Carboxylic acids, C5-9

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Fd Chem. Tox. Vol 31, No 12. Year 1993

Author:

Webb D.R., Wood F.E., Bertram T.A. and Fortier N.E.

Year:

1993

Bibliographic source:

Fd Chem. Tox. Vol 31, No 12. Year 1993

Reference 2

Reference Type:

publication

Title:

Estimation of NOAEL values for fatty acids from Caprenin rat feeding experiments

Author:

Hillesheim W

Year:

2014

Bibliographic source:

GUBDH Consultancy

Report date:

2014

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Caprenin

IUPAC Name:

Caprenin

Test material form:

not specified

Details on test material:

- Name of test material (as cited in study report): caprenin
- Molecular formula (if other than submission substance): triglyceride; fatty acid composition

Fatty acid composition of Caprein, MCT, and vehicle corn oil (% w/w) (Webb et al. 1993)

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 - -




- Molecular weight (if other than submission substance): approx. ; calculated from fatty acid composition

- Substance type: triglyceride
- Physical state: no data
- Analytical purity: analytical results see table above
- Impurities (identity and concentrations): analytical results see table above
- Composition of test material, percentage of components: analytical results see table above
- Stability under test conditions: no data; fresh diets were prepared weekly
- Storage condition of test material: no data

Test animals

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;
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


- 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

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: semi-purified corn oil control diet (min3% corn oil; contained mineral an dvitamine mix, carbohydrates, protein; publication table 4)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): weeks
- Mixing appropriate amounts with (Type of food): semi-purified diet a sdescribed above
- 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.

VEHICLE
- Justification for use and choice of vehicle (if other than water): diet is the vehicle of choice in feeding studies
- Concentration in vehicle: 5.23, 10.23, and 15.0% (w/w)
- Purity: cf. publication, tables 3 and 4

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

91 days

Frequency of treatment:

7 days/week

Doses / concentrations

Remarks:

Doses / Concentrations:
5.23, 10,23, 15% (w/w)
Basis:
nominal in diet

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.
- Rationale for animal assignment (if not random): n.a.
- Rationale for selecting satellite groups: n.a.
- Post-exposure recovery period in satellite groups: n.a.
- Section schedule rationale (if not random): n.a.

Positive control:

MCT 11.21%

Examinations

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

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data
- Time schedule for examinations:

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: Yes / No / No data
- How many animals: 20/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, perirenl 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 list provided . liver, heart, kidneys, , bone marrow, possibly more organs (see above)

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.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

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

Mortality:

no mortality observed

Description (incidence):

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

Body weight and weight changes:

no effects observed

Description (incidence and severity):

no statistical significant diferences in body weight gain at termination

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

in both sexes increased with caprenin dose, compared to corn oil and MCT controls

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

decreased in both sexes at the top caprenin dose, compared with corn oil and MCT controls

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):

marginal changes were seen

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

several statistical significant changes, but mild with no toxicological relevance

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

relative colon weight stat. signif. increased in both sexes, but not considered to be of toxicological relevance

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

significantly increased number of granular/rough kidneys in high dose females (9/20) compared to corn oil controls (9/20). No histopathological correlate noted.

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

Details on results:

CLINICAL SIGNS AND MORTALITY
No clinical signs noted. One corn oil control male and one high-dose female died in wk. 6 an wk. 8 respectively. Deaths were not related to treatment.

BODY WEIGHT AND WEIGHT GAIN
no statistical significant differences in body weight gain across groups at termination.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
in both sexes increased with caprenin dose, compared to corn oil and MCT controls

FOOD 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).


OPHTHALMOSCOPIC EXAMINATION
No effect in either group

HAEMATOLOGY
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 CHEMISTRY
Caprenin treatment: 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.

MCT treatment: similarly, several statistically significant changes were noted compared to corn oil controls. Examples include glucose, AP, total protein, albumin, phosphorous, calcium, and triglycerides. With the exception of triglycerides the magnitude was generally low and within the historical control range. Hence, the changes were not considered to represent toxicological responses.

URINALYSIS no data

NEUROBEHAVIOUR no data

ORGAN WEIGHTS
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 gained statistical significance dose especially in males. 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 relationship to treatment, 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 PATHOLOGY
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.

HISTOPATHOLOGY: NON-NEOPLASTIC
Vacuolisation of the liver was seen in all treated and control rats. This was considered as a common finding in rats maintained at high-fat, semi-purified diets (Webb eta. 1991). Nephrocalcinosis was also seen in all rats, but there was no histopathological correlate that demonstrated a relationship to treatment.

OTHER FINDINGS
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.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: see 'Remark'

2

Dose descriptor:

NOAEL

Effect level:

11.21 other: % (w/w) in diet

Based on:

other: MCT;

Sex:

male/female

Basis for effect level:

other: Absence of advers effects. During metabolism, MCT liberates octanoic (content 70.6%, w/w) and decanoic acid (content 25.9%, w/w).

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

 

 

 

 

Applicant's summary and conclusion

Conclusions:

No adverse effect was seen in a subacute feeding study using male and female rats exposed to caprenin (up to 15% in diet) and MCT (11.21% in diet).
For nonanoic acid NOAEL values of >3000 and 5000 mg/kg bw and day were derived from experiments using two different types of triglycerides.Hence, the NOAEL(rat, oral feed, 90d) for nonanoic acid is set at > 3000 mg/kg bw and 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 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 (5700 mg/kg bw and day) and decanoic acid (3100 mg/kg bw and day) of the animals fed Caprenin or MCT it is concluded that the NOAEL(rat, oral feeed, 90d) for nonanoic acid is > 3000 mg/kg bw and day. 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 or C10 acid)	3122 mg/kg bw/day	5074 mg/kg bw/day