacked a histopathological correlate. Hence, the observations lack toxicological relevance. The weight of testes and ovaries was not changed by any treatment.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

A significantly increased number of granular/rough kidneys in high-dose caprenin females (9/20) compared to corn oil controls (9/20) was noted. As there was no histopathological correlate, this was not considered to be adverse.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Liver vacuolisation in all treated and control rats; considered as a common finding in rats maintained at high-fat, semi-purified diets. Nephrocalcinosis was also seen in all rats, but histopathology revealed no treatment-relationship

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

The total fat content in heart, liver and perirenal fat was comparable across all groups, i.e. feeding caprenin or MCT had no effect compared to corn oil feeding.
Behenic acid (C22:0) was not detectable in fat from heart and liver. Low quantities of C22:0 acid were only found in the perirenal fat of caprenin fed males and females in the range of 0.66 to 1.75 % of total fat. The content was higher in females, but there was no relation to dose in either sex. The observation of only trace amounts indicates that absorbed Behenic acid undergoes degradation and is not integrated as such into fat tissue.

Key result

Dose descriptor:

NOAEL

Effect level:

13 200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks:

corresponds to 15% in diet

Sex:

male

Basis for effect level:

other:

Remarks on result:

other: Highest dose tested, which was above limit dose, did not induce effects

Critical effects observed:

not specified

 

 

 

 

Conclusions:

No adverse effect was seen in a subacute feeding study using male and female rats exposed to caprenin (up to 15% in diet). The high dose corresponds to 13200 mg/kg bw/day. Hence, the NOAEL according to this study is >13200 mg/kg bw/day.

Executive summary:

The subchronic toxicity of caprenin was examined in a 90-day feeding study in rats using a protocol that was comparable to OECD TG 408. Caprenin represents a triglyceride containing primarily C8, C10, and C22 saturated fatty acids. The dose levels were the same as those in a preceding 28-day rat study (NOAEL 15 % in diet), i.e. 5.23, 10.23, and 15.0% in diet. Control groups were fed a corn oil diet (12.4%) or a diet containing 11.21% MCT, i.e. triglycerides containing primarily C6, C8, and C10 fatty acids. For clarity, the fatty acid content of the various oils is given in table 1 below.

 

Table 1 Fatty acid composition of Caprein, MCT, and vehicle corn oil (% w/w)

Fatty acid	Caprenin	Corn oil	MCT
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	-	-

 

The animals received isocaloric diets during Day 1 through day 91 of the study. The fat composition is given in table 2. Content of proteins, carbohydrates, vitamin mix, minerals, fibre was comparable across the diets; for practical reasons not tabulated.

 

Table 2 Composition (%, w/w) of Caprenin and control diets

Ingredient	Corn oil control	MCT control	Caprenin
			Low dose	Mid dose	High dose
Caprenin	-	-	5.23	10.23	15.00
Corn oil	12.14	3.13	8.96	5.91	3.00
MCT oil	-	11.21	-	-	-

 

Thus, all diets contained at least 3.13 % corn oil and other fatty acids from either MCT or Caprenin. Fresh diets were weekly prepared and food consumption of the animals (singly housed; 25 rats per sex and group, total of 125 male and 125 female rats) was recorded. Examinations were similar to OECD 408, urinalysis was, however, omitted, and histopathology was performed in less organs than in the OECD 408 test guideline.

 

The results obtained in 20 rats per sex and group indicate that no treatment-related adverse effects were noted in any of the test groups. In brief, treatment-related clinical signs and mortality were not seen; body weight development of treated groups was comparable to the corn oil controls with a slightly decreased food efficiency in the high-dose groups compared to the corn oil and MCT controls; Ophthalmoscopic examination towards the end of the study revealed no abnormalities; changes of few haematological and clinical chemistry parameters gained a level of statistical significance when compared with the corn oil or the MCT controls, but the changes were generally small, not dose-related, occurred inconsistently across sexes, or were within or close to the historical control range and were, therefore, not considered to be of toxicological relevance. Similarly, absolute and/or relative organ weight changes in liver, kidneys, spleen, heart and colon gained in some instances a level of statistical significance. Increased colon weights in Caprenin groups was attributed to a decreased absorption of the long chain Behenic acid (C22:0). The changes in the other organs were small, within the historical control range, not related to dose, or lacked a histopathological correlate and were, therefore, not considered to be adverse. The weight of the reproductive organs, testes and ovaries, were not changed in any of the treated groups.

 

In 5 rats per sex and group the total fat content in heart, liver, and perirenal fat was comparable across all groups, i.e. treatment with Caprenin or MCT had no influence. The concentration of Behenic acid was also examined in these fat tissues. Concentrations were undetectable in fat from heart and liver (traces in caprenin groups, below detection limit of 0.5%), whereas small amounts (0.66 to 1.75%) were detected in all male and female groups fed Caprenin. The concentrations were not related to the dose. Anyhow, this finding suggests that absorbed Behenic acid is metabolised and is not integrated into fat as such.

 

To conclude, no adverse effects were seen in any of the groups receiving cornoil, Caprenin, or MCT. The 90-day oral NOAEL values (rat) were therefore as follows:

 

NOAEL Caprenin:           15.0 % in the diet.

Equivalent to males: 13,200 ± 6,700 mg/kg bw and day
females: 14,600 ± 5,700 mg/kg bw and day

The calculations are described in the attached document (Hillesheim, 2014). In addition calculations relative to octanoic acid and decanoci acid content were performed:

 

 

Test material	Caprenin	MCT
Exposure	feed	feed
Max dose	15% in diet	11.21% in diet
NOAEL Test material	15% in diet; M: 13200 mg/kg bw/day F: 14600 mg/kg bw/day	11.21% in diet M: 9865 mg/kg bw/day (estimated)
NOAEL C8 acid (estimated)	2723 mg/kg bw/day	5704 mg/kg bw/day
NOAEL C10 acid (estimated)	3122 mg/kg bw/day	2092 mg/kg bw/day

Reference 3

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

Study design not comparable to current guideline. Not enough animals/group. Not all of the examinations performed (e.g. microscopic pathology apparently only for liver and intestine).

