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REACH

Carboxylic acids, C5-9

EC number: 271-676-4 | CAS number: 68603-84-9

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Description of key information

Valid dermal and inhalation studies are not available.
Systemic toxicity is low, as evidenced by NOAEL values up to 5000 mg expressed of octanoic or decanoic acid. These data were obtained in oral studies using triglycerides instead of the free acid.

Key value for chemical safety assessment

Toxic effect type:: dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference 1

Endpoint:

chronic toxicity: oral

Type of information:

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

Adequacy of study:

supporting study

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Hypothesis for the analogue approach: This read-across is based on the hypothesis that source and target substances have similar toxicological properties because, following oral intake, the source substances hydrolyse in the gut to non-common products predicted to have no toxicological effect (metabolic approach). The target substance is n-nonanoic acid, a saturated linear, medium-chain length carboxylic acid. The prediction is limited for saturated linear, medium-chain length carboxylic acids as source substances and for systemic toxicity endpoints, e.g. repeated dose toxicity and toxicity to reproduction. The prediction is supported by valid toxicological data on the substances and their hydrolysis products, based on known rapid and extensive hydrolysis and subsequent metabolism. For read across justification, see the atteched file in endpoint 13 ( Analogue approach justification): Pre-guideline study. Relevant methodological deficiencies compared to current test guidelines.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Pre-guideline study. A diet containing19.5% 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:

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

Remarks:

Doses / Concentrations:
19.5% MCT in diet
Basis:
nominal in diet

No. of animals per sex per dose:

Control animals:

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

Positive control:

not needed

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
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

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
- fatyt acids composition of body fat (carcass, liver, epiddidymal)
- histopthology: instestine, liver
- organ weights: liver, kidneys, spleen, heart, adrenals, femurs, testes, epididymal fat

Sacrifice and pathology:

GROSS PATHOLOGY: No data
HISTOPATHOLOGY: Yes
- histopthology: instestine, liver
- organ weights: liver, kidneys, spleen, heart, adrenals, femurs, testes, epididymal fat

Other examinations:

absortion of dietary fat

Statistics:

no data

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

at terminationin in week 47 slighty lower in rats fed MCT (ca. 5%)

Food consumption and compound intake (if feeding study):

not specified

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:

no effects observed

Description (incidence and severity):

no clear effect

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

only liver and intestine examined

Histopathological findings: neoplastic:

not specified

Critical effects observed:

not specified

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

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. The combined NOAEL for octanoic and decanoic acid was >8000 mg/kg bw and day. This result can be read across to nonanoic acid..

Executive summary:

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 onions available at the ECHA website.

Reference 2

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

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:

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

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

Remarks:

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

No. of animals per sex per dose:

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%

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.

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.

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'

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).

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) 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
Ingredient	Corn oil control	MCT control	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

Reference 3

Endpoint:

sub-chronic toxicity: oral

Type of information:

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

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

Hypothesis for the analogue approach: This read-across is based on the hypothesis that source and target substances have similar toxicological properties because, following oral intake, the source substances hydrolyse in the gut to non-common products predicted to have no toxicological effect (metabolic approach). The target substance is n-nonanoic acid, a saturated linear, medium-chain length carboxylic acid. The prediction is limited for saturated linear, medium-chain length carboxylic acids as source substances and for systemic toxicity endpoints, e.g. repeated dose toxicity and toxicity to reproduction. The prediction is supported by valid toxicological data on the substances and their hydrolysis products, based on known rapid and extensive hydrolysis and subsequent metabolism. For read across justificationsee the atteched file in endpoint 13 ( Analogue approach justification). Comparable to guideline studies with acceptable restrictions. Restrictions: no urinalysis conducted; limited histopathology compared to OECD 408).
See section 13 for the read across justification.

Reason / purpose for cross-reference:

read-across source

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:

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

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:

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

91 days

Frequency of treatment:

7 days/week

Remarks:

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

No. of animals per sex per dose:

Control animals:

yes, concurrent vehicle

other: MCT 11.21%

Details on study design:

Positive control:

MCT 11.21%

Observations and examinations performed and frequency:

Sacrifice and pathology:

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.

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):

Histopathological findings: neoplastic:

not examined

Details on results:

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'

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).

Critical effects observed:

not specified

Conclusions:

Executive summary:

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	-	-

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

Ingredient	Corn oil control	MCT control	Caprenin
Ingredient	Corn oil control	MCT control	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	-	-	-

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

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

Reference 4

Endpoint:: short-term repeated dose toxicity: oral
Type of information:: experimental study
Adequacy of study:: other information
Study period:: 1971
Reliability:: 3 (not reliable)
Rationale for reliability incl. deficiencies:: other: Relevant methodological deficiences
Principles of method if other than guideline:: Subacute feeding study, only one parameter examined (growth)
GLP compliance:: no
Limit test:: no
Species:: rat
Strain:: Wistar
Sex:: male
Route of administration:: oral: feed
Vehicle:: unchanged (no vehicle)
Analytical verification of doses or concentrations:: no
Duration of treatment / exposure:: 4 wks
Frequency of treatment:: continuous, via food
Remarks:: Doses / Concentrations:
4.17 % in diet (approx. 2100 mg/(kg bw*d)
Basis:
nominal in diet
No. of animals per sex per dose:: 8 males
Control animals:: other: diet with 1.95% propionic acid
Details on study design:: Post-exposure period: no
Dose descriptor:: NOAEL
Effect level:: ca. 2 100 mg/kg bw/day (nominal)
Sex:: male
Basis for effect level:: other: calculated from a concentration of 4.17% in diet, only parameter examined: growth
Critical effects observed:: not specified
Conclusions:: No definite conclusion can be drawn due to the limited range of examinations
Executive summary:: 8 male Wistar rats were fed a diet containing 4.17% of the test substance (purity not stated) for 4 weeks (body dose approx. 2100 mg/kg bw/day. The control group was represented by animals, fed with a diet containing 1.95% propionic acid. The treatment produced only a slight, not significant reduction in body weight gain (4%) compared to the controls. No further parameters were examined. Therefore the NOAEL of this study was approx. 2100 mg/kg bw/day (Dryden and Hartman, 1971).

Reference 5

Endpoint:: short-term repeated dose toxicity: oral
Type of information:: experimental study
Adequacy of study:: supporting study
Study period:: 1959
Reliability:: 3 (not reliable)
Rationale for reliability incl. deficiencies:: significant methodological deficiencies
Qualifier:: no guideline followed
Principles of method if other than guideline:: Five male rats were fed lauric acid at the 10% level of their diet for 18 weeks. A control group of 5 males was fed concurrently.
GLP compliance:: not specified
Limit test:: no
Species:: rat
Strain:: other: Albino rat of Osborne-Mendel strain
Sex:: male
Route of administration:: oral: feed
Details on route of administration:: The rats were housed in individual cages and were allowed food and water ad libitum.
Vehicle:: unchanged (no vehicle)
Details on oral exposure:: Diets were prepared i by blending the basal diet of a ground commercial rat biscuit with the test material so that the test material was the desired percentage by weight of the total diet (10%).
Analytical verification of doses or concentrations:: no
Dose / conc.:: 10 other: %
Remarks:: 10% by weight on total diet
No. of animals per sex per dose:: 5
Control animals:: yes, concurrent no treatment
Positive control:: No
Observations and examinations performed and frequency:: Albino rats of Osborne-Mendel strain weighing between 40 and 50 g were used in the experiments. All animals were randomized in the various experimental groups and their controls on a littermate basis except for the lauric acid study. The rats were housed in individual cages and were allowed food and water ad libitum. Animal weights were recorded weekly, and records of mortalities and abnormalities of the rats in regard to general physical condition, appearance, and behavior were kept. Complete blood counts were taken from 5 or more animals from each group toward the end of the experimental period. At the end of the test period, survivors were sacrificed by exsanguination and autopsied.
Sacrifice and pathology:: Organ weights and gross pathology were recorded for the sacrificed animals.
Other examinations:: None
Statistics:: None
Clinical signs:: no effects observed
Mortality:: no mortality observed
Body weight and weight changes:: no effects observed
Food consumption and compound intake (if feeding study):: no effects observed
Food efficiency:: not specified
Water consumption and compound intake (if drinking water study):: not examined
Ophthalmological findings:: not specified
Haematological findings:: not specified
Clinical biochemistry findings:: not examined
Urinalysis findings:: not examined
Behaviour (functional findings):: no effects observed
Immunological findings:: not examined
Organ weight findings including organ / body weight ratios:: no effects observed
Gross pathological findings:: no effects observed
Neuropathological findings:: not examined
Histopathological findings: non-neoplastic:: no effects observed
Histopathological findings: neoplastic:: no effects observed
Other effects:: not specified
Details on results:: Five male rats were fed lauric acid at the 10% level of their diet for 18 weeks. A control group of 5 males was fed concurrently. There were no observable clinical effects, no adverse effects on weight gain, nor was there any mortality. Gross organ pathology and comparison of individual organ weights showed no significant differences between the controls and test animals.
Dose descriptor:: conc. level:
Effect level:: > 10 other: % by weight on total diet
Based on:: test mat.
Sex:: male
Conclusions:: Five male rats were fed lauric acid at the 10% level of their diet for 18 weeks. A control group of 5 males was fed concurrently. There were no observable clinical effects, no adverse effects on weight gain, nor was there any mortality. Gross organ pathology and comparison of individual organ weights showed no significant differences between the controls and test animals.

Reference 6

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:

other: see 'Remark'

Qualifier:

no guideline followed

Principles of method if other than guideline:

Groups of 4 male and 4 female Beagle dogs were fed diets containg 0, 5, 10 or 15% of MCT for 90 days. The beagles were monitored for signs of
toxicity by clinical observations, body weight measurements, food consumption level, physical examin ations, hematology and serum chemistry, ophthalmlc examinations, and urinalysis

GLP compliance:

not specified

Limit test:

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;estimate: 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
- Concentration in vehicle: 0, 5, 10, 15%

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:

90 days

Frequency of treatment:

daily, 7 days/week

Remarks:

Doses / Concentrations:
0, 5, 10, 15%
Basis:
nominal in diet

No. of animals per sex per dose:

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

Positive control:

not required

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: Yes / No / No data

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

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
- Parametersexamined: 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.

GROSS PATHOLOGY: No
HISTOPATHOLOGY: No

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; reduce dfood 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 15% fed groups as compared to controls; nonsignificant increase of cholesterol in both sexes)

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

urinary volume decreased in the 10% and 15% groups

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Dose descriptor:

NOAEL

Effect level:

ca. 15 other: % in diet

Based on:

other: MCT

Sex:

male/female

Basis for effect level:

other: no adverse effect noted

Dose descriptor:

NOAEL

Effect level:

ca. 2 295 mg/kg bw/day (actual dose received)

Based on:

other: octanoic acid

Sex:

male

Basis for effect level:

other: NOAEL calculated. Males 2295 (1720-2870) mg octanoic acid/kg and day; females 3385 (2470-4300) mg octanoic acid/kg bw and day

Dose descriptor:

NOAEL

Effect level:

ca. 984 mg/kg bw/day (actual dose received)

Based on:

other: Decanoic acid

Sex:

male

Basis for effect level:

other: NOAEL calculated. Males 984 (738-1230) mg decanoic acid/kg and day; females 1451 (1058-1845) mg decanoic acid/kg bw and day

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.
For nonanoic acid a NOAEL(dog, oral feed, 90d) of > 2000 mg/kg bw and day was derived.

Executive summary:

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

> 2000 mg/kg bw and day. The true NOAEL may be up to 3000 or 5000 mg/kg bw and day (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.

Reference 7

Endpoint:

sub-chronic toxicity: oral

Type of information:

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

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Justification for type of information:

Hypothesis for the analogue approach: This read-across is based on the hypothesis that source and target substances have similar toxicological properties because, following oral intake, the source substances hydrolyse in the gut to non-common products predicted to have no toxicological effect (metabolic approach). The target substance is n-nonanoic acid, a saturated linear, medium-chain length carboxylic acid. The prediction is limited for saturated linear, medium-chain length carboxylic acids as source substances and for systemic toxicity endpoints, e.g. repeated dose toxicity and toxicity to reproduction. The prediction is supported by valid toxicological data on the substances and their hydrolysis products, based on known rapid and extensive hydrolysis and subsequent metabolism. For read across justificationsee the atteched file in endpoint 13 ( Analogue approach justification). Comparable to guideline studies with acceptable restrictions. Restrictions: no urinalysis conducted; limited histopathology compared to OECD 408).
See section 13 for the read across justification.

Reason / purpose for cross-reference:

read-across source

Qualifier:

no guideline followed

Principles of method if other than guideline:

GLP compliance:

not specified

Limit test:

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

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:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

no data

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily, 7 days/week

Remarks:

Doses / Concentrations:
0, 5, 10, 15%
Basis:
nominal in diet

No. of animals per sex per dose:

Control animals:

yes, concurrent vehicle

Details on study design:

Positive control:

not required

Observations and examinations performed and frequency:

Sacrifice and pathology:

Animals were not sacrificed at termination.

GROSS PATHOLOGY: No
HISTOPATHOLOGY: No

Statistics:

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; reduce dfood 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 15% fed groups as compared to controls; nonsignificant increase of cholesterol in both sexes)

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

urinary volume decreased in the 10% and 15% groups

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Dose descriptor:

NOAEL

Effect level:

ca. 15 other: % in diet

Based on:

other: MCT

Sex:

male/female

Basis for effect level:

other: no adverse effect noted

Dose descriptor:

NOAEL

Effect level:

ca. 2 295 mg/kg bw/day (actual dose received)

Based on:

other: octanoic acid

Sex:

male

Basis for effect level:

other: NOAEL calculated. Males 2295 (1720-2870) mg octanoic acid/kg and day; females 3385 (2470-4300) mg octanoic acid/kg bw and day

Dose descriptor:

NOAEL

Effect level:

ca. 984 mg/kg bw/day (actual dose received)

Based on:

other: Decanoic acid

Sex:

male

Basis for effect level:

other: NOAEL calculated. Males 984 (738-1230) mg decanoic acid/kg and day; females 1451 (1058-1845) mg decanoic acid/kg bw and day

Critical effects observed:

not specified

Conclusions:

Executive summary:

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

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

> 2000 mg/kg bw and day. The true NOAEL may be up to 3000 or 5000 mg/kg bw and day (comparable to the experimental results for octanoic and decanoic acid obtained in this study and described above).

Reference 8

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes

Specific details on test material used for the study:

Test substance octanoic acid, alternate name: n-caproic acid; English chemical name: octanoic acid, CAS No. 124-07-2, Ministerial Circular Publication Adjustment Number (Chemical Evaluation Act) :2-608, Fig.1]; chemical structure:CH3(CH2)6COOH, molecular weight: 144.21, physical properties, characteristics, extremely faint yellow clear liquid with distinctive odor; does not dissolve in water, readily dissolves in ethanol and acetone; ignition point: 109°C; density (20°C): 0.910g/mL

Species:

rat

Strain:

other: Crl:CD (SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

The types of animals used in this study were generally those used in toxicity tests and we used CrF(SD) female and male rats (SPF) on a clear systematic basis. We obtained 62 male and 42 female animals aged 7 weeks in the main test group on October 20, 2010 and 62 female parents aged 7 weeks as the mating group. The body weight range one 1 after we obtained them was 230 to 250 g for the males in the main test group, 165 to 193 g for the females in the main test group and 155 to 181 g for the female parents in the mating group.

The animals were bred at a set temperature of 23oC, a set humidity of 55 %, 12 hours in the light and in the dark (illumination: 6 AM to 6 PM), number of ventilations 12 times/hour (fresh air through neutral performance filter) in an animal breeding room (wing E, No. 10 room).

Route of administration:

oral: gavage

Details on route of administration:

Octanoic acid is thought to be potentially ingested by humans continually by mouth so that we selected an oral administration route.
Administration was carried out forcefully using a disposable polypropylene injection tube (Termo Ld.) to which was attached a disposable metal oral-gastric probe (Fuchigami Kikai Ltd.) for rats. When administration was carried out, we suctioned each administration specimen into the while stirring using a magnetic stirrer.

Vehicle:

methylcellulose

Details on oral exposure:

he test dose was 62.5, 250 and 1000 mg/kg/day. In the main test group, the dose was administered for 28 days, and a recovery period of 14 days was set. In the mating group, the dose was administered for 42 to 46 days: up to 14 days before mating, during the mating period, during the gestation period and day 4 of lactation.

Duration of treatment / exposure:

28days

Dose / conc.:

62.5 mg/kg bw/day (actual dose received)

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

There were 12 each males and females in the control group and the 62.5, 250 and 1000 mg/kg groups.

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

Confirmation of deaths and observation of the general conditions were carried out once a day and on the day of necropsy during the administration period and before administration and after administration (2-145 minutes after administration (when detailed observation was carried out during the recovery period and during the administration period twice a day, the general condition after administration was observed after the detailed observation (FOB) was terminated. However, when salivation was confirmed before making a detailed observation (FOB), we recorded this as general condition (see that this is not in accord with 15.2 Test Plan). When we carried out measurement of the amount of spontaneous motion, we observed the general condition after administration after we finished measuring the amount of spontaneous motion.

Mortality:

no mortality observed

Description (incidence):

Males During Administration Period:
There were no cases of deaths or moribund animals in any of the groups. After administration in the 1000 mg/kg group, there were 10 cases of transitory salivation. No aberrations in the general condition were noted in the 250 and 62.5 mg/kg group or in the control groups.

Females During Administration Period:
There were no cases of death or moribund animals in any of the groups. After administration in the 100 mg/kg group, there were 4 cases of transitory salivation in the 1000 mg/kg group. No aberrations in the general condition were noted in the 250 and 62.6 mg/g group or in the control groups.

Males During Recovery Period:
There were no cases of deaths or moribund animals in any of the groups.
No aberrations were seen in the general condition in any of the groups.

Females During Recovery Period:
There were no cases of deaths or moribund animals in any of the groups. No aberrations in the general condition were noted in the 1000 mg/kg group and in the control groups.

Mating Group Female Parents:
There were no cases of deaths, moribund animals, salivation or premature births in any of the groups. There were 6 cases of transitory salivation in the 1000 mg/kg group before mating started and during the mating period and 3 cases during the gestation period. There were no aberrations in the general conditions in the 250 and 62.5 mg/kg group and in the control groups before mating started, during the mating period, during the gestation period and during the lactation period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Males During Administration Period :
No significant difference was seen in the body weight on each measuring day in all of the administration groups compared to the control groups.

Females During Administration Period:
There were no significant differences in body weight on each measuring day for all of the administration groups compared to the control groups.

Males During Recovery Period:
There were no significant differences in body weight for all of the administration groups compared to the control groups.

Females During Recovery Period:
There were no significant differences body weight on each measuring day for the 1000 mg/kg group compared to the control group.

Mating Group Female Parents:
There were no significant differences in body weight on each measuring day for all of the administration groups compared to the control groups

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Males During Administration Period:
There were no significant differences in the amount ingested on each measuring day for all of the administration groups compared to the control groups.

Females During Administration Period:
No significant high values were seen in the 62.5 mg/kg group compared to the control groups, however, there were no changes which were dose dependent. As a result, there were no significant differences in the amount ingested on each measuring day in the 1000 and 250 mg/kg groups which could be thought to be toxicologically influential as compared to the control groups.

Males During Recovery Period:
There were no significantly high values in the amount ingested on day 2 of recovery in the 62.5mg/kg group compared to the control groups, however, there were no dose-dependent changes so that there were no significant differences in the amount ingested on each measuring day in the 1000 and 250 mg/kg group which could be thought to be toxicologically influential as compared to the control groups.

Females During Recovery Period:
The 1000mg / kg group showed a significantly lower food intake on the 5th day of recovery compared to the control group.
No abnormalities were observed on other measurement days, and this was not considered a toxic biological effect.

Mating Group Female Parents:
No significant differences were seen in the amount ingested on each measuring day for all of the administration groups compared to the control groups.

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

no effects observed

Description (incidence and severity):

Males During Administration Period:

No significant differences were seen in the amount of water ingested on each measuring day for all of the administration groups compared to the control groups.
Females During Administration Period:
No significant differences were seen in the amount of water ingested on each measuring day for all of the administration groups compared to the control groups.
Males During Recovery Period:
No significant differences were seen in the amount of water ingested on each measuring day in all of the administration groups compared to the control groups.
Females During Recovery Period:
No significant differences were seen in the amount of water ingested on each measuring day in the 1000 mg/kg group compared to the control groups.

Mating Group Female Parents:
No significantly high values were seen in the amount of water ingested on administration day 9 in the 1000 mg/kg group compared to the control groups, however, as no aberrations were noted in the main test group, there were no significant differences in the amount of water ingested on each measuring day in the 250 and 62.5 mg/kg group compared to the control group which could be thought to have a toxicological effect.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Males When Administration Period Terminated:
No significant difference was seen in any of the measuring items for any of the administration groups compared to the control groups.

Females When Administration Period Terminated:
No significant difference was seen in any of the measuring items for any of the administration groups compared to the control groups.

Males When Recovery Period Terminated:
No significant difference was seen in any of the measuring items in any of the administration groups compared to the control groups.

Female When Recovery Period Terminated:
The 1000 mg / kg group showed significantly higher MCHC than the control group, but because the difference from the control group was slight, the red blood cell count and hemoglobin level were unchanged, and because it was within the range of background data of the laboratory [MCHC: 36.1 Sat 0.5 (g / dL); Attachment 11] this was not thought to be affected by the test substance.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Males When Administration Period Terminated:
No significantly low values were seen in the urea nitrogen and no significantly high values were seen in the inorganic phosphorus in the1000 mg/kg group compared to the control group. No significant difference was seen in any of the measuring items in the 250 and 62.5 mg/kg groups compared to the control groups.

Females When Administration Period Terminated:
No significantly high potassium values were seen in the 1000 mg/kg group compared to the control groups. No significant difference was seen in any of the measuring items in the 250 and 62.5 mg/kg groups compared to the control groups.

Males When Recovery Period Terminated:
No significant difference was seen in any of the measuring items in any of the administration groups compared to the control groups.

Females When Recovery Period Terminated:
No significant difference was noted in any of the measuring items in the 1000 mg/kg group compared to the control groups.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Males When Administration Period Terminated:
There were no significant differences in urine volume or urine specific gravity in each treatment group compared to the control group.
The color tone, pH, protein, glucose, ketones, bilirubin, occult blood, urobilinogen and sediment were approximately the same for the control groups.

Females When Administration Group Terminated:
No significant difference was seen in the amount of urine and the urine specific weight for any of the administration groups compared to the control groups. The color tone, pH, protein, glucose, ketones, bilirubin, occult blood, urobilinogen and sediment were approximately the same for all of the administration groups.

Males When Recovery Period Terminated:
No significantly high values were seen in the urine specific weight in the 1000 mg/kg group compared to the control groups.
The color tone, pH, protein, glucose, ketones, bilirubin, occult blood, urobilinogen and sediment were approximately the each for all of the administration groups compared to the control groups.

Female When Recovery Period Terminated:
No significant differences were seen in the amount of urine and the urine specific weight in the 1000 mg/kg group compared to the control groups.
The color tone, pH, protein, glucose, ketones, bilirubin, occult blood, urobilinogen and sediment were approximately the same in the 1000 mg/kg compared to the control groups.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Males When Administration Period Terminated:
There were no significantly low values in the number of times of general standing up measured at 10 day intervals in the 62.5 mg/kg group compared to the control groups, however, there were no dose dependent changes so that these were not thought to be toxicologically influential.

Females When Administration Period Terminated:
There were no significant differences in any of the items for any of the administration groups compared to the control groups.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Males When Administration Period Terminated:
No significant difference was seen in the body weight on the day of necropsy in any of the administration groups compared to the control groups. No significant difference was seen in absolute body weight and relative body weight for any of the administration groups compared to the control groups.

Females When Administration Period Terminated:
No significant difference was seen in body weight on the day of necropsy in any of the groups compared to the control groups.
No significantly high values were seen in the absolute weight of the brain in the 62.5 mg/kg group compared to the control groups, however, as this was not a dose-dependent change, it was not thought to be toxicologically influential.
No significant difference was seen in absolute and relative organ weight in the 1000 and 250 mg/kg groups compared to the control groups.

Males When Recovery Period Terminated:
No significant difference was seen in the body weight on the day of necropsy in any of the administration groups compared to the control groups.
No significant difference was seen for the absolute and relative organ weight in any of the administration groups compared to the control groups.

Females When Recovery Period Terminated:
No significant differences were seen in body weight on the day of necropsy in the 1000 mg/kg group compared to the control groups. No significant differences were seen in the absolute body weight and relative body weight of the organs in the 1000 mg/kg compared to the control group.

Mating Group Female Parents:
There was no significant difference in body weight on the day of necropsy in any of the administration groups compared to the control groups. There was no significant difference in absolute weight and relative weight of the ovaries and uterus in any of the groups compared to the control groups.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Males When Administration Period Terminated:
Lungs: one-sided foreign matter granuloma was seen in 1 case. Stomach：hyperplasia of the anterior stomach squamous epithelium was seen in 6 case in the 1000 and 250 mg/kg groups and in 2 cases in the 62.5 mg/kg group. The degree was moderate in the 1000 mg/kg group, mild in the 250 mg/kg group and extremely mild in the 62. 5 mg/kg group. In addition, stomach ulcers were seen in 3 cases in the 250 mg/kg group and the degree was extremely mild. Of these findings, a significant difference in the hyperplasia of the anterior stomach squamous was confirmed in the 1000 and 250 mg/kg groups compared to the control groups and a dose responsiveness was confirmed.
Other changes were obtained in the following findings.
Pancreas: lobule atrophy was seen in 1 case in the control group.
Testes: there was 1 case of double-side atrophy of the seminiferous tubule at time of necropsy, however, this was a case in which there was a diminution and softening of the testes and a diminution of the epididymis.
Epididymis: there was 1 case of double-sided atrophy and cellular residue inside the lumen, however, this was in the control group. However, this was a case of diminution and softening of the testes and diminution of the epididymis during the necropsy.
Prostate gland ventral lobe: there was 1 case of cell infiltration in the control group and 2 cases in the 1000 mg/kg group. The 1 case in the control group was a case in which there was a diminution and a softening of the testes and a diminution of the epididymis during necropsy.
Pituitary gland: there was 1 case of a cyst in the 1000 mg/kg group.
Ophthalmus: there was 1 case of one-sided localized retinal atrophy in the 1000 mg/kg group.
Furthermore, the changes seen in the 1000 mg/kg group were findings which were usually observed in the control groups. There was no difference in the incidence with the control group and it was determined to be an incidental change. In addition, there were no aberrations seen in the heart, trachea, sublingual gland, submandibular gland, esophagus, duodenum, colon, ileum (including Peyer’s patch), the appendix, the intestine, the rectum, the thymus, the spleen, the submandibular lymph nodes, the mesenteric lymph nodes
the kidneys, the scrotal sac (including the hard gland), the adrenal gland, the thyroid (including the parathyroid gland), the cerebrum, the cerebellum, the pons, the spinal cord, the sciatic nerve, the Harderian gland, the sternum (including the medulla ossium), the femur (including the medulla ossium), the musculus rectus femoris and the mammary glands.

Females At Termination of Administration Period:
Stomach: hyperplasia on anterior stomach squamous epithelium was seen in all 5 animals in the 1000, 250 and 62.5 mg/kg group. The degree was moderate in the 1000 mg/kg group, mild in the 250 mg/kg group and very mild or mild in the 62.5 mg/kg group. In addition, an ulcer in the anterior stomach was seen in one animal in the 250 mg/kg group and the degree was mild. In these findings, hyperplasia of the anterior stomach squamous epithelium was confirmed at a significant difference compared to the control group in the 1000, 250 and 62.5 mg/kg groups and the dose reactivity was also confirmed.
In addition, no aberrations were seen in the 1000 mg/kg group and the control group in the heart, lungs, trachea, liver, pancreas, sublingual gland, submandibular gland, esophagus, duodenum, intestine, ileum (including Peyer’s patch), appendix, colon, rectum, thymus, spleen, submandibular lymph nodes, mesenteric lymph nodes, kidneys, bladder, ovaries, uterus, pituitary gland, adrenal gland, thyroid (including parathyroid), cerebrum, cerebellum, pons, spinal cord, sciatic nerve, eyeballs, Harderian gland, sternum (including medulla ossium), femur (including medulla ossium), musculus rectus femoris (including medulla ossium) and the mammary glands.

Males after Recovery Period Terminated:
Stomach: hyperplasia of the anterior stomach squamous epithelium was seen in 6 animals in the 1000 and 250 mg/kg group; the degree was very mild or mild in the 1000 mg/kg group and extremely mild in the 250 mg/kg group. The findings showed a significant difference compared to the control groups in the 1000 and 250 mg/kg groups and no aberrations were noted in the stomach in the 62.5 mg/kg group even for dose reactivity.

Findings were obtained as indicated below as other changes, however, these were determined to be one-sided incidental changes.
Testes: one-sided atrophy in the seminiferous tubule was seen in the 1000 mg/kg group, however, during the necropsy, this was a case where a diminution of the testes and the epididymis was seen.
Epididymis: Although one-sided atrophy was seen in one case in the 1000 mg/kg group, during the necropsy, this turned out to be a case where a diminution of the testes and epididymis was seen.

Females after Recovery Period Terminated:
Stomach: Hyperplasia of the anterior stomach squamous epithelium was observed in all 5 cases in the 1000 mg/kg group. The degree was mild or very mild.
This finding was significantly different for the 1000 mg/kg group compared to the control group.
In the control group, there were no abnormalities in the stomach.

Female Parents in Mating Group:
Stomach: Hyperplasia of the anterior stomach squamous epithelium was seen in 6 cases in the 1000 mg/kg group and the degree was mild or moderate. In addition, stomach ulcers were seen in 3 cases in the 1000 mg/kg group and the degree was extremely mild or mild. In the 1000 mg/kg group and the control groups, no aberrations were seen in the ovaries, uterus and mammary glands.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

no hyperplasia of the anterior stomach mucus membrane was seen in the males and females in the 1000 and 250 mg/kg groups and in the males and females in the 1000 mg/kg group when the administration period terminated.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: genital

Key result

Critical effects observed:

System:

male reproductive system

Key result

Critical effects observed:

System:

female reproductive system

Octanoic acid is one of the main constituents of this substance. The obtained results can be also applied to the target substance

Executive summary:

We carried out repeated toxicity and genital development toxicity combined tests on rats using octanoic acid and studied the general toxicological effect on female and male rats. At the same time, we studied the effect on sexual gland function, mating behavior, development of conceived animals and parturition and other genital development on female and male animals. The high dose was set at 1000 mg/kg/day, the medium dose was set at 250 mg/kg/day and the low dose was set at 62.5 mg/kg/day.

No toxicity caused by repeated administration resulting in death or moribund animals was noted in any of the male groups or in the groups of female parents.

Although there was salivation in both male, female parent groups in the 1000 mg/kg group as a change in general condition, these were noted only as transitory phenomena after administration and no weight in the salivary gland and no histopathological changes were noted such as clonic and tonic convulsions so that these were determined to be changes based on stimulus of the test substance and not toxic symptoms.. No changes were seen in body weight, amount ingested or amount of water ingested caused by the test substance in the male, female or female parent groups.

In detailed observation (FOB), no changes caused by the test substance were seen in the males, female or female adults. No changes caused by the test substance were seen in the females or males when the administration period terminated in the sensitivity response, the grip and the amount of spontaneous motion.

In urine tests, no high values were seen in urine specific weight in the male 1000 mg/kg group when the repeated administration period terminated. However, as there was a change in the range of the background data of the test institution [urine specific weight: 1.046 土 0.011; Attachment 16], this was not thought to be caused by the test substance.

In hematological tests, no changes caused by the test substance were seen in the females and males when the administration period terminated and when the repeated administration period terminated.

In biochemical tests, low values for urea nitrogen, high values for inorganic phosphorus were seen in the male 1000 mg/kg group and high values for potassium were seen in the female 1000 mg/kg group when the administration period terminated. However, in other tests, no findings related to these were obtained. Furthermore, the urea nitrogen and inorganic phosphorus in the male 1000 mg/kg group and the potassium in the female 1000 mg/kg group were thought to be mild changes as they were within the range of the background data of the test institution [urea nitrogen in males; 16.3 土2.0 (mg/dL), inorganic phosphorus in males: 7.5 土 0.7 (mg/dL); Attachment 12; potassium in females: 4.01 土 0.24; Attachment 13].

In measuring the hormones in the blood (T3, T4 and TSH), no changes caused by the test substance were seen in the females and males when the administration period terminated.

In the necropsies, no hyperplasia of the anterior stomach mucus membrane was seen in the males and females in the 1000 and 250 mg/kg groups and in the males and females in the 1000 mg/kg group when the administration period terminated. In histopathological tests, hyperplasia pf the anterior stomach squamous epithelium was seen in females and males in the 62.5 mg/kg group when the administration period terminated and dose reactivity was noted to that extent. The same type of histopathological findings were seen in the anterior stomach in the female parents in the 1000 mg/kg group. In addition, ulcers of the anterior stomach were seen in the males and females in the 250 mg/kg group and in the female parents in the 1000 mg/kg group when the administration period terminated. Octanoic acid has a pH of approximately 4 and is corrosive to the flesh11). Necrosis, sores and ulcers developed in the rat anterior stomach due to the strongly acidic corrosive substance. As time passes, reproductive hyperplasia occurs12) so that in this study, the findings seen in the anterior stomach are assumed to be a biological response to the irritating nature of the octanoic acid. However, no aberrations were seen with the naked eye in the anterior stomach for both females and males in the 1000 mg/kg group when the administration was suspended for 2 weeks. Histopathologically, although hyperplasia of the anterior stomach squamous epithelium was seen in males in the 1000 and 250 mg/kg groups, the degree was weak and there were no findings in the 62.5 mg/kg group. Although this was seen only in the 1000 mg/kg repeated administration female group, the degree of histopathological findings seen when the repeated administration terminated

was clearly weaker compared to when the administration period terminated. Based on the abovementioned necropsies and histopathological findings, the reaction relative to the irritation when the administration was suspended for 2 weeks diminished and become weaker and it is thought that this was due to the repeated tendency.

In terms of organ weight， no abnormalities due to the test substance were observed in males, females and female parents at the end of the administration period, and in males and females at the end of the recovery period.

In histopathological tests, aside from the abovementioned findings seen in the stomach, there were no changes brought about by the test substance for the females and males.

No changes involving genital development toxicity of the parent animals which were caused by the test substance were seen in the number of ruttings, mating rate, number of days required for mating, number of females conceiving, conception rate, gestation period, delivery rate, number of luteal bodies, number of nidations, nidation rate, parturition condition and lactation condition in the administration period (14 days) before mating began.

No changes among the animal young which were caused by the test substance were seen in the total number of births of animal young, number of deaths of animal young, number neonatals on lactation day 0, the parturition rate, the young animal delivery rate, the birth rate, the gender ratio, the number of neonatals on lactation day 4, the survival rate on lactation day 4, the general condition, the body weight on lactation days 0 and 4 and the epicuticle and necropsy findings. As indicated above, the non-effective dose of the octanoic acid is thought to be less than 62.5 mg/kg/day as hyperplasia of the anterior stomach squamous epithelium was confirmed for both females and males in the 62.5 mg/kg group. A genital developmentally toxicologically non-effective dose was confirmed to be influential in all of the items for both females and males in the 1000 mg/kg group so that it is thought to be 1000 mg/kg/day.

The non-effective dose on the animal young was such that no effect was seen in any of the items in the 1000 mg/kg group so that the non-effective dose is believed to be 1000 mg/kg/day.

Endpoint conclusion

Endpoint conclusion:: no adverse effect observed
Dose descriptor:: NOAEL
: 2 000 mg/kg bw/day
Study duration:: subchronic
Species:: rat
Quality of whole database:: sufficient
Estimated NOAEL from Matulka study for octanoic and nonanoic acid (see the attached document by GUBDH Dr. W. Hillesheim, 2014-04-30

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:: no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:: no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:: no study available

Additional information

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

Oral exposure:

Read-across from Matulka (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

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

> 2000 mg/kg bw and day. The true NOAEL may be up to 3000 or 5000 mg/kg bw and day (comparable to the experimental results for octanoic and decanoic acid obtained in this study and described above).

Read across from Webb (1993)

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	-	-

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

Ingredient	Corn oil control	MCT control	Caprenin
Ingredient	Corn oil control	MCT control	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	-	-	-

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, Carboxylic acids, C5 -9, 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 the target substance 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

Read-across from Harkins (1968)

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

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 the target substance. 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.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Subchronic rat study with high systemic dose of surrogates:estimated NOAEL up to 5074 mg/kg/d for octanoic acid and up to 3122 mg/kg/d for decanoic acid. Read-across to Nonanoic acid: 5074 mg/kg/d

Justification for classification or non-classification

Classification criteria of (EC) 1272/2008 and Council Directive 67/548/EEC are not met.

Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.

Registration Dossier

Registration Dossier

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Diss Factsheets

Carboxylic acids, C5-9

Endpoint summary

Administrative data

Description of key information

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

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Endpoint conclusion

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Additional information

Justification for classification or non-classification

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