Octanoic acid, monoester with glycerol

Endpoint summary

Description of key information

Studies on oral repeated dose toxicity were available for the following Category members (CAS No.):

73398-61-5, 8001-79-4, 91845 -19-1 and for medium- and long-chain triglyceride mixtures.

All available studies resulted in oral NOAELs of 1000 mg/kg bw/d or greater than 1000 mg/kg bw/d.

Studies on dermal repeated dose toxicity were available for the following Category member (CAS No.): 73398-61-5.

A subacute (28 days) dermal NOAEL of 2000 mg/kg bw/d for rabbits was reported.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Basic data given.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

lack of details on test substance, only limited examination parameters

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 120 - 150 g
- Housing: 2 animals
- Diet (e.g. ad libitum): standard pellet diet (ssniff R, Intermast GmbH, Germany)
- Water (e.g. ad libitum): yes

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

30 days

Frequency of treatment:

once a day

Remarks:

Doses / Concentrations:
test group I: 1 mL / rat
Basis:
actual ingested

Remarks:

Doses / Concentrations:
test group II: 3 mL/rat
Basis:
actual ingested

Remarks:

Doses / Concentrations:
test group I: 3.58 - 7.6 mL/ kg bw
Basis:
actual ingested

Remarks:

Doses / Concentrations:
test group II: 10.8 - 21.3 mL/kg bw
Basis:
other: actual ingested, equivalent to 10206-20128.5 mg/kg bw/d

No. of animals per sex per dose:

10

Control animals:

yes, concurrent no treatment

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes, daily

BODY WEIGHT: Yes
- Time schedule for examinations: every 8 days

FOOD CONSUMPTION: Yes

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of study
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters checked: Protein, Glucose, Urobilinogen, Sediment

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, at the end of the study
HISTOPATHOLOGY: Yes (see table) / No / No data

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):

effects observed, treatment-related

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
Experimental group I: no signs of toxicity, no mortality
Experimental group II: Within the first days the animals showed reduced food consumption, softened faeces, ruffled fur. These clinical signs disappeared after a few days of adaption.

BODY WEIGHT AND WEIGHT GAIN
Experimental group I: average body weights increased from 130 to 279 g (control group: 125 g - 256 g).
Experimental group II: average body weights increased from 140 to 275 g (control group: 125 g - 256 g).

FOOD CONSUMPTION:
Experimental group I: 19-21 g / day - no significant differences to control group.
Experimental group II: 17-19 g / day

URINALYSIS
Experimental group I: E. coli and tripel phosphates found
Experimental group II: E. coli and tripel phosphates found,
Urine was negative for protein, glucose and urobilinogen.

GROSS PATHOLOGY
Experimental group I: no abnormalities, no signs of inflammation in the intestinal mucosa
Experimental group II: no abnormalities found.

Dose descriptor:

NOAEL

Effect level:

ca. 10 000 mg/kg bw/day (actual dose received)

Sex:

male

Basis for effect level:

other: based on clinical signs, body weight increase, food consumption, urine analysis and gross pathology

Critical effects observed:

not specified

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Basic data given. Dosing concentration unclear.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

lack of details on test substance, only limited examination parameters.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 120 - 150 g
- Housing: 2 animals
- Diet: standard pellet diet (ssniff R, Intermast GmbH, Germany), ad libitum
- Water: ad libitum

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily, 7 days/week

Remarks:

Doses / Concentrations:
10000 and 50000 ppm
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
approx. 1000 and 5000 mg/kg bw/day
Basis:
other: assuming a mean body weight of 200 g and a mean food intake of 20 g/day over the study period

No. of animals per sex per dose:

20

Control animals:

yes, plain diet

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes, daily

BODY WEIGHT: Yes
- Time schedule for examinations: every 14 days

FOOD CONSUMPTION: Yes

URINALYSIS: Yes
- Time schedule for collection of urine: at the middle and end of study period
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters checked:

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the middle and end of study period
- Animals fasted: No data
- How many animals: all animals
- Parameters checked: GOT, GPT, determination of free fatty acids and fatty acid esters

ORGAN WEIGHTS: Yes

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: No

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 examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
No signs of systemic toxicity and no deaths occured.

BODY WEIGHT AND WEIGHT GAIN
No significant differences in the body weight gain of treated and control animals occured.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Food consumption increased from 15 - 17 g/ day to 20 - 22 g/ day during the study period and did not differ significantly from the untreated control group.

HAEMATOLOGY
Hemoglobin, Erythrocyte and Leukocyte parameters did not differ significantly from the control group.

CLINICAL CHEMISTRY
GOT and GPT values did not differ significantly from the control group.
Blood contents on free fatty acids and fatty acid esters did not differ significantly from the control group, indicating that the treatment did not influence the lipid metabolism.

URINALYSIS
The urine did not contain protein, glucose or urobilinogen. pH and urine sediment analysis were not changed.

ORGAN WEIGHTS
No significant differences between treated and control groups observed.

GROSS PATHOLOGY
No abnormalities observed.

Dose descriptor:

NOAEL

Effect level:

5 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

Reference 3

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

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

Adequacy of study:

supporting study

Study period:

February 17th, 2010 - April 7th, 2010 (end of in-life phase)

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP - Guideline study, tested with the source substance Glycerides, C8-18 and C18-unsatd. mono- and di-, acetates (CAS 91052-13-0). In accordance to the ECHA guidance document ¿Practical guide 6: How to report read-across and categories (March 2010)¿, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance. Read-across from Glycerides, C8-18 and C18-unsatd. mono- and di-, acetates (CAS No. 91052-13-0) for systemic mammalian toxicity endpoints was judged to be justified for the following reasons: This read across substance is also a glyceride, containing mainly C12-14 fatty acid and acetate moiety as well as glycerol. It¿s an organic liquid with a pour point of -8 °C and a melting point of 357.85 °C and a vapour pressure < 0.01 Pa at room temperature. In contrast to the glycerides of the fatty acid glyceride category, it has a higher water solubility of 8.75 mg/L, which might influence its environmental distribution, but not the mammalian metabolism upon systemic uptake. Therefore it is expected to feed into the same mammalian physiological pathways as the members of the fatty acid glyceride category, like citric acid cycle, sugar synthesis and lipid synthesis.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

After acclimatisation, four Main groups of 10 male and 5 female Wistar Han rats were exposed by oral gavage to the test substance at 0, 100, 300 and 1000 mg/kg/day. An additional 5 Recovery group males and females in the control and high dose group were allowed 14 days of recovery.
An additional 10 females were added to each group for the assessment of reproduction and developmental toxicity. Main and Recovery animals were exposed for at least 28 days from start of treatment up to termination or start of recovery. Females used for the assessment of reproduction/developmental toxicity were exposed for 41-49 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:WI(Han) (outbred, SPF-Quality)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories France, L'Arbresle Cedex, France.
- Age at study initiation: Approximately 12 weeks.
- Weight at study initiation:
- Fasting period before study:
- Housing : 5 animals/sex/cage in Macrolon cages (MIV type, height 18 cm). This was also applicable for Main females and Recovery animals throughout the complete treatment period. Sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap-water.
- Acclimation period: At least 5 days prior to start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3°C (actual range: 19.7 - 21.9°C)
- Humidity (%): 40 - 70% (actual range: 22 - 71%)
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. NOTOX BV has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level.
Adjustment was made for the density of the test substance (0.944), and the specific gravity of the vehicle (1.125).

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at NOTOX.
- Purity: polyethylene glycol 400, specific gravity 1.125 (Merck, Darmstadt, Germany)

Dose volume: 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chemical analyses of formulations were conducted once during the study to assess accuracy, homogeneity and stability. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% for suspensions. Homogeneity was demonstrated if the coefficient of variation was ¿ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
The concentrations analyzed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ¿ 10%) and formulations at the entire range were stable when stored at room temperature for at least 6 hours.

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily for 7 days per week

Remarks:

Doses / Concentrations:
0, 100, 300 and 1000 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

10 male and 5 females
(additionally 5 male and females in the recovery groups dosed with 0 and 1000 mg/kg bw/d)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the results of a 10-Day dose range finding study
- Post-exposure recovery period in satellite groups: 14 days

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once prior to start of treatment and at weekly intervals this was also performed outside the home cage in a standard arena. Arena observations were conducted during the treatment phase only. Arena observations were not performed when the animals were mating, or housed individually. The time of onset, degree and duration was recorded. All symptoms were recorded and graded according to fixed scales:
Maximum grade 1: grade 0 = absent, grade 1 = present
Maximum grade 3 or 4: grade 1 = slight, grade 2 = moderate, grade 3 = severe, grade 4 = very severe

BODY WEIGHT: Yes
Males and females were weighed on the first day of exposure and weekly thereafter. Mated Repro females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum, and during lactation on Days 1 and 4.