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Pre-guideline study. A diet containing 19.5% MCT was fed to Wistar rats (15 per sex) for 47 weeks. Examinations included mortality, body and organ weights, histopathology (liver and intestine), absorption of fat from the diet and examination of body fats, cholesterol, and phospholipids in blood.

GLP compliance:

no

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Route of administration:

oral: feed

Vehicle:

other: casein diet with 2.5% safflower oil

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

Fatty acids were examined by gas chromatography after methylation in oils, faeces, carcass, liver, and epididymides. No explicit information was noted that the dose was analytically examined.

Duration of treatment / exposure:

47 weeks

Frequency of treatment:

7 days/week

Dose / conc.:

19.5 other: %

No. of animals per sex per dose:

15

Control animals:

other: yes, groups receiving 5 other diets with differing fatty acid composition

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: yes

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: weeks 7, 14, 21, 35, and 47 after study initiation
- Animals fasted: No data
- How many animals: all
- Parameters examined: cholesterol

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
- Feces: collected daily and pooled in weekly samples, analysed for fat, total nitrogen, and calcium
- fatty acids composition of body fat (carcass, liver, epididymal)

Sacrifice and pathology:

GROSS PATHOLOGY: No data. organ weights determined for: liver, kidneys, spleen, heart, adrenals, femurs, testes, epididymal fat

HISTOPATHOLOGY: Yes
- histopthology: instestine, liver

Other examinations:

absortion rates of dietary fat, protein and calcium

Statistics:

no information

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

at termination in in week 47 slighty lower body weigth in rats fed MCT (ca. 5%)

Food consumption and compound intake (if feeding study):

not specified

Description (incidence and severity):

Not reported, but no diffeence in food efficiency.

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not examined

Haematological findings:

not specified

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Cholesterol levels were lower in male rats during the study. At termination, cholesterol levels were higher. The differences may have been attributable to differences in the conditions under which blood samples were taken or reflect changes in age of the animals or in length of time on the diets.

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

Description (incidence and severity):

only liver and intestine examined

Histopathological findings: neoplastic:

not specified

Other effects:

no effects observed

Details on results:

Dietary fat was completely absorbed (98%) in rats fed MCT

Key result

Dose descriptor:

NOAEL

Effect level:

19.5 other: %

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

not specified

 

 

 

 

 

Conclusions:

No adverse effect were seen in rats (Wistar, 15/sex) fed a 19.5% MCT diet for 47 weeks. Dietary fat was completely absorbed (98%) in rats fed MCT. According to this study, the NOAEL was >19.5% MCT in the diet.

Executive summary:

In this study, 15 Wistar rats per sex were fed a 19.5% MCT diet for 47 weeks. This diet was compared to similar diets containing conventional dietary fats.

The study predates current guidelines and does not follow a protocol comparable to a guideline. Main evaluations of a repeated dose toxicity study are included.

MCT supported normal growth and development. There was no difference in food efficiency. No toxicologically relevant effects were observed. At autopsy, carcass protein and ash levels, vital organ weights and composition were similar to those of rats fed the conventional diets. MCT was completely absorbed.

According to this study, the NOAEL was considered to be > 19.5% MCT.

I

Reference 4

Endpoint:

sub-chronic toxicity: oral

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

For details on endpoint specific justification please see read-across report in section 13 or find a link in cross-reference “assessment report”.

Reason / purpose for cross-reference:

assessment report

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 15 other: % in diet

Based on:

other: MCT

Remarks:

corresponds to octanoic acid/kg and day: males 2295 (1720-2870) mg, females 3385 (2470-4300) mg; corresponds to decanoic acid/kg and day: males 984 (738-1230) mg , females 1451 (1058-1845) mg

Sex:

male/female

Basis for effect level:

other: no adverse effect noted

Remarks on result:

other: Highest dose tested, which was above limit dose, did not induce effects

Critical effects observed:

not specified

The NOAEL for n-nonanoic acid has been calculated on basis of the NOAEL for MCT using the dose of MCT (4000 mg/kg bw/d) and the molecular weights 495 g/mol (MCT) and 158.24 g/mol (n-nonanoic acid).

Conclusions:

Based on the findings of this 91-day feeding study, there are no indications of toxicological effects in dogs fed up to 15% MCT in the diet. For nonanoic acid a NOAEL (dog, oral feed, 91d) of ca. 3300 mg/kg bw and day was derived.

Executive summary:

In this 91 day study, 4 male and 4 female beagle dogs were treated with 0, 5, 10, or 15% MCT via the diet. The study followed a protocol similar to OECD TG 409. No statement regarding GLP is given.

 

A reduced food intake an several consecutive days in the groups with MCT exposure was noted and attributed to palatibility of MCT. Bodyweight developed normal.

There was a singificant increase of blood urea nitrogen in the 10% and 15% groups of both sexes and a non-significant increase of cholesterol in both sexes of the 10% and 15% group.

The changes were not considered to be adverse. The same was concluded for the observed reduction of urine volumes in the mid- and high-dose groups.

In this study animals were not sacrificed at termination. No tissue samples were taken, nor were any animals terminated.

 

No other effects were observed. The NOAEL was therefore 15% MCT in the diet in the beagle dog.

 

The estimated NOAEL-values in the beagle dog are as follows, based on calculations described in the attached document (Hillesheim, 2014):

 

octanoic acid:      

males           2295 (1700 – 2900) mg/kg bw and day                            

females        3385 (2500 – 4300) mg/kg bw and day

decanoic acid:       

males            984 (740 – 1200) mg/kg bw and day

females        1451 (1060 – 1850) mg/kg bw and day

Based on these calculations a NOAEL of ca. 3300 mg nonanoic acid/kg bw/d can be derived for male dogs.

Reference 5

Endpoint:

sub-chronic toxicity: oral

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

For details on endpoint specific justification please see read-across report in section 13 or find a link in cross-reference “assessment report”.

Reason / purpose for cross-reference:

assessment report

Reason / purpose for cross-reference:

read-across source

Clinical signs:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

13 200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks:

corresponds to 15% in diet

Sex:

male

Basis for effect level:

other:

Remarks on result:

other: Highest dose tested, which was above limit dose, did not induce effects

Critical effects observed:

not specified

 

 

 

 

Conclusions:

No adverse effect was seen in a subacute feeding study using male and female rats exposed to caprenin (up to 15% in diet). The high dose corresponds to 13200 mg/kg bw/day. Based on the results of the rat study with caprenin a NOAEL of ca. 5800 mg nonanoic acid/kg bw/day can be derived

Executive summary:

The subchronic toxicity of caprenin was examined in a 90-day feeding study in rats using a protocol that was comparable to OECD TG 408. Caprenin represents a triglyceride containing primarily C8, C10, and C22 saturated fatty acids. The dose levels were the same as those in a preceding 28-day rat study (NOAEL 15 % in diet), i.e. 5.23, 10.23, and 15.0% in diet. Control groups were fed a corn oil diet (12.4%) or a diet containing 11.21% MCT, i.e. triglycerides containing primarily C6, C8, and C10 fatty acids. For clarity, the fatty acid content of the various oils is given in table 1 below.

 

Table 1 Fatty acid composition of Caprein, MCT, and vehicle corn oil (% w/w)

Fatty acid	Caprenin	Corn oil	MCT
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	-	-

 

The animals received isocaloric diets during Day 1 through day 91 of the study. The fat composition is given in table 2. Content of proteins, carbohydrates, vitamin mix, minerals, fibre was comparable across the diets; for practical reasons not tabulated.

 

Table 2 Composition (%, w/w) of Caprenin and control diets

Ingredient	Corn oil control	MCT control	Caprenin
			Low dose	Mid dose	High dose
Caprenin	-	-	5.23	10.23	15.00
Corn oil	12.14	3.13	8.96	5.91	3.00
MCT oil	-	11.21	-	-	-

 

Thus, all diets contained at least 3.13 % corn oil and other fatty acids from either MCT or Caprenin. Fresh diets were weekly prepared and food consumption of the animals (singly housed; 25 rats per sex and group, total of 125 male and 125 female rats) was recorded. Examinations were similar to OECD 408, urinalysis was, however, omitted, and histopathology was performed in less organs than in the OECD 408 test guideline.

 

The results obtained in 20 rats per sex and group indicate that no treatment-related adverse effects were noted in any of the test groups. In brief, treatment-related clinical signs and mortality were not seen; body weight development of treated groups was comparable to the corn oil controls with a slightly decreased food efficiency in the high-dose groups compared to the corn oil and MCT controls; Ophthalmoscopic examination towards the end of the study revealed no abnormalities; changes of few haematological and clinical chemistry parameters gained a level of statistical significance when compared with the corn oil or the MCT controls, but the changes were generally small, not dose-related, occurred inconsistently across sexes, or were within or close to the historical control range and were, therefore, not considered to be of toxicological relevance. Similarly, absolute and/or relative organ weight changes in liver, kidneys, spleen, heart and colon gained in some instances a level of statistical significance. Increased colon weights in Caprenin groups was attributed to a decreased absorption of the long chain Behenic acid (C22:0). The changes in the other organs were small, within the historical control range, not related to dose, or lacked a histopathological correlate and were, therefore, not considered to be adverse. The weight of the reproductive organs, testes and ovaries, were not changed in any of the treated groups.

 

In 5 rats per sex and group the total fat content in heart, liver, and perirenal fat was comparable across all groups, i.e. treatment with Caprenin or MCT had no influence. The concentration of Behenic acid was also examined in these fat tissues. Concentrations were undetectable in fat from heart and liver (traces in caprenin groups, below detection limit of 0.5%), whereas small amounts (0.66 to 1.75%) were detected in all male and female groups fed Caprenin. The concentrations were not related to the dose. Anyhow, this finding suggests that absorbed Behenic acid is metabolised and is not integrated into fat as such.

 

To conclude, no adverse effects were seen in any of the groups receiving cornoil, Caprenin, or MCT. The 90-day oral NOAEL values (rat) were therefore as follows:

 

NOAEL Caprenin:           15.0 % in the diet.

Equivalent to males: 13,200 ± 6,700 mg/kg bw and day
females: 14,600 ± 5,700 mg/kg bw and day

The calculations are described in the attached document (Hillesheim, 2014). In addition calculations relative to octanoic acid and decanoci acid content were performed:

 

 

Test material	Caprenin	MCT
Exposure	feed	feed
Max dose	15% in diet	11.21% in diet
NOAEL Test material	15% in diet; M: 13200 mg/kg bw/day F: 14600 mg/kg bw/day	11.21% in diet M: 9865 mg/kg bw/day (estimated)
NOAEL C8 acid (estimated)	2723 mg/kg bw/day	5704 mg/kg bw/day
NOAEL C10 acid (estimated)	3122 mg/kg bw/day	2092 mg/kg bw/day
NOAEL C9 acid (read across from C8 and C10 acid)	ca. 5800 mg/kg bw/day	ca. 7800 mg/kg bw/day

Reference 6

Endpoint:

chronic toxicity: oral

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

For details on endpoint specific justification please see read-across report in section 13 or find a link in cross-reference “assessment report”.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

assessment report

Urinalysis findings:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

19.5 other: %

Based on:

test mat.

Remarks:

corresponds to ca. 8000 mg/kg bw/d nonanoic acid

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

not specified

 

 

 

 

 

Conclusions:

No adverse effect were seen in rats (Wistar, 15/sex) fed a 19.5% MCT diet for 47 weeks. Dietary fat was completely absorbed (98%) in rats fed MCT. According to this study, the NOAEL was >19.5% MCT in the diet (ca. 8000 mg nonanoic acid/kg bw/d).

Executive summary:

In this study, 15 Wistar rats per sex were fed a 19.5% MCT diet for 47 weeks. This diet was compared to similar diets containing conventional dietary fats.

The study predates current guidelines and does not follow a protocol comparable to a guideline. Main evaluations of a repeated dose toxicity study are included.

 

MCT supported normal growth and development. There was no difference in food efficiency. No toxicologically relevant effects were observed. At autopsy, carcass protein and ash levels, vital organ weights and composition were similar to those of rats fed the conventional diets. MCT was completely absorbed.

 

According to this study, the NOAEL was considered to be > 19.5% MCT. 

According to the calculations in the CLH-report for nonanoic acid this corresponds to a NOAEL of 4700 mg/kg bw/d for octanoic acid and 3200 mg/kg bw/day for decanoic acid, i.e. combined approximately 8000 mg/kg bw/d nonanoic acid. 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

3 300 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

sufficient

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

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

other information

Study period:

1 Dec 1980 to 29 Dec 1980

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Does not meet important criteria of today standard methods: Low animal numbers, only one dose tested, treatment period only 14 days, limited parameters examined (no organ weights, no hematology, clinical biochemistry, or urinalysis)

Principles of method if other than guideline:

14-day repeated dose dermal toxicity study using 5 male and 5 female rabbits at one dose level. A satellite group of 3 animals was allowed to recover for 14 days

GLP compliance:

not specified

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Dutchland Laboratory Animals, Denver, PA, USA
- Age at study initiation: young adult
- Weight at study initiation: 2.1-3.2 kg (males); 2.3-3.2 kg (females)
- Housing: individually
- Diet (ad libitum): Purina rabbit chow
- Water (ad libitum): tap water
- Acclimation period: 21 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15.5-21.1
- Humidity (%): monitored daily
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

other: open, animals with collar

Vehicle:

other: mineral oil

Details on exposure:

TEST SITE
- Area of exposure: dorsal and lateral, about 10% of the total body surface
- % coverage: about 10%
- Type of wrap if used: application sites not covered
- Time intervals for shavings or clipplings: prior to first dose, reclipping as nessesary during the dosing period
REMOVAL OF TEST SUBSTANCE
- Washing (if done): no

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 500 mg/kg/day (about 2 mL of test substance solution); test solution and vehicle (for controls) were applied directly onto skin and spread evenly over the entire area with a glass rod.
- Concentration (if solution): 25% w/w in mineral oil; to 100 g of test substance, vehicle was added to acchieve a total weight of 400 g. The mixture was stirred slowly to achieve a homogeneous mixture. Fresh mixtures were prepared weekly.
- Constant volume or concentration used: yes

VEHICLE
- Justification for use and choice of vehicle (if other than water): suitable solvent
- Amount(s) applied (volume or weight with unit): 2 mL
- Concentration (if solution): 25% w/w test substance in vehicle

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes; Elisabethan collars

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

14 d

Frequency of treatment:

1x/d, 5d/wk, 10x

Remarks:

Doses / Concentrations:
500 mg/(kg bw day) as 25 % solution in mineral oil
Basis:
nominal per unit body weight

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: no data
- Rationale for animal assignment (if not random): random by random numbers table
- Rationale for selecting satellite groups: 3 animals (from 10 of the dosed group) were selected as satellite animals for the recovery group
- Post-exposure recovery period in satellite groups: 2 weeks
- Section schedule rationale (if not random): no data
-Skin treatment: The skin of half of the animals (3 males, 2 females) was abraded prior to the first, sixth and eighth substance application. If the integrity of the skin was interrupted because of response to test material application, additional abrasions were not made.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: mortality twice daily, cage side observations daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: prior to first dosing and daily thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: prior to first application, weekly thereafter

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Time schedule: 6 animals after 2 weeks (3 each with abraded and intact skin regardless of sex, end of treatment period), remaining 3 animals after 4 weeks (end of post exposure recovery period).
- Preserved tissues: adrenals (2), brain, eyes, gonads, heart, intestine (colon, duodenum, ileum), kidneys (2), liver (2), lungs (2) lymph nodes (mesenteric), mammary gland, pancreas, pituitary, salivary gland, skeletal muscle, skin (treated, untreated), spinal cord (cervical), spleen, stomach, thyroid, urinary bladder, uterus/prostate, gross lesions tissue masses. Tissues were preserved in 10% neutral buffered formalin.

HISTOPATHOLOGY: Yes
- Tissues: brain, heart, kidneys, liver, lungs, skin treated and untreated. Tissue slides were prepared, stained with hematoxylin and eosin, and examined microscopically

Clinical signs:

no effects observed

Dermal irritation:

effects observed, treatment-related

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

no effects observed

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
No mortality and no overt clinical signs occured.

DERMAL IRRITATION
Most animals showed slight to severe erythema and edema without necrosis or eschar formation during the first week of the study. Necrosis and eschar occured subsequently in all animals during the second week of treatment. Further effects were atonia, desquamation, fissuring and exfoliation. These effects subsided during the recovery period. One animal exhibited hair loss during the recovery period.

BODY WEIGHT AND WEIGHT GAIN
Most animals treated with the test substance exhibited slicht body weight losses (0.1-0.4 kg) after one week and/or two weeks of study. Animals held for the two weeks recovery period gained weight during this interval.

FOOD CONSUMPTION
Several aminals exhibited a decreased food consumption during the second and third week of the study.

GROSS PATHOLOGY
Mostly, morphological abnormalities of the treated skin were observed (see Dermal Irritation). Other gross morphological and microscopic alterations occured sporadically in treated and control animals and do not appear to be related to the exposure. Other changes were too inconclusively to demonstrate the presence or absence of systemic effect of the test substance.

HISTOPATHOLOGY: NON-NEOPLASTIC
Microscopic examination showed epidermal necrosis, epidermal hyperplasia, hyperkeratosis as well as diffuse and perifollicular dermal inflammation at the test sites. The application sites appeared to be healed at the end of the recovery period (re-epitheliazed and continuous, mild to moderate epidermal hyperplasia and hyperkeratosis, normal follicular structure and population). Treatment-related tissue reactions occured in abraded as well as non-abraded sites.

Dose descriptor:

LOAEL

Effect level:

500 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: reduced food consumption and slight weight losses during exposure, no clearly treatment-related systemic effects, local effects: skin irritation and necrosis

Critical effects observed:

not specified

Conclusions:

Under the conditions of this repeated dermal toxicity study on rabbits the most prominent effect was irritation and necrosis of the skin. The effects subsided in a recovery group of 3 animals within 14 days. Systemic toxic responses were restricted to unspecific effects (reduced food consumption, body weight loss).

Executive summary:

5 male and female New Zealand rabbits each were exposed dermally to 0 or 500 mg/kg bw of the test item (purity not stated) 5 d/w for 2 weeks. The skin of 50% of the animals (3 males, 2 females) has been abraded. Six animals (three with abraded and three with intact skin) were examined at the end of exposure. 3 of 10 animals were selected as satellite group for a 2 weeks recovery period.

Several aminals exhibited a decreased food consumption during the second and third week of the study.

Most animals exhibited weight loss (0.1 - 0.4 kg) after 1 or/and 2 weeks of  the study. Animals regained weight during the recovery period.

Most animals showed slight to severe erythema and edema during the first week of the study. Necrosis and eschar occured subsequently in all animals during the second week of treatment. Further effects were atonia, desquamation, fissuring and exfoliation. Microscopic examination showed epidermal necrosis, epidermal hyperplasia, hyperkeratosis as well as diffuse and perifollicular dermal inflammation at the test sites. The skin application sites appeared to be healed at the end of recovery period. No clearly treatment-related macroscopic or microscopic lesions were observed in other organs.

The LOAEL in this study is 500 mg/kg bw/day, based on body weight reduction and skin damage, a NOAEL was not derived (Bio/dynamics, 1981). 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

500 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

poor, study reliability 3 (invalid)

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

other information

Study period:

1 Dec 1980 to 29 Dec 1980

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Does not meet important criteria of today standard methods: Low animal numbers, only one dose tested, treatment period only 14 days, limited parameters examined (no organ weights, no hematology, clinical biochemistry, or urinalysis)

Principles of method if other than guideline:

14-day repeated dose dermal toxicity study using 5 male and 5 female rabbits at one dose level. A satellite group of 3 animals was allowed to recover for 14 days

GLP compliance:

not specified

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Dutchland Laboratory Animals, Denver, PA, USA
- Age at study initiation: young adult
- Weight at study initiation: 2.1-3.2 kg (males); 2.3-3.2 kg (females)
- Housing: individually
- Diet (ad libitum): Purina rabbit chow
- Water (ad libitum): tap water
- Acclimation period: 21 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15.5-21.1
- Humidity (%): monitored daily
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

other: open, animals with collar

Vehicle:

other: mineral oil

Details on exposure:

TEST SITE
- Area of exposure: dorsal and lateral, about 10% of the total body surface
- % coverage: about 10%
- Type of wrap if used: application sites not covered
- Time intervals for shavings or clipplings: prior to first dose, reclipping as nessesary during the dosing period
REMOVAL OF TEST SUBSTANCE
- Washing (if done): no

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 500 mg/kg/day (about 2 mL of test substance solution); test solution and vehicle (for controls) were applied directly onto skin and spread evenly over the entire area with a glass rod.
- Concentration (if solution): 25% w/w in mineral oil; to 100 g of test substance, vehicle was added to acchieve a total weight of 400 g. The mixture was stirred slowly to achieve a homogeneous mixture. Fresh mixtures were prepared weekly.
- Constant volume or concentration used: yes

VEHICLE
- Justification for use and choice of vehicle (if other than water): suitable solvent
- Amount(s) applied (volume or weight with unit): 2 mL
- Concentration (if solution): 25% w/w test substance in vehicle

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes; Elisabethan collars

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

14 d

Frequency of treatment:

1x/d, 5d/wk, 10x

Remarks:

Doses / Concentrations:
500 mg/(kg bw day) as 25 % solution in mineral oil
Basis:
nominal per unit body weight

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: no data
- Rationale for animal assignment (if not random): random by random numbers table
- Rationale for selecting satellite groups: 3 animals (from 10 of the dosed group) were selected as satellite animals for the recovery group
- Post-exposure recovery period in satellite groups: 2 weeks
- Section schedule rationale (if not random): no data
-Skin treatment: The skin of half of the animals (3 males, 2 females) was abraded prior to the first, sixth and eighth substance application. If the integrity of the skin was interrupted because of response to test material application, additional abrasions were not made.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: mortality twice daily, cage side observations daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: prior to first dosing and daily thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: prior to first application, weekly thereafter

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Time schedule: 6 animals after 2 weeks (3 each with abraded and intact skin regardless of sex, end of treatment period), remaining 3 animals after 4 weeks (end of post exposure recovery period).
- Preserved tissues: adrenals (2), brain, eyes, gonads, heart, intestine (colon, duodenum, ileum), kidneys (2), liver (2), lungs (2) lymph nodes (mesenteric), mammary gland, pancreas, pituitary, salivary gland, skeletal muscle, skin (treated, untreated), spinal cord (cervical), spleen, stomach, thyroid, urinary bladder, uterus/prostate, gross lesions tissue masses. Tissues were preserved in 10% neutral buffered formalin.

HISTOPATHOLOGY: Yes
- Tissues: brain, heart, kidneys, liver, lungs, skin treated and untreated. Tissue slides were prepared, stained with hematoxylin and eosin, and examined microscopically

Clinical signs:

no effects observed

Dermal irritation:

effects observed, treatment-related

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

no effects observed

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
No mortality and no overt clinical signs occured.

DERMAL IRRITATION
Most animals showed slight to severe erythema and edema without necrosis or eschar formation during the first week of the study. Necrosis and eschar occured subsequently in all animals during the second week of treatment. Further effects were atonia, desquamation, fissuring and exfoliation. These effects subsided during the recovery period. One animal exhibited hair loss during the recovery period.

BODY WEIGHT AND WEIGHT GAIN
Most animals treated with the test substance exhibited slicht body weight losses (0.1-0.4 kg) after one week and/or two weeks of study. Animals held for the two weeks recovery period gained weight during this interval.

FOOD CONSUMPTION
Several aminals exhibited a decreased food consumption during the second and third week of the study.

GROSS PATHOLOGY
Mostly, morphological abnormalities of the treated skin were observed (see Dermal Irritation). Other gross morphological and microscopic alterations occured sporadically in treated and control animals and do not appear to be related to the exposure. Other changes were too inconclusively to demonstrate the presence or absence of systemic effect of the test substance.

HISTOPATHOLOGY: NON-NEOPLASTIC
Microscopic examination showed epidermal necrosis, epidermal hyperplasia, hyperkeratosis as well as diffuse and perifollicular dermal inflammation at the test sites. The application sites appeared to be healed at the end of the recovery period (re-epitheliazed and continuous, mild to moderate epidermal hyperplasia and hyperkeratosis, normal follicular structure and population). Treatment-related tissue reactions occured in abraded as well as non-abraded sites.

Dose descriptor:

LOAEL

Effect level:

500 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: reduced food consumption and slight weight losses during exposure, no clearly treatment-related systemic effects, local effects: skin irritation and necrosis

Critical effects observed:

not specified

Conclusions:

Under the conditions of this repeated dermal toxicity study on rabbits the most prominent effect was irritation and necrosis of the skin. The effects subsided in a recovery group of 3 animals within 14 days. Systemic toxic responses were restricted to unspecific effects (reduced food consumption, body weight loss).

Executive summary:

5 male and female New Zealand rabbits each were exposed dermally to 0 or 500 mg/kg bw of the test item (purity not stated) 5 d/w for 2 weeks. The skin of 50% of the animals (3 males, 2 females) has been abraded. Six animals (three with abraded and three with intact skin) were examined at the end of exposure. 3 of 10 animals were selected as satellite group for a 2 weeks recovery period.

Several aminals exhibited a decreased food consumption during the second and third week of the study.

Most animals exhibited weight loss (0.1 - 0.4 kg) after 1 or/and 2 weeks of  the study. Animals regained weight during the recovery period.

Most animals showed slight to severe erythema and edema during the first week of the study. Necrosis and eschar occured subsequently in all animals during the second week of treatment. Further effects were atonia, desquamation, fissuring and exfoliation. Microscopic examination showed epidermal necrosis, epidermal hyperplasia, hyperkeratosis as well as diffuse and perifollicular dermal inflammation at the test sites. The skin application sites appeared to be healed at the end of recovery period. No clearly treatment-related macroscopic or microscopic lesions were observed in other organs.

The LOAEL in this study is 500 mg/kg bw/day, based on body weight reduction and skin damage, a NOAEL was not derived (Bio/dynamics, 1981). 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

3.33 mg/cm²

Study duration:

subacute

Species:

rabbit

Quality of whole database:

Sufficient, confirms irritating properties seen in the acute toxicity tests. Single high dose was used and does not allow an exact estimation of the LOAEL or NOAEL value. This is, however, not required, because exposure in industrial settings is low.

Additional information

Information from supporting substances has been used to fulfil the informationrequirementsfor this endpoint. For Read-across justification , see the atteched file in endpoint 13 ( Analogue approach justification):

 

 

Oral exposure:

 

Notox (2002)

The repeated dose toxicity of pelargonic acid (0, 50, 150, 1000 mg/kg bw/day; oral gavage; frequency 7 doses per week) was examined in a subacute 28-day study according to OECD guideline 407 and under GLP conditions in Wistar rats (5 rats/sex/dose).

No substance-related mortality occurred. Slight to moderate breathing difficulties (rales and/or gasping) were seen in several high dose animals on some days during week 3. These signs subsided and were absent during week 4. No signs were noted in the low and intermediate dose groups. Body weights and body weight gain were similar across controls and treated groups.

There was no effect on hematology and clinical chemistry parameters. No changes were noted in hearing ability, pupillary reflex, static righting reflex and grip strength, or in motor activity in neurobehavioral functional tests.

At termination, absolute and relative organ weights were similar between control and treated animals. An irregular surface of the forestomach was noted in all high dose animals at necropsy. A thickened (glandular mucosa of the ) stomach was observed among the treated animals. Since none of these cases could be confirmed microscopically, they were considered to be of no toxicological relevance. Histopathology revealed no other findings than slight to marked hyperplasia of the squamous epithelium of the forestomach in all high dose animals, and at a minimal degree in 3 intermediate dose group animals.

 

Based on the above, and taking into account that there is no correlate for the rat’s forestomach in humans the sub-acute NOAEL for systemic toxicity is considered to be 1000 mg/kg bw/day.

 

Read-across from Matulka (2009)

Based on the findings of this 91-day MCT feeding study, there are no indications of toxicological effects in dogs fed 5, 10, or 15% MCT in the diet. Changes in blood regarding cholesterol (increase) and urea nitrogen (significant increase at 10% and 15%) were noted but not considered to be adverse. The same was concluded for the observed reduction of urine volumes in the mid- and high-dose groups. The NOAEL was therefore 15% MCT in the diet in the Beagle dog (Matulka et al., 2009).

 

The estimated NOAEL-values in the Beagle dog are as follows, based on calculations described in the attached document (Hillesheim, 2014):

 

C₈fatty acid:       males           2295 (1700 – 2900) mg/kg bw and day
                            females        3385 (2500 – 4300) mg/kg bw and day

C₁₀fatty acid:   males            984 (740 – 1200) mg/kg bw and day

                  females        1451 (1060 – 1850) mg/kg bw and day

 

It is concluded that in this subchronic canine study even very high dietary doses of Medium Chain Triglycerides did not exert adverse effects. Following absorption the triglycerides are cleaved and liberate the respective amounts of fatty acids. The vast majority represents octanoic and decanoic acid since only trace amounts of hexanoic and dodecanoic acid are present in MCT. The known relative proportion which allows calculating NOAEL values in terms of “mg acid/kg body weight and day”. The resulting NOAEL-values for octanoic acid is far beyond 1000 mg/kg bw and day, and that of decanoic acid is approx. 1000 mg/kg bw and day. Calculations are described in the attached document (Hillesheim, 2014)

 

It should further be mentioned that the lower NOAEL ofC₁₀fatty acid results entirely from the lower content in the test article compared to C₈fatty acid, i.e. there is no reason to assume that the longer C₁₀fatty acid would be more toxic than the C₈fatty acid. In contrast, the NOAEL of the sum of both fatty acids was 3280 mg/kg bw and day in males, and 4836 mg/kg bw and day in females, i.e. even high amounts of fatty acids were nontoxic, which may be expected for a food constituent that is ingested in high amounts during normal life.

 

As to n-nonanoic acid, an equally rapid absorption and metabolism is expected. Therefore, a similar NOAEL-value is expected, i.e. in the range of

ca. 3300 mg/kg bw and day or even higher (comparable to the experimental results for octanoic and decanoic acid obtained in this study and described above).

 

Overall, it is concluded that, despite the study was not conducted in accordance with current subchronic test guidelines, the publication provides valid information on octanoic and decanoic acid that may be used as weight of evidence to assess the toxicity of the closely related target substance, nonanoic acid.

 

Read across from Webb (1993)

The subchronic toxicity of caprenin was examined in a 90-day feeding study in rats using a protocol that was comparable to OECD TG 408. Caprenin represents a triglyceride containing primarily C8, C10, and C22 saturated fatty acids. The dose levels were the same as those in a preceding 28-day rat study (NOAEL 15 % in diet), i.e. 5.23, 10..23, and 15.0% in diet. Control groups were fed a corn oil diet (12.4%) or a diet containing 11.21% MCT, i.e. triglycerides containing primarily C6, C8, and C10 fatty acids. For clarity, the fatty acid content of the various oils is given in table 1 below.

 

Table 1 Fatty acid composition of Caprein, MCT, and vehicle corn oil (% w/w)

Fatty acid	Caprenin	Corn oil	MCT
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	-	-

 

The animals received isocaloric diets during Day 1 through day 91 of the study. The fat composition is given in table 2. Content of proteins, carbohydrates, vitamin mix, minerals, fibre was comparable across the diets; for practical reasons not tabulated.

Table 2 Composition (%, w/w) of Caprenin and control diets

Ingredient	Corn oil control	MCT control	Caprenin
			Low dose	Mid dose	High dose
Caprenin	-	-	5.23	10.23	15.00
Corn oil	12.14	3.13	8.96	5.91	3.00
MCT oil	-	11.21	-	-	-

 

Thus, all diets contained at least 3.13 % corn oil and other fatty acids from either MCT or Caprenin. Fresh diets were weekly prepared and food consumption of the animals (singly housed; 25 rats per sex and group, total of 125 male and 125 female rats) was recorded. Examinations were similar to OECD 408, urinalysis was, however, omitted, and histopathology was performed in less organs than in the OECD 408 test guideline.

The results obtained in 20 rats per sex and group indicate that no treatment-related adverse effects were noted in any of the test groups. In brief, treatment-related clinical signs and mortality were not seen; body weight development of treated groups was comparable to the corn oil controls with a slightly decreased food efficiency in the high-dose groups compared to the corn oil and MCT controls; Ophthalmoscopic examination towards the end of the study revealed no abnormalities; changes of few haematological and clinical chemistry parameters gained a level of statistical significance when compared with the corn oil or the MCT controls, but the changes were generally small, not dose-related, occurred inconsistently across sexes, or were within or close to the historical control range and were, therefore, not considered to be of toxicological relevance. Similarly, absolute and/or relative organ weight changes in liver, kidneys, spleen, heart and colon gained in some instances a level of statistical significance. Increased colon weights in Caprenin groups was attributed to a decreased absorption of the long chain Behenic acid (C22:0). The changes in the other organs were small, within the historical control range, not related to dose, or lacked a histopathological correlate and were, therefore, not considered to be adverse. The weight of the reproductive organs, testes and ovaries, were not changed in any of the treated groups.

In 5 rats per sex and group the total fat content in heart, liver, and perirenal fat was comparable across all groups, i.e. treatment with Caprenin or MCT had no influence. The concentration of Behenic acid was also examined in these fat tissues. Concentrations were undetectable in fat from heart and liver (traces in caprenin groups, below detection limit of 0.5%), whereas small amounts (0.66 to 1.75%) were detected in all male and female groups fed Caprenin. The concentrations were not related to the dose. Anyhow, this finding suggests that absorbed Behenic acid is metabolised and is not integrated into fat as such.

To conclude, no adverse effects were seen in any of the groups receiving cornoil, Caprenin, or MCT. The 90-day oral NOAEL values (rat) were therefore as follows:

NOAEL Corn oil:                           males and females: 12.14 % in the diet

NOAEL Caprenin:           15.0 % in the diet.

Equivalent to males: 13,200 ± 6,700 mg/kg bw and day
females: 14,600 ± 5,700 mg/kg bw and day

NOAEL MCT:                    males and females: 11.21 % in the diet

 

The study (Webb et al. 1993) is considered to be valid and useful for assessment for the target substance, nonanoic acid, in a Weight of Evidence approach because of the similarity of the medium chain fatty acids (C8, C9, C10) with regard to the absorption and metabolism in the fatty acid cycle.

It is therefore considered that the NOAEL of nonanoic acid is comparable to that of octanoic or decanoic acid. The NOAEL might also be set at the sum of the NOAELs of octanoic and decanoic acid because the rats were simultaneously exposed to both fatty acids without exerting adverse effects, and because all fatty acids must be metabolised in the fatty acid cycle. It could be mentioned that this applies also to fatty acids that are ingested in large amounts with food or feed without overt adverse effects.

On the basis of the estimated NOAEL values for octanoic and decanoic acid of the animals fed Caprenin or MCT it is concluded that the NOAEL(rat, oral feeed, 90d) for nonanoic acid is 5800 mg/kg bw and day orhigher.The calculations are described in the attached document (Hillesheim, 2014).

 

Test material	Caprenin	MCT
Exposure	feed	feed
Max dose	15% in diet	11.21% in diet
NOAEL Test material	15% in diet; M: 13200 mg/kg bw/day F: 14600 mg/kg bw/day	11.21% in diet M: 9865 mg/kg bw/day (estimated)
NOAEL C8 acid (estimated)	2723 mg/kg bw/day	5704 mg/kg bw/day
NOAEL C10 acid (estimated)	3122 mg/kg bw/day	2092 mg/kg bw/day
NOAEL C9 acid (read across from C8 and C10 acid)	ca. 5800 mg/kg bw/day	ca. 7800 mg/kg bw/day

 

 

 

Read-across from Harkins (1968)

The summary of the CLH report for nonanoic acid (2011; p 42/89) reads as follows:

 

“Harkins et Sarett 1968 published a nutritional evaluation of a medium chain triglyceride (MCT) preparation. A casein diet, containing 18.5% MCT and 2.5% safflower oil, the latter to supply essential fatty acids, was compared with similar diets containing conventional

dietary fats. The MCT contained about 51% octanoic acid and 35% decanoic aridresulting in an octanoic acid dietary dose of about 4700 mg/kg bw day and a decanoic acid dietary dose of about 3200 mg/kg bw day.Data obtained in a 47-week study showed that the MCT diet supported normal growth and development. At autopsy carcass composition (without liver, heart, epididymal fat pads, GI) in terms of weight, fat, protein and ash levels were similar to those in rats fed with conventional fats. Also organ weights of liver, kidney, spleen, heart, adrenals, femurs and testes were similar in all groups. Histological study showed that intestinal and liver sections were normal after 47 weeks on the MCT-containing diet. In general, rats fed MCT had slightly lower growth rates and caloric efficiency values, less carcass fat and smaller epididymal fat pads than animals fed conventional dietary fats. Little C8 and Cl0 were found in depot fat that is 0.5 and 4.9%, respectively, though these fatty acids comprised about 85% of the dietary fat. The MCT diet also supported normal reproduction, as indicated by litter size and number. For Decanoic acid and Octanoic acid a common NOAEL of ≥ 8000 mg/kg bw day is apparent in this study. “

 

It could further be mentioned that

- MCT fatty acids were completely absorbed

- MCT fatty acids were digested since the fatty acid composition of body fats including that in breast milk was different from that in MCT

- the NOAEL was 4700 mg/kg bw and day for octanoic acid, and 3200 mg/kg bw and day for decanoic acid, combined approx. 8000 mg/kg bw and day

 

The study is considered to be valid for assessment of nonanoic acid. A low reliability was assigned because this early pre-guideline study does not comply in several regards with the current test guidelinee, but basic information is well decomuneted and may be used for assessment. The metabolism is similar for all fatty acids, the NOAEL can therefore be read across to Nonanoic acid: ca. 4000 mg/kg bw and day. This was also acknowledged in the CLH reports on octanoic, nonanoic, and decanoic acids, and also in the respective RAC opinions available at the ECHA website.

 

 

Dermal Exposure:

Low reliability studies exist as follows. It should be noted, however, that repeated dermal studies with corrosive test substances could lead to false results through the formation of toxic reactions due to necrosis, rather than the test substance itself. Moreover, such studies are obsolete because of animal welfare considerations.

 

New Zealand rabbits (5 per sex) were exposed dermally to 500 mg/kg bw of the test item (purity not stated) 5 d/w for 2 weeks. Three animals per sex were examined at the end of exposure, the remaining animals after 2 weeks of recovery.

Several aminals exhibited a decreased food consumption during the second and third week of the study. Most animals exhibited weight loss during the treatment, but regained weight during the recovery period.

Most animals showed signs of marked irritation, necrosis and eschar at the treated sites, but appeared to be healed at the end of recovery period. No treatment-related macroscopic or microscopic organ lesions were observed. The LOAEL of this study was 500 mg/kg bw/day, based on body weight reduction and skin damage (Bio/dynamics, 1981). This study was judged to be not reliable due to poor data reporting , as no complete study report is available (RL3).

 

50 male C3H mice were dermally exposed to the test item (purity not stated) for 80 weeks (twice weekly, 50 mg per treatment, maximum tolerated dose based on a pre-test). Compared to the control groups, the treatment did not reduce survival and caused no clearly treatment-related non-carcinogenic or carcinogenic effects. The substance was irritating to the skin. The NOAEL of this study for systemic effects was approx. 400 mg/kg bw/day, but this dose is a LOAEL for local effects (Celanese/Kettering, 1985). Due to the limited range of examinations on non-carcinogenic toxicity endpoints this study is judged to be not reliable (RL3).

 

Inhalation exposure:

In accordance with REACH, Annex VIII, No. 8.6.1, column 2, repeated dose inhalation testing is not required

Justification for classification or non-classification

Classification criteria of (EC) 1272/2008 are not met.