FOOD CONSUMPTION: Yes
Weekly, for males and females. Food consumption was not recorded during the mating period (except for males). Food consumption of mated Repro females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to scheduled post mortem examination
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes, overnight
- How many animals: Blood samples were collected from the first 5 Main males (randomly selected at allocation), all Recovery animals and the 5 Main females from each group. In addition to blood collection prior to necropsy, blood samples were collected from the Recovery animals at the end of treatment.
- Parameters checked: White blood cells, Differential leucocyte count, neutrophils, lymphocytes, monocytes, eosinophils, basophils, Red blood cells, Reticulocytes, Red blood cell distribution width, Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelets, Prothrombin time, Activated Partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to scheduled post mortem examination
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes, overnight
- How many animals: Blood samples were collected from the first 5 Main males (randomly selected at allocation), all Recovery animals and the 5 Main females from each group. In addition to blood collection prior to necropsy, blood samples were collected from the Recovery animals at the end of treatment.
- Parameters checked: ALAT, ASAT, ALP, Total Protein, Albumin, Total Bilirubin, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate, Bile acids

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
The following tests were performed on the first Main males (randomly selected at allocation), all Recovery animals and the 5 Main females from each group:
- hearing ability, pupillary reflex, static righting reflex and grip strength (score 0 = normal/present, score 1 = abnormal/absent).
- motor activity test (recording period: 1-hour for individual animals, using a computerized monitoring system; Pearson Technical Services, Suffolk, Great Britain).
During the motor activity test, animals were caged individually. The assigned animals were tested during week 4 of treatment (all before blood sampling). Since no treatment-related findings were noted, the functional observation tests and motor activity measurements were not extended to all animals at the end of the recovery phase.

ORGAN WEIGHTS:
The following organ weights and terminal body weight were recorded from the following animals on the scheduled day of necropsy:

- From the first 5 Main males (randomly selected at allocation), the 5 Main females and all
Recovery animals per group:
Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Spleen, Testes, Thymus, Ovaries, Uterus (including cervix), Prostate, Seminal vesicles including coagulating glands, Thyroid including parathyroid.

- From all remaining males:
Epididymides, Testes

Since no toxicologically relevant effect was noted on organ weights of Main females, no organ weights were collected from Repro females.

Sacrifice and pathology:

Day of necropsy:
- Main animals: Following completion of a minimum of 28 days of dose administration.
- Recovery animals: Following completion of a minimum of 28 days of dose administration and a recovery period of 14 days.
One animal was euthanized in extremis.

GROSS PATHOLOGY: Yes, all animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.

HISTOPATHOLOGY: Yes

- from the first 5 Main animals/sex/group (randomly selected at allocation), all Recovery animals, the selected Repro females/group (with live offspring) and animal no.117 that was killed in extremis (Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination):

Adrenal glands, (Aorta), Brain (cerebellum, mid-brain, cortex), Caecum, Cervix, Clitoral gland, Colon, Duodenum, Epididymides, Eyes (including optic nerve and Harderian gland), Female mammary gland area, Femur including joint, Heart, Ileum, Jejunum, Kidneys, (Lacrimal gland, exorbital), (Larynx), Liver, Lung, infused with formalin, Lymph nodes - mandibular, mesenteric, (Nasopharynx), (Oesophagus), Ovaries, (Pancreas), Peyer's patches (jejunum, ileum) if detectable, Pituitary gland, Preputial gland, Prostate gland, Rectum, (Salivary glands - mandibular, sublingual), Sciatic nerve, Seminal vesicles including coagulating gland, Skeletal muscle, (Skin), Spinal cord -cervical, midthoracic, lumbar, Spleen, Sternum with bone marrow, Stomach, Testes1, Thymus, Thyroid including parathyroid (if detectable), (Tongue), Trachea, Urinary bladder, Uterus, Vagina, All gross lesions.

- from all remaining animals and females which failed to deliver:

Cervix, Clitoral gland, Coagulation gland, Epididymides, Ovaries, Preputial gland, Prostate gland, Seminal vesicles, Testes, Uterus, Vagina, All gross lesions.

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
An attempt was made to transform the number of corpora lutea by using 1/x, log x, x2 and ¿x.
However, a normal distribution was not obtained. Therefore, the number of corpora lutea was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine inter-group differences, followed by the Wilcoxon test to compare the treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
No statistical analysis was performed on histopathology findings.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

non-adverse effects

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

non-adverse effects

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

non-adverse effects

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

non-adverse effects

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

non-adverse effects

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

non-adverse effects

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

non-adverse effects

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS
No clinical signs of toxicity were noted that were attributable to treatment.

MORTALITY
No mortality occurred during the study period that was considered to be related to treatment with the test substance. Female no. 117 (1000 mg/kg/day) was killed in extremis on Day 17 post-coitum. Microscopic examination revealed a marked granuloma in the bronchus region containing macrophages surrounding food particles and with central areas of necrosis. These findings were indicative of a gavage accident.

BODY WEIGHT AND WEIGHT GAIN
No toxicologically relevant changes in body weights and body weight gain were noted up to 1000 mg/kg.
Any statistically significant changes in body weight gain observed among males and females during the treatment or recovery period were considered to be of no toxicological relevance since the changes occurred in the absence of a dose-related trend and/or were of a slight and/or temporary nature. These changes consisted of a higher body weight gain for males at 1000 mg/kg/day during the recovery phase, a lower body weight gain of Repro females at 1000 mg/kg/day on Day 11 of the post coitum period, and a higher body weight gain of Main females at 100 mg/kg/day over treatment Days 8-22.

FOOD CONSUMPTION
Food consumption before or after allowance for body weight was similar between treated and control animals.

HAEMATOLOGY
No toxicologically relevant changes occurred in haematological parameters of treated rats.
The statistically significant lower prothrombin time (PT) in males at 1000 mg/kg/day and lower relative lymphocyte counts in females at 300 mg/kg/day at the end of treatment were considered to be of no toxicological relevance. These changes were absent at the end of the recovery period, occurred in the absence of a (clear) treatment-related trend and remained within the range considered normal for rats of this age and strain.

CLINICAL CHEMISTRY
No toxicologically relevant changes occurred in clinical biochemistry parameters of treated rats.
Any statistically significant changes in clinical biochemistry parameters were considered to be of no toxicological relevance as these occurred in the absence of a (clear) treatment-related trend, remained within the range considered normal for rats of this age and strain and/or were present at the end of the recovery period only. At the end of treatment these changes consisted of lower alanine aminotransferase in males at 300 and 1000 mg/kg/day, and higher chloride levels in males at 100 mg/kg/day. Changes at 1000 mg/kg/day at the end of the recovery phase included lower albumin and higher glucose levels in females, and higher inorganic phosphate levels in males.

NEUROBEHAVIOUR
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals.
The variation in motor activity did not indicate a relation with treatment. Males at 300 mg/kg had significantly lower high sensor counts. Since the range of high sensor values encountered at 300 mg/kg/day was similar to that observed in the control group and no dose-related trend was noted, no toxicological relevance was ascribed to this variation. A notable variation in high sensor counts was recorded for females at 1000 mg/kg/day. Since the range of values at this dose was essentially similar to that observed in the control group, it was considered that no toxicologically significant effect on high sensor counts had occurred in females at 1000 mg/kg/day.

ORGAN WEIGHTS
No toxicologically relevant changes were noted in organ weights and organ to body weight
ratios.
Any statistically significant changes in organ weights and organ to body weight ratios were considered to be of no toxicological relevance as these occurred in the absence of a (clear) treatment-related trend, remained within the range considered normal for rats of this age and strain and/or were present at the end of the recovery period only. Also, no histopathological correlates were noted to support these changes. These changes consisted of a lower spleen to body weight ratio in males at 1000 mg/kg/day at the end of the recovery phase, a higher spleen weight and spleen to body weight ratio in females at 100 mg/kg/day and the end of treatment, lower heart weight in females at 1000 mg/kg/day, higher heart to body weight ratio in females at 1000 mg/kg/day at the end of treatment, lower adrenal weight and/or adrenal to body weight ratio in females at 300 and 1000 mg/kg/day at the end of treatment, and a lower ovary and ovary to body weight ratio in females at 1000 mg/kg/day at the end of treatment. Other organ weights and organ to body weight ratios among the dose groups were similar to control levels.

GROSS PATHOLOGY
Macroscopic observations at necropsy did not reveal any toxicologically relevant alterations.
The female at 1000 mg/kg/day euthanized in extremis (no. 117) showed a hard, greenish nodule on the right medial lobe of the lung, dark red discoloration of the left ovary, enlarged adrenal glands, reduced size of the thymus, enlargement and greenish discoloration of the bronchial lymph node and pleura grown together with the lungs. A watery-clear cyst was found for a single control Repro female (no. 89). This finding was corroborated with a cyst found in the cervix found upon histopathological examination, which likely contributed to this animal¿s suspected infertility.
Other incidental findings among control and treated animals at the end of the treatment and/or recovery period included alopecia, red foci on the thymus, reddish discolouration of the thymus or mesenteric lymph node, pelvic dilation of the kidney, reduced size of the testes, epididymides or seminal vesicles, yellowish hard nodules, a red-brown focus or tan discolouration of the clitoral glands, and fluid in the uterus. The incidence of these findings remained within the background range of findings that are encountered among rats of this age and strain, and the their incidence did not show a dose-related trend. These necropsy findings were therefore considered to be of no toxicological relevance.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no treatment-related microscopic findings.
One control female (no. 89) had a marked cyst present in the cervix which correlated to the macroscopic finding in this animal and likely accounted for the infertility. One male rat at 100 mg/kg/day (no. 18) had an extensive bilateral seminiferous tubular atrophy in the testes with a subsequent extensive epididymal oligospermia, which accounted for its infertility. This was considered to be a spontaneous abnormality with no likely relationship to the test item. No other abnormalities were seen in the reproductive organs of the remaining suspected nonfertile animals which could account for their infertility. All microscopic findings recorded were considered to be within the normal range of background pathology encountered in Wistar-Han rats of this age and strain. The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis.

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: clinical signs, functional observations, body weights, food consumption, clinical pathology, macroscopy, organ weights, and histopathology.

Critical effects observed:

not specified

Reference 4

Endpoint:

chronic toxicity: oral

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Study period:

04 Nov 2008 - 12 Feb 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP - Guideline study. According to the ECHA guidance document "Practical guide 6: How to report read-across and categories (March 2010)", the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Qualifier:

according to guideline

Guideline:

OECD Guideline 452 (Chronic Toxicity Studies)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.4100 (Chronic Toxicity)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.30 (Chronic Toxicity Studies)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF, Testing Guidelines for Toxicology Studies (2-1-14), 12 NohSan No.8147, November 24, 2000

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Wistar HsdHan™:WIST

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Oxon, UK
- Age at study initiation: 6 weeks
- Weight at study initiation: 178-234 g (males) and 138-183 g (females)
- Housing: animals were housed in groups of up to 3 by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). Environmental enrichment was provided in form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK).
- Diet: ground diet (Rat and Mouse SQC Ground Diet No.1, Special Diet Services Ltd, Witham, UK), ad libitum
- Water: mains drinking water, ad libitum
- Acclimation period: at least 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 14
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet: prior to treatment and then twice monthly
- Mixing appropriate amounts with: basal laboratory diet
- Storage of food: stored in labelled, double plastic bags in labelled, covered plastic bins

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples were taken of each dietary admixture and were analysed for concentration twice monthly for the first two months, monthly for the next two months and thereafter every three months. The concentration of the test material in the dietary admixtures was determined by gas chromatography using an external standard technique. The results indicate that the mean prepared dietary admixture concentrations were within ± 18% of the nominal concentration.

Duration of treatment / exposure:

52 weeks

Frequency of treatment:

daily, 7 days/week

Remarks:

Doses / Concentrations:
1500, 6000 and 15000/25000 (from Week 10)/30000 (from Week 41) ppm
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
98, 392 and 1333 mg/kg bw/day
Basis:
other: Group mean achieved dose level calculated on actual values for food consumption and body weight with calculated test material intake.

Remarks:

Doses / Concentrations:
87.1, 348.5 and 1116.5 mg/kg bw/day
Basis:
other: Group mean achieved dose level of males calculated on actual values for food consumption and body weight with calculated test material intake.

Remarks:

Doses / Concentrations:
109.3, 435.0 and 1549.8 mg/kg bw/day
Basis:
other: Group mean achieved dose level of females calculated on actual values for food consumption and body weight with calculated test material intake.

No. of animals per sex per dose:

21

Control animals:

other: diet treated with Arachis oil to achieve comparable calorific intake

Details on study design:

- Dose selection rationale: The dietary concentrations were chosen based on toxicity data and consultation with the Study Sponsor (no further information).
- Dietary concentration: In order to achieve a high dose level that approximated a test material intake equivalent to 1000 mg/kg bw/day, the dietary concentration of the test material in the diet was reviewed and periodically adjusted. High-dose concentrations were changed at Week 10 (25000 ppm) and Week 41 (30000 ppm).

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once per week

BODY WEIGHT: Yes
- Time schedule for examinations: at Day 1 and at weekly intervals for the first 13 weeks and subsequently, every 4 weeks until study termination.

FOOD CONSUMPTION: Yes
- Food consumption for each cage group was determined at weekly internals from Week 1 to Week 13 and subsequently for one week in each four week period until termination.

FOOD EFFICIENCY: Yes
- Food efficiency and chemical intake was calculated for the food consumption periods.

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily, for each cage group by visual inspection of the water bottles for any overt changes.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to testing and during Week 50
- Dose groups that were examined: all animals prior to testing and 10 females and 10 males from the control and high-dose groups during Week 50 were observed.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at 3, 6 and 12 months
- How many animals: 10 females and 10 males from each group
- Parameters checked: Haemoglobin (Hb), Erythrocyte count (RBC), Haematocrit (Hct), Erythrocyte indices, Total leucocyte count (WBC), Differential leucocyte count, Neutrophils (Neut), Lymphocytes (Lymph), Monocytes (Mono), Eosinophils (Eos), Basophils (Bas), Platelet count (PLT), Reticulocyte count (Retic), Prothrombine time (CT), Activated partial thromboplastin time (APPT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at 6 and 12 months
- How many animals: 10 females and 10 males from each group
- Parameters checked: Urea, Glucose, Total protein (Tot. Prot.), Albumin, Albumin/Globulin (A/G) ratio (by calculation), Sodium (Na+), Potassium (K+), Chloride (Cl-), Calcium (Ca++), Inorganic phosphorus (P), Aspartate aminotransferase (ASAT), Alanine aminotransferase (ALAT), Alkaline phosphatase (AP), Creatinine (Creat), Total cholesterol (Chol), Total bilirubin (Bili), Triglycerides (Tri), Gamma glutamyltranspeptidase (γGT)

URINALYSIS: Yes
- Time schedule for collection of urine: at 3, 6 and 12 months
- Parameters checked: Specific gravity, volume, pH, protein, glucose, ketones, bilirubin, urobilinogen, reducing substances, blood, microscopic examination of sediment

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: prior to the start of treatment and at monthly intervals and during Week 51
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity / grip strength / motor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. Adrenals, brain, epididymides, heart, kidneys, liver, ovaries, pituitary, prostate, spleen, testes, thyroid with parathyroid (post fixation), uterus.
HISTOPATHOLOGY: Yes. Adrenals, aorta (thoracic), bone & bone marrow (femur including stifle joint), bone & bone marrow (sternum), brain, caecum, colon, duodenum, epididymides, eyes (with optic nerve), gross lesions including palpable masses, harderian gland, head (to include pharynx, nasopharynx and, paranasal sinuses), heart, ileum, jejunum, kidneys, larynx, liver, lungs (with bronchi), lymph nodes (cervical and mesenteric), mammary glands, muscle (skeletal), nose, oesophagus, ovaries, pancreas, pharynx, pituitary, prostate, rectum, salivary glands (submaxillary), sciatic nerve, seminal vesicles, skin (hind limb), spinal cord (cervical, mid-thoracic and lumbar), spleen, stomach, testes, thymus, thyroid/parathyroid, tongue, trachea, urinary bladder, uterus.

Statistics:

Data were processed to give group mean values and standard deviations where appropriate. Where appropriate, quantitative data were analysed by the Provantis™ Tables and Statistics Module. For each variable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogeneity of means assessed using ANOVA or ANCOVA and Bartlett’s test. The transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams test for parametric data or the Shirley test for non-parametric data. If no dose response was found, but the data showed non-homogeneity of means, the data were analysed by a stepwise Dunnett (parametric) or Steel (non-parametric) test to determine significant differences from the control group. Finally, if required, pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric). Histopathology data were analysed using the following methods to determine significant differences between control and treatment groups for the individual sexes. Chi squared analysis for differences in the incidence of lesions occurring with an overall frequency of 1 or greater. Kruskal-Wallis one way non-parametric analysis of variance for the comparison of severity grades for the more frequently observed graded conditions. Probability values (P) are presented as follows: P < 0.001(+++ or --- or ***), P < 0.01 (++ or -- or **), P < 0.05 ( + or - or *), P < 0.1 ((+), (-), (*)), p > 0.1 (N.S. (not significant)). With plus signs indicating positive differences from the control group and minus signs indicating negative differences. Asterisks refer to overall between group variation which is non-directional.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

High-dose group: one male was found dead on Day 280, multifocal necrosis in the liver, not treatment related

Mortality:

mortality observed, treatment-related

Description (incidence):

High-dose group: one male was found dead on Day 280, multifocal necrosis in the liver, not treatment related

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

higher mean body weight in males and females in different groups and time points (see Table 1 under "Any other information on results incl. tables"), non-adverse

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

all treatment groups males: reduction in PLT and Neut; high- and mid-dose males: reduction in WBC (see Table 2 under "Any other information on results incl. tables"), non-adverse

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

all treatment groups males: increased urea, reductions in Tri; mid-dose group males: decreased AP; low-dose group males: increased ALAT; all treatment groups females: reduction in Cl-; mid-dose group females: increase in K+, non-adverse

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

all treatment groups males and low dose-group females: increased kidney weight, all treatment groups females: increased thyroid/parathyroid weight (see Table 3 under "Any other information on results incl. tables"), non-adverse

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no toxicologically significant clinical signs detected throughout the treatment period. Episodes of fur loss, fur staining, scab formation, mass formations and wounds were evident throughout the treatment and control animals during the course of the study. Observations of this nature are commonly observed in group housed animals or in ageing rodents and are not considered to be related to treatment.
One male treated at the high dose level was found dead on Day 280. Histopathological examination of this animal revealed a marked severity of multifocal necrosis in the liver which would have significantly contributed towards death. Acanthosis and hyperkeratosis of the fore stomach were also observed. In the absence of similar effects being detected in terminal kill animals the microscopic changes identified in the decedent were considered not to be an effect of test material toxicity. There were no further unscheduled deaths.

BODY WEIGHT AND WEIGHT GAIN
Overall bodyweight gain for males from all treatment groups was comparable to control values. Overall bodyweight gain for females from all treated groups was higher than controls. For males treated at the high dose group and at 6000 and 1500 ppm overall bodyweight gain was 94%, 99% and 97% of control values respectively. Occasional statistically significant differences in bodyweight gain were evident during Weeks 3 (mid-dose group) and 7 (high-dose group) and between Weeks 22-25 (mid- and high-dose groups), 35-38 (high-dose group) and 39-43 (mid- and high-dose group) for males and between Weeks 48-51 (low-dose group) for females. There was no obvious treatment related trend in the intergroup differences and in the absence of an overall effect on bodyweight the intergroup differences were considered not to be of toxicological importance.

FOOD CONSUMPTION/EFFICIENCY AND COMPOUND INTAKE
There were no treatment related effects on food consumption values or food efficiency for animals of either sex from any treatment group throughout the course of the study.

OPHTHALMOSCOPIC EXAMINATION
There were no treatment related ocular effects detected.

HAEMATOLOGY
During Month 3 evaluations, males in the low-dose group showed a statistically significant reduction in platelet count (p < 0.05). The effect on platelet count continued into the Month 6 assessments and extended to males in the remaining treatment groups (mid-and high-dose group, p < 0.05). During the final evaluations during Month 12 males treated at the high- and mid-dose level showed a statistically significant reduction in total leucocyte count (p < 0.05). Males from all treatment groups also showed a statistically significant reduction in neutrophil count (p < 0.05-0.01). There were no dose related responses in the intergroup differences detected and therefore were considered not to be of toxicological significance (see Table 2 under “Any other information on results incl. tables”).

CLINICAL CHEMISTRY
During Month 6 evaluations, males from all treatment groups showed statistically significant increases in urea (p < 0.05 - < 0.01) and statistically significant reductions in triglycerides (p < 0.05). Males in the mid-dose group showed a statistically significant reduction in alkaline phosphatase and males in the low-dose group showed a statistically significant increase in alanine aminotransferase. The effect on urea in males (p < 0.01) continued during the final evaluations at Month 12. At Month 12, females from all treatment groups showed statistically significant reductions in chloride concentration (p < 0.05). Females in the mid-dose group showed a statistically significant increase in potassium concentration (p < 0.05). There were no dose related responses in any of the parameters measured and therefore were considered not to be of toxicological significance (see Table 2 under “Any other information on results incl. tables”).

URINALYSIS
There were no treatment related findings detected in urine volume, urine specific gravity, urine sediment or the remaining urinalytical parameters examined.

NEUROBEHAVIOUR
Monthly open field arena observations did not reveal any treatment-related effects for treated animals when compared to controls. There were no treatment related changes in functional performance and in sensory reactivity.

ORGAN WEIGHTS
In the study, there were no toxicologically significant effects detected in the organ weights measured. Males from all treatment groups and females in the low-dose group showed statistically significant increases in kidney weight both absolute and relative to terminal body weight. Females from all treatment groups also showed statistically significant increases in absolute and relative thyroid/parathyroid weight (see Table 3 under “Any other information on results incl. tables”). In the absence of a dose related response or any associated histology correlates the intergroup differences were considered not to be of toxicological importance.

GROSS PATHOLOGY
There were no treatment-related macroscopic changes observed for either sex amongst animals at scheduled kill or the decedent animal. Macroscopic observations were identified as reddened lungs, pale or dark foci on the lungs, masses in various tissues, sloughing in the stomach or an enlarged thyroid. Observations of this nature are those typically observed for laboratory animals of this type and the occurrences of these observations were recorded for both control and treated animals. In the absence of any significant histology correlates the findings were considered to be associated with ageing animals and of no toxicological importance.

HISTOPATHOLOGY
There were no treatment related microscopic abnormalities detected. All morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed and, since there were no meaningful differences in incidence or severity between control and treatment groups, all were considered not to be treatment related. In Table 4 under “Any other information on results incl. tables” conditions that warrant specific mention are given.

Dose descriptor:

NOAEL

Effect level:

>= 1 333 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: NOAEL corresponding to the highest dose tested

Critical effects observed:

not specified

Table 1. Significant increases in body weight gains in comparison to the respective control group (g).

Group	Week 3	Week 7	Week 25	Week 35	Week 39	Week 48
Mid-dose group (M)	17.3 ± 9.1**	-	4.2 ± 3.5*	-	7.9 ± 6.2*	-
High-dose group (M)	-	11.1 ± 4.8*	-	9.5 ± 5.9**	7.1 ± 7.3*	-
Low-dose group (F)	-	-	-	-	-	9.7 ± 8.7*

*: P < 0.05

**: P < 0.01

-: no significant change in comparison to the respective control group.

Table 2. Significant changes of haematology/clinical examinations.

Group	PLT 10^9/ L	WBC 10^9/ L	Neut 10^9/ L	Urea mg/dL	ALAT IU/L	AP IU/L	Tri mg/dL	K+ mmol/L	Cl- mmol/L
Males
Low-dose group	558.5 ± 79.4*(M3) 615.5 ± 59.8* (M6)	-	1.005 ± 0.356* (M12)	35.0 ± 2.0 *(M6) 30.8 ± 2.9** (M12)	55.7 ± 20.7* (M6)	79.1 ± 11.3* (M6)	153.4 ± 33.1* (M6)	-	-
Mid-dose group	632.5 ± 51.6* (M6)	4.11 ± 0.49** (M12)	0.860 ± 0.274** (M12)	37.0 ± 3.9** (M6) 29.7 ± 4.0** (M12)	-	-	183.6 ± 59.8* (M6)	-	-
High-dose group	638.7 ± 56.4* (M6)	4.73 ± 0.74* (M12)	0.879 ± 0.341** (M)	35.6 ± 5.4** (M6) 29.5 ± 4.0** (M12)	-	-	168.7 ± 39.4* (M6)	-	-
Females
Low-dose group	-	-	-	-	-	-	-	-	103.5 ± 1.3* (M12)
Mid-dose group	-	-	-	-	-	-	-	4.428 ± 0.190* (M12)	103.8 ± 1.0* (M12)
High-dose group	-	-	-	-	-	-	-	-	103.6 ± 1.8* (M12)

*: P < 0.05

**: P < 0.01

M: Month

-: no significant change in comparison to the respective control group.

Table 3. Group mean kidney/thyroid weights with corresponding relative (% of Body weight) organ weights.

Group	Low-dose	Mid-dose	High-dose
Organ	Males
Kidney Mean ± S.D. (g) Mean ± S.D. (%)	2.45836 ± 0.25241** 0.453 ± 0.051*	2.55051 ± 0.25241** 0.462 ± 0.038**	2.46595 ± 0.35788** 0.461 ± 0.037**
	Females
Kidney Mean ± S.D. (g) Mean ± S.D. (%)	1.87500 ± 0.22877* 0.572 ± 0.059*	-	-
Thyroid/Parthyroid Mean ± S.D. (g) Mean ± S.D. (%)	0.02643 ± 0.00551* 0.008 ± 0.002*	0.02479 ± 0.00475* 0.008 ± 0.002*	0.02648 ± 0.00776* 0.008 ± 0.002*

*: P < 0.05

**: P < 0.01

-: no significant change in comparison to the respective control group.

Table 4. Summary of incidence of histopathological findings.

Organ/Tissue	Histological Finding
Bone marrow	Adipose infiltration of the marrow is an indicator of changes in marrow cellularity (higher grades of severity among ageing rats). No differences were seen between control and treated groups.
Adrenal glands	Cortical vacuolation is commonly seen among laboratory rats (especially males). Foci of altered cortical cells are also commonly seen among ageing rats and are characteristically hypertrophic and vacuolated. Isolated instances of haemorrhagic foci were seen among female rats. No evidence of a treatment-related distribution of incidence for any adrenal pathology.
Eyes	Retinal atrophy was observed for a few animals. No treatment-related distribution was observed.
Heart	Focal myocarditis was seen frequently among male rats and rarely among female rats. This is a common background entity in laboratory rats; the severity of the condition was never greater than 1 or 2 foci and should not be interpreted as being indicative of any significant ongoing myocardial disease.
Liver	Scattered mononuclear cell foci were observed in the majority of animals examined in the study. Such are commonly observed in the rodent liver and the severity was rarely greater than minimal or 1 or 2 foci. Centrilobular hepatocyte lipid vacuolation was seen among male rats without a treatment-related distribution of incidence and hepatocyte enlargement and periportal lipid vacuolation were seen occasionally among female rats. Foci of altered hepatocytes are commonly seen in the liver of ageing rats and foci in this investigation foci were typically clear-cell and basophilic. There was no neoplastic liver pathology in this study.
Lymph nodes	Isolated instances of spontaneously arising conditions were seen. Lymphoid neoplasia was not observed in this investigation.
Spleen	Extramedullary haemopoiesis is a normal background condition in the rat spleen. The severities observed were considered to be within normal limits for maintained rats of this age and strain and there was no treatment-related distribution of incidence or severity grades in this investigation.
Kidney	Isolated groups of basophilic/dilated tubules are frequently encountered in the renal cortex as spontaneous change in laboratory maintained rats of this age and strain and have no pathological significance at the severities or frequencies reported in this study. Similarly pelvic corticomedullary mineralisation is a commonly observed background condition amongst ageing female rats and was of no toxicological significance. Hyperplasia of the pelvic/papillary epithelium was observed for a few animals.
Lung	A minimal severity of perivascular/peribronchiolar lymphoid tissue was reported for all animals examined in the study and is not indicative of respiratory disease. Accumulations of alveolar macrophages, cuboidal cell metaplasia, haemorrhage/oedema, and congestion were also seen without significant group distribution of incidence or severity.
Mammary gland	Glandular and secretory hyperplasia were seen among control and high dose female rats and there was no evidence that the incidence or severity of either condition was related to treatment. A mammary fibro-adenoma was seen for one control female rat and a mammary adenocarcinoma was seen for one high dose female rat; these are relatively common spontaneous neoplasms in ageing female rats.
Ovary	Follicular/fluid-filled cysts, cystic corpora lutea, and haemorrhagic cysts are seen with increasing frequency among ageing female rats as spontaneous conditions. There was no relationship to treatment for ovarian cysts in this investigation.
Pancreas	Exocrine atrophy and islet cell hyperplasia were seen for several rats as spontaneous change and without toxicological significance.
Pituitary	Benign adenomas of the pars anterior were seen for a few control and treated rats of either sex. These are relatively common spontaneous neoplasms of laboratory rats (incidence increasing with age). Isolated instances of focal hyperplasia, haemorrhage, and developmental cysts were also seen.
Prostate	Interstitial chronic inflammatory cell infiltrates and prostatitis are commonly observed spontaneous conditions in laboratory rats. Variations in secretory content are also seen relatively frequently.
Thyroid	Parafollicular or C-cell hyperplasia was observed for 3 control and 3 high-dose male rats and follicular cell hyperplasia was seen for 1 control male rat. A few thyroid neoplasms were seen, notably a follicular cell adenoma for 2 animals and a follicular cell carcinoma for 2 animals. There was no treatment-related distribution of incidence.
Seminal vesicles	Variations in secretory content are seen relatively frequently.
Thymus	Lymphoid atrophy is common in the thymus of ageing rats (especially males) and there was no evidence of a treatment-related distribution of incidence or severity in this study.
Uterus/Vagina	Dilatation of the uterine horns and keratinisation of the cervical epithelium are commonly observed cyclical conditions in laboratory female rats, as is keratinisation of the vaginal epithelium. Isolated instances of a few other age-related conditions were also seen without toxicological significance.
Tissue masses	Nodules of fat necrosis were observed for a few animals; these are relatively commonly seen among ageing laboratory rats.

Reference 5

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Study period:

17 Jan - 20 May 2008

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP - Guideline study. According to the ECHA guidance document "Practical guide 6: How to report read-across and categories (March 2010)", the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Umwelt und Naturschutz, Landwirtschaft und Verbraucherschutz des Landes Nordrhein-Westfalen, Düsseldorf, Germany

Species:

rat

Strain:

other: Hsd Cpb:WU

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, Germany
- Age at study initiation: 5 weeks
- Weight at study initiation: 145-171 g (males), 134-153 g (females)
- Housing: in groups with 2 or 3 animals into Makrolon cages Type IV. For environmental enrichment wooden blocks were provided to each cage and renewed as necessary. During the acclimatization and experimental period animals were kept under controlled environmental conditions on low-dust wood granules. Cages and bedding were replaced weekly by new ones.
- Diet: fixed-formula standard diet (Provimi Kliba SA, Kaiseraugst, Switzerland), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 5
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 17 Jan 2008 To: 14 Feb 2008

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test item (test substance) was formulated with corn oil at the appropriate concentrations at room temperature and maximally used over the stability period of eight days.

VEHICLE
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The correct concentrations in the formulations given to the rats were determined twice by analytical examination during the study.
A gas chromatographic method for quantifying the test item in liquid formulations was developed. For analytical investigations, representative samples from the test item formulation covering the test item concentration range used in the study, were taken. These samples were diluted with ethanol into the calibration range and subsequently quantified by gas chromatography (GC) with FID-detection (flame ionization detection). Standard solutions of the authentic test item were used for calibration. Linearity, Precision, Specificity, Robustness and Accuracy of the analytical method were evaluated apart from this GLP-study and fulfil the predefined acceptance criteria. Additional system suitability tests in terms of specificity, precision and linearity indicated that qualified analytical procedures were followed during the study.
Analysis of blank samples (0 mg/mL) revealed no measurable traces of test item.
The content checks assure that during the study appropriate and equal mixture procedures were followed (see Table 1).

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily, 7 days/week

Remarks:

Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: dose levels were selected according to results obtained in a previous oral study performed in rats. In this study the test substance was administered to 3 males and 3 females per group for 2 weeks orally by gavage in daily doses of 0, 250, 500, 750 and 1000 mg/kg.
Survival was not affected by the treatment with the test substance. At clinical observations no treatment-related findings were recorded. Body weight development in both sexes was comparable to that of the respective control group up to 1000 mg/kg. The food and water intake was not affected by the treatment. Determination of organ weights (liver, kidneys, spleen) might indicate a slight increase at 1000 mg/kg in males.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations included: morbidity and mortality (twice daily, once on weekends and public holidays), general clinical examinations (once daily).

DETAILED CLINICAL OBSERVATIONS: Yes, including Open Field Observation (OFO)
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: daily

FOOD CONSUMPTION:
- Food consumption determined per groups and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: weekly

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 28
- Anaesthetic used for blood collection: Yes (CO2/O2 (from room air))
- Animals fasted: No
- How many animals: all
- Parameters examined: differential blood count, erythrocyte morphology, erythrocyte count, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, mean corpuscular volume, haemoglobin concentration, haematocrit, leucocyte count, reticulocyte count, thrombocyte count, thromboplastin time (Hepato-Quick).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 28
- Animals fasted: No
- How many animals: all
- Parameters examined: alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, albumin, cholesterol, creatinine, total protein, urea, glucose, potassium, sodium.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Day 21 (males), Day 22 (females)
- Dose groups that were examined: all
- Battery of functions tested:

Functional Observational Battery

The following observations/examinations were performed:
- home cage observation: posture, piloerection, gait abnormalities, involuntary motor movements, vocalization, others
- observations during handling: ease of removing, reaction to being handled, muscle tone, palpebral closure, lacrymation, nasal discharge, salivation and stains
- open field observations: piloerection, respiratory abnormalities, posture, involuntary motor movements, stereotypy, bizarre behaviour, gait abnormalities, vocalization, arousal, rearing, defecation, urination, others
- the following manipulative tests were additionally performed: approach response, touch response, auditory response, tail pinch response, righting reflex, grip strength, landing foot splays, body temperature, pupil size and pupil response.

Motor Activity ("Figure 8 Maze")
Motor activity (MA) and locomotor activity (LMA) were examined as activity for the entire 60-minute session (Summary Session MA and LMA) and activity during each 10-minute interval (Summary Interval MA and LMA). Motor activity was measured as the number of beam interruptions that occurred during the test session. Locomotor activity was measured by eliminating consecutive counts for a given beam. Thus, for locomotor activity, only one interruption of a given beam was counted until the animal relocated in the maze and interrupted one of the other beams. Habituation was evaluated as a decrement in activity during the test session.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, the necropsy was a systematic gross examination of each animal's general physical condition, body orifices, external and internal organs and tissues.
The following organs of the animals killed at the end of the treatment period were weighed before fixation: brain, heart, liver, spleen, kidneys (both), thymus, adrenal glands (both), epididymides and testes (both).
HISTOPATHOLOGY: Yes (see Table 2)

Statistics:

See "Any other information on material and methods incl. tables".

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw/day: one female showed increased motility (and loss of hair which is considered as incidental) during detailed clinical observation (non-adverse).

Mortality:

mortality observed, treatment-related

Description (incidence):

1000 mg/kg bw/day: one female showed increased motility (and loss of hair which is considered as incidental) during detailed clinical observation (non-adverse).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

300 and 1000 mg/kg bw/day: mean body weight values of females in these groups were marginally to slightly lower than in the respective control group (non-adverse).

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw/day: Foot splay landing slightly higher in females (non-adverse). In males, motor activity slightly increased over the 60-minute observation period as well as within the first two 10-minute intervals (non-adverse).

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Necropsy revealed only a few findings in control and treated animals. None of them had to be attributed to the treatment with the test substance (see also Details on results).

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

All findings in the examined organs were evenly distributed among the groups and are known as spontaneous findings in similar incidence and severity from other studies with rats of this source (see also Details on results).

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
During the treatment period there was no evidence of intercurrent morbidity and mortality related to dosing with the test substance.
No clinical findings could be observed in males up to 1000 mg/kg bw/day and in females up to 300 mg/kg bw/day.
Within the last two weeks of treatment, one 1000 mg/kg bw/day female showed increased motility (and loss of hair which is considered as incidental) during detailed clinical observation.

BODY WEIGHT AND WEIGHT GAIN
Body weight development was comparable to controls in treated males up to 1000 mg/kg bw/day and in treated females at 100 mg/kg bw/day (see Table 3).
In general, mean body weight values were marginally to slightly lower in females at 300 and 1000 mg/kg bw/day than in the respective control group. This finding is most presumably based on the fact that mean body weights of these both groups were already lower than controls before the first administration of the test substance (day 1: 6% lower than control).
Therefore, a toxicologically relevant effect on the body weight is not considered in both sexes up to 1000 mg/kg bw/day, which is supported by the fact that the body weight gain during the study was comparable with that of controls.

FOOD CONSUMPTION
Food intake per animal and per kg body weight of treated animals was comparable to that of controls up to 1000 mg/kg bw/day.

WATER CONSUMPTION
Water intake per animal and per kg body weight of all dose groups was not toxicologically relevantly changed compared to the respective controls up to 1000 mg/kg bw/day.

HAEMATOLOGY
Haematological investigation revealed no toxicologically relevant changes in red and white cell parameters as well as in blood coagulation in any dose group of male and female rats (see Table 4).
Mean erythrocytes, haemoglobin and haematocrit values of the 300 mg/kg bw/day males were only slightly but statistically significantly increased (by ca. 7, 6 and 7%, respectively) compared to control values.

CLINICAL CHEMISTRY
Determinations of enzyme activities in peripheral blood evidenced no toxicologically relevantly changes in males and females up to 1000 mg/kg bw/day.
Substrates in peripheral blood revealed few statistically significant changes in 300 mg/kg bw/day males: a ca. 5% increase in total protein and albumin concentration, respectively; without dose dependency, no toxicologically relevant changes were considered (see Table 5).
Determination of serum electrolyte concentrations showed no changes which were attributable to the treatment with the test substance.

NEUROBEHAVIOUR
- Functional Observation Battery (FOB):
Corresponding to the clinical observation, one 1000 mg/kg bw/day female showed increased motility during the FOB investigation on Day 22 of the study; however, the values of this animal quantified in the MA and LMA (see below) were not indicative of any treatment-induced effect.
Additionally in this rat, the number of rearing was relatively high; alopecia is not considered as treatment-related.
Foot splay landing revealed slightly higher mean values in 1000 mg/kg females compared to control. With increasing dosages the mean values were: 77 ± 5; 85 ± 10 ; 79 ± 11; 94 ± 16 mm. The difference to the control mean was not statistically significant and the value of 94 ± 16 mm was within the range of historical data determined in other studies (mean values: 83-98 mm) (see Table 6).

- Motor and Locomotor Activity (MA and LMA):
The activity determination over the entire 60-minute observation period as well as of the 10-minute intervals did not reveal any significant effect on locomotor activity in treated rats at any dose and on motor activity in males up to 300 mg/kg bw/day and in females up to 1000 mg/kg bw/day (see Table 7).
In males at 1000 mg/kg bw/day, motor activity appeared slightly but not statistically significantly increased over the entire 60-minute observation period as well as within the first two 10-minute intervals. The differences between 0 and 1000 mg/kg bw/day males were comparable to historical values measured in former studies.
The motility observed during detailed clinical observation and FOB (see above) of one female (1000 mg/kg bw/day) appeared higher than in control animals; the individual values of this animal quantified in the MA and LMA were not indicative of any treatment-induced effect.

ORGAN WEIGHTS
Absolute and relative organ weights were inconspicuous in any dose group; no toxicologically relevant changes were observed in both sexes. The only change noted was a ca. 15% decrease in the mean relative spleen weight in females dosed 100 mg/kg bw/day compared to control (200 vs. 236 mg/100 g bw, respectively).

GROSS PATHOLOGY
Necropsy revealed only a few findings. None of them had to be attributed to the treatment with the test substance (see Table 8).

HISTOPATHOLOGY: NON-NEOPLASTIC
Histopathology revealed no findings that had to be attributed to the treatment. All findings in the examined organs were evenly distributed among the groups and are known as spontaneous findings in similar incidence and severity from other studies with rats of this source (see Table 9).

HISTORICAL CONTROL DATA
Historical reference mean values for foot splay of control females and motor activity of control males are given Tables 6 and 7, respectively.

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: overall effects; NOAEL corresponding to the highest dose tested

Critical effects observed:

not specified

Table 3. Group mean body weights and body weight gains.

 

Dose (mg/kg bw/day)	Body weight Day 1 (g)	Body weight Day 29, prior to necropsy (g)	Body weight gain Days 1-29 (g)
Males
0	158	294	136
100	163	313	150
300	162	283	121
1000	156	291	135
Females
0	152	212	60
100	148	217	69
300	143++	205	62
1000	143+	201	58

 

+ p ≤ 0.05

++ p ≤ 0.01

 

 

Table 4. Haematology.

 

Dose (mg/kg bw/day)	ERY (10E12/L)	HB (g/L)	HCT (L/L)	MCV (fl)	MCH (pg)	MCHC (g/L ERY)	RETI (‰)	THRO (10E9/L)	HQUICK (sec)
Males Day 28
0	7.64	150	0.480	62.9	19.7	313	23	1251	41.7
100	7.56	153	0.490	64.9	20.2	311	24	1337	39.4
300	8.17++	159++	0.514++	62.9	19.5	309	19	1251	40.4
1000	7.74	154	0.490	63.3	19.9	314	21	1167	40.5
Females Day 28
0	7.88	148	0.466	59.1	18.8	318	17	1244	35.2
100	7.81	149	0.471	60.4	19.2	317	20	1336	35.4
300	7.86	150	0.476	60.6	19.1	314	21	1254	35.1
1000	7.76	147	0.466	60.1	18.9	315	22	1228	36.6

 

ERY = Erythrocytes; HB = Haemoglobin; HCT = Haematocrit; MCV = Mean corpuscular volume erythrocytes; MCH = Mean corpuscular haemoglobin; MCHC = Mean corpuscular haemoglobin concentration; RETI = Reticulocytes; THRO = Thrombocytes/Platelets; HQUICK = Hepato Quick.

++ p ≤ 0.01

 

 

Table 5. Clinical chemistry: Substrates.

 

Dose (mg/kg bw/day)	GLUCOSE (mmol/L)	CHOL (mmol/L)	CREA (mcmol/L)	UREA (mmol/L)	PROT (g/L)	ALBUMIN (g/L)
Males Day 28
0	5.70	1.59	56	7.70	65.7	34.5
100	5.76	1.66	57	7.87	66.3	34.6
300	5.14	1.76	57	7.51	69.2+	36.3+
1000	5.51	1.93	55	7.53	67.5	35.4
Females Day 28
0	5.40	1.65	56	7.16	68.5	37.3
100	5.16	1.65	59	7.54	68.4	37.2
300	5.17	1.75	56	7.44	68.2	36.8
1000	5.29	1.63	55	7.31	64.9	35.5

 

CHOL = Cholesterol; CREA = Creatinine; PROT = Protein

+ p ≤ 0.05

 

 

Table 6. Footsplay.

 

Dose (mg/kg bw/day)	Footsplay (mm ± SD)	Reference values (mm) / Date
Males Day 21
0	81 ± 10	-
100	87 ± 12	-
300	98 ± 9	-
1000	92 ± 9	-
Females Day 22
0	77 ± 5	98 ± 15 / 07 Apr 2008 91 ± 13 / 05 May 2008 83 ± 9 / 27 Feb 2007
100	85 ± 10	-
300	79 ± 11	-
1000	94 ± 16	-

 

 

Table 7. Motor activity (males).

 

Dose (mg/kg bw/day)	Interval 1	Interval 2	Interval 3	Interval 4	Interval 5	Interval 6	60 min period	Reference values Interval 1	Reference values 60 min period	Dates of reference values
Males Day 22
0	132 ± 26	94 ± 61	52 ± 24	39 ± 21	23 ± 12	8 ± 12	347 ± 98	172 ± 51 272 ± 104 209 ± 85 179 ± 36	394 ± 178 549 ± 215 431 ± 195 367 ± 91	20 Aug 2007 07 Apr 2008 20 May 2008 26 May 2008
100	110 ± 29	85 ± 24	42 ± 10	25 ± 14	16 ± 9	13 ± 11	282 ± 30	-	-	-
300	132 ± 27	86 ± 43	61 ± 39	28 ± 34	39 ± 38	25 ± 22	364 ± 150	-	-	-
1000	187 ± 61	127 ± 44	50 ± 12	28 ± 11	24 ± 21	26 ± 24	427 ± 96	-	-	-

 

 

Table 8. Number of animals with necropsy findings by organ/group/sex.

 

Organ / Finding	Dose group (mg/kg bw/day)	0		100		300		1000
	Sex	m	f	m	f	m	f	m	f
	Animals examined	5	5	5	5	5	5	5	5
Heart: - coloured area(s)		-	-	1	-	-	-	-	-
Lungs: - coloured area(s)		-	-	-	-	-	1	-	-
Liver: -discolouration(s) - distinct lobulation		- 1	- -	- -	1 1	1 1	- -	- -	- 2
Kidneys: - retraction(s) - surface change(s)		- -	- -	- 1	1 -	- -	- -	- -	- -
Spleen: - surface change(s)		-	-	-	-	2	-	-	-

 

 

Table 9. Number of animals with microscopic findings by organ/group/sex.

 

Organ / Finding	Dose group (mg/kg bw/day)	0		100		300		1000
	Sex	m	f	m	f	m	f	m	F
	Animals examined	5	5	5	5	5	5	5	5
HEART
- Mononucl. Infiltrat.		-	2	1	-	-	-	2	3
- Hyperemia/Congestion		-	-	1	-	-	-	-	-
LUNGS
- Hemorrhage,(m-) focal		4	2	-	-	-	1	3	2
- Inflammat. Infiltr.		1	3	-	-	-	1	1	1
ESOPHAGUS
- Mononuc. Infiltrat.		1	-	-	-	-	-	-	-
LIVER
- Periport. Infiltrat.		-	2	-	-	-	-	-	1
- Kupffer Cell Focus/i		4	3	-	-	2	-	3	3
LIVER (ORO)
- Fat-pos. Reaction/PP		5	5	-	-	-	-	4	5
- Fat-pos. Reaction/SC		1	2	-	-	-	-	3	3
- Fat-pos. Reaction/CL		-	-	-	-	-	-	1	-
KIDNEYS
- Basophilic Tubules		3	2	1	-	-	-	2	3
- Cystic Tub. Dilation		-	1	-	-	-	-	-	-
- Mineralization/CMT		-	2	-	-	-	-	-	3
- Pelvic Dilation		-	-	-	-	-	-	-	1
- Pyelonephritis		-	-	-	1	-	-	-	-
- Trans. Cell Hyperpl.		-	-	-	1	-	-	-	-
URINARY BLADDER
- Foc. Simple Hyperpl.		1	-	-	-	-	-	1	-
- Mononucl. Infiltrat.		-	-	-	-	-	-	1	-
EPIDIDYMIDES
- Sperm Granuloma		1	-	-	-	-	-	-	-
PROSTATE
- Interst. Inflammat.		2	-	-	-	-	-	2	-
OVIDUCTS
- Debris/Oocyte Lumen		-	-	-	-	-	-	-	1
UTERUS
- Dilation/Horns		-	1	-	-	-	-	-	-
- Keratinization/Cerv.		-	1	-	-	-	-	-	2
- Mucification/Cervix		-	2	-	-	-	-	-	1
THYROID GLAND
- Foil. Cell Hypertr.		2	-	-	-	-	3	1
- Ectopic Thymus Tiss.		1	2	-	-	-	-	1	2
ADRENAL GLANDS
- Incr. Vacuolation		1	-	-	-	-	-	-	-
- Access. Cort. Nodule		-	-	-	-	-	-	-	1
MANDIB.LYMPH NODES
- Blood Resorption		2	1	-	-	-	-	2	1
STERNUM
- Increased Fat Marrow		-	-	-	-	-	-	1	1

Reference 6

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Study period:

17 Feb - 07 April 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP - Guideline study. According to the ECHA guidance document "Practical guide 6: How to report read-across and categories (March 2010)", the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:WI (Han) (outbred, SPF-Quality)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories France, L'Arbresle Cedex, France
- Age at study initiation: approximately 12 weeks
- Weight at study initiation: 277 - 353 g (males) and 183 - 225 g (females)
- Housing: 5 animals of the same sex per cage in Macrolon cages (MIV type). Sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, UK) were supplied.
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap-water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3
- Humidity (%): 40-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

OTHER
- This species and strain of rat has been recognised as appropriate for general and reproduction toxicity studies. The testing laboratory has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 h prior to dosing and were homogenised to a visually acceptable level.
Adjustment was made for the density of the test substance (0.944), and the specific gravity of the vehicle (1.125).

VEHICLE:
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at the testing laboratory.
- Purity: polyethylene glycol 400, specific gravity 1.125 (Merck, Darmstadt, Germany)

DOSE VOLUME:
5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chemical analyses of formulations were conducted once during the study to assess accuracy, homogeneity and stability. Samples of formulations were analysed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 h at room temperature was also determined (highest and lowest concentration).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% for suspensions. Homogeneity was demonstrated if the coefficient of variation was ≤10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%) and formulations at the entire range were stable when stored at room temperature for at least 6 h.

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily, 7 days/week

Remarks:

Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10 males and 5 females (main study)
5 males and 5 females (recovery group)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on the results of a 10-day dose range finding study.
- Post-exposure recovery period in satellite groups: 14 days

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once prior to start of treatment and at weekly intervals this was also performed outside the home cage in a standard arena. Arena observations were conducted during the treatment phase only. Arena observations were not performed when the animals were mating, or housed individually. The time of onset, degree and duration was recorded. All symptoms were recorded and graded according to fixed scales:
Maximum grade 1: grade 0 = absent, grade 1 = present
Maximum grade 3 or 4: grade 1 = slight, grade 2 = moderate, grade 3 = severe, grade 4 = very severe

BODY WEIGHT: Yes
Males and females were weighed on the first day of exposure and weekly thereafter. Mated Repro females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum, and during lactation on Days 1 and 4.

FOOD CONSUMPTION: Yes
Weekly, for males and females. Food consumption was not recorded during the mating period (except for males). Food consumption of mated Repro females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to scheduled post mortem examination
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes
- How many animals: Blood samples were collected from the first 5 Main males (randomly selected at allocation), all Recovery animals and the 5 Main females from each group. In addition to blood collection prior to necropsy, blood samples were collected from the Recovery animals at the end of treatment.
- Parameters checked: White blood cells, Differential leucocyte count, neutrophils, lymphocytes, monocytes, eosinophils, basophils, Red blood cells, Reticulocytes, Red blood cell distribution width, Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelets, Prothrombin time, Activated Partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to scheduled post mortem examination
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes, overnight
- How many animals: Blood samples were collected from the first 5 Main males (randomly selected at allocation), all Recovery animals and the 5 Main females from each group. In addition to blood collection prior to necropsy, blood samples were collected from the Recovery animals at the end of treatment.
- Parameters checked: ALAT, ASAT, ALP, Total Protein, Albumin, Total Bilirubin, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate, Bile acids.

NEUROBEHAVIOURAL EXAMINATION: Yes
The following tests were performed on the first Main males (randomly selected at allocation), all Recovery animals and the 5 Main females from each group:
- hearing ability, pupillary reflex, static righting reflex and grip strength (score 0 = normal/present, score 1 = abnormal/absent).
- motor activity test (recording period: 1-hour for individual animals, using a computerized monitoring system; Pearson Technical Services, Suffolk, Great Britain).
During the motor activity test, animals were caged individually. The assigned animals were tested during week 4 of treatment (all before blood sampling). Since no treatment-related findings were noted, the functional observation tests and motor activity measurements were not extended to all animals at the end of the recovery phase.

ORGAN WEIGHTS:
The following organ weights and terminal body weight were recorded from the following animals on the scheduled day of necropsy:

- From the first 5 Main males (randomly selected at allocation), the 5 Main females and all
Recovery animals per group: Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Spleen, Testes, Thymus, Ovaries, Uterus (including cervix), Prostate, Seminal vesicles including coagulating glands, Thyroid including parathyroid.

- From all remaining males: Epididymides, Testes

Since no toxicologically relevant effect was noted on organ weights of Main females, no organ weights were collected from Repro females.

Sacrifice and pathology:

DAY OF NERCROPSY:
- Main animals: Following completion of a minimum of 28 days of dose administration.
- Recovery animals: Following completion of a minimum of 28 days of dose administration and a recovery period of 14 days.
One animal was euthanised in extremis.

GROSS PATHOLOGY: Yes
All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.

HISTOPATHOLOGY: Yes
From the first 5 Main animals/sex/group, all Recovery animals, the selected Repro females/group (with live offspring) and animal no.117 that was killed in extremis (Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination):
Adrenal glands, (Aorta), Brain (cerebellum, mid-brain, cortex), Caecum, Cervix, Clitoral gland, Colon, Duodenum, Epididymides, Eyes (including optic nerve and Harderian gland), Female mammary gland area, Femur including joint, Heart, Ileum, Jejunum, Kidneys, (Lacrimal gland, exorbital), (Larynx), Liver, Lung, infused with formalin, Lymph nodes - mandibular, mesenteric, (Nasopharynx), (Oesophagus), Ovaries, (Pancreas), Peyer's patches (jejunum, ileum) if detectable, Pituitary gland, Preputial gland, Prostate gland, Rectum, (Salivary glands - mandibular, sublingual), Sciatic nerve, Seminal vesicles including coagulating gland, Skeletal muscle, (Skin), Spinal cord -cervical, midthoracic, lumbar, Spleen, Sternum with bone marrow, Stomach, Testes1, Thymus, Thyroid including parathyroid (if detectable), (Tongue), Trachea, Urinary bladder, Uterus, Vagina, All gross lesions.

From all remaining animals and females which failed to deliver: Cervix, Clitoral gland, Coagulation gland, Epididymides, Ovaries, Preputial gland, Prostate gland, Seminal vesicles, Testes, Uterus, Vagina, All gross lesions.

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
An attempt was made to transform the number of corpora lutea by using 1/x, log x, x² and √x.
However, a normal distribution was not obtained. Therefore, the number of corpora lutea was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine inter-group differences, followed by the Wilcoxon test to compare the treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
No statistical analysis was performed on histopathology findings.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw: higher body weight gain during recovery phase (males, non adverse); 100 mg/kg bw: higher body weight gain over treatment Days 8-22 (Main females; non adverse)

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw: lower prothrombin time (males, non adverse); 300 mg/kg bw: lower relative lymphocyte counts (females, non adverse)

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

300, 1000 mg/kg bw: lower alanine aminotransferase; 100 mg/kg bw: higher chloride leves (males, non adverse); 1000 mg/kg bw: lower albumin and higher glucose levels (females, non adverse); higher inorganic phosphate levels (males, non adverse)

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

300 mg/kg bw: lower high sensor counts (males, non adverse); 1000 mg/kg bw: effect on high sensor counts (females, non adverse)

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

no adverse effects (see "Details on results")

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

no adverse effects (see "Details on results")

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

no adverse effects (see "Details on results")

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS
No clinical signs of toxicity were noted that were attributable to treatment.

MORTALITY
No mortality occurred during the study period that was considered to be related to treatment with the test substance. Female no. 117 (1000 mg/kg bw/day) was killed in extremis on Day 17 post-coitum. Microscopic examination revealed a marked granuloma in the bronchus region containing macrophages surrounding food particles and with central areas of necrosis. These findings were indicative of a gavage accident.

BODY WEIGHT AND WEIGHT GAIN
No toxicologically relevant changes in body weights and body weight gain were noted up to 1000 mg/kg bw/day.
Any statistically significant changes in body weight gain observed among males and females during the treatment or recovery period were considered to be of no toxicological relevance since the changes occurred in the absence of a dose-related trend and/or were of a slight and/or temporary nature. These changes consisted of a higher body weight gain for males at 1000 mg/kg bw/day during the recovery phase, a lower body weight gain of Repro females at 1000 mg/kg bw/day on Day 11 of the post coitum period, and a higher body weight gain of Main females at 100 mg/kg bw/day over treatment Days 8-22.

FOOD CONSUMPTION
Food consumption before or after allowance for body weight was similar between treated and control animals.

HAEMATOLOGY
No toxicologically relevant changes occurred in haematological parameters of treated rats.
The statistically significant lower prothrombin time in males at 1000 mg/kg bw/day and lower relative lymphocyte counts in females at 300 mg/kg bw/day at the end of treatment were considered to be of no toxicological relevance. These changes were absent at the end of the recovery period, occurred in the absence of a (clear) treatment-related trend and remained within the range considered normal for rats of this age and strain.

CLINICAL CHEMISTRY
No toxicologically relevant changes occurred in clinical biochemistry parameters of treated rats.
Any statistically significant changes in clinical biochemistry parameters were considered to be of no toxicological relevance as these occurred in the absence of a (clear) treatment-related trend, remained within the range considered normal for rats of this age and strain and/or were present at the end of the recovery period only. At the end of treatment these changes consisted of lower alanine aminotransferase in males at 300 and 1000 mg/kg bw/day, and higher chloride levels in males at 100 mg/kg bw/day. Changes at 1000 mg/kg bw/day at the end of the recovery phase included lower albumin and higher glucose levels in females, and higher inorganic phosphate levels in males.

NEUROBEHAVIOUR
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals.
The variation in motor activity did not indicate a relation with treatment. Males at 300 mg/kg bw/day had significantly lower high sensor counts. Since the range of high sensor values encountered at 300 mg/kg bw/day was similar to that observed in the control group and no dose-related trend was noted, no toxicological relevance was ascribed to this variation. A notable variation in high sensor counts was recorded for females at 1000 mg/kg bw/day. Since the range of values at this dose was essentially similar to that observed in the control group, it was considered that no toxicologically significant effect on high sensor counts had occurred in females at 1000 mg/kg bw/day.

ORGAN WEIGHTS
No toxicologically relevant changes were noted in organ weights and organ to body weight
ratios.
Any statistically significant changes in organ weights and organ to body weight ratios were considered to be of no toxicological relevance as these occurred in the absence of a (clear) treatment-related trend, remained within the range considered normal for rats of this age and strain and/or were present at the end of the recovery period only. Also, no histopathological correlates were noted to support these changes. These changes consisted of a lower spleen to body weight ratio in males at 1000 mg/kg bw/day at the end of the recovery phase, a higher spleen weight and spleen to body weight ratio in females at 100 mg/kg bw/day and the end of treatment, lower heart weight in females at 1000 mg/kg bw/day, higher heart to body weight ratio in females at 1000 mg/kg bw/day at the end of treatment, lower adrenal weight and/or adrenal to body weight ratio in females at 300 and 1000 mg/kg bw/day at the end of treatment, and a lower ovary and ovary to body weight ratio in females at 1000 mg/kg bw/day at the end of treatment. Other organ weights and organ to body weight ratios among the dose groups were similar to control levels.

GROSS PATHOLOGY
Macroscopic observations at necropsy did not reveal any toxicologically relevant alterations.
The female at 1000 mg/kg bw/day euthanized in extremis (no. 117) showed a hard, greenish nodule on the right medial lobe of the lung, dark red discolouration of the left ovary, enlarged adrenal glands, reduced size of the thymus, enlargement and greenish discolouration of the bronchial lymph node and pleura grown together with the lungs. A watery-clear cyst was found for a single control Repro female (no. 89). This finding was corroborated with a cyst found in the cervix found upon histopathological examination, which likely contributed to this animal’s suspected infertility.
Other incidental findings among control and treated animals at the end of the treatment and/or recovery period included alopecia, red foci on the thymus, reddish discolouration of the thymus or mesenteric lymph node, pelvic dilation of the kidney, reduced size of the testes, epididymides or seminal vesicles, yellowish hard nodules, a red-brown focus or tan discolouration of the clitoral glands, and fluid in the uterus. The incidence of these findings remained within the background range of findings that are encountered among rats of this age and strain, and their incidence did not show a dose-related trend. These necropsy findings were therefore considered to be of no toxicological relevance.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no treatment-related microscopic findings.
One control female (no. 89) had a marked cyst present in the cervix which correlated to the macroscopic finding in this animal and likely accounted for the infertility. One male rat at 100 mg/kg bw/day (no. 18) had an extensive bilateral seminiferous tubular atrophy in the testes with a subsequent extensive epididymal oligospermia, which accounted for its infertility. This was considered to be a spontaneous abnormality with no likely relationship to the test item. No other abnormalities were seen in the reproductive organs of the remaining suspected nonfertile animals which could account for their infertility. All microscopic findings recorded were considered to be within the normal range of background pathology encountered in Wistar-Han rats of this age and strain. The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis.

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: clinical signs, functional observations, body weights, food consumption, clinical pathology, macroscopy, organ weights, and histopathology.

Critical effects observed:

not specified

Reference 7

Endpoint:

chronic toxicity: oral

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well documented study report which meets basic scientific principles.

Principles of method if other than guideline:

Male rats were exposed to corn oil, safflower oil and tricaprylin via gavage. After 15 months (interim evaluation) and 2 years, animals were sacrificed for gross and histopathological examinations to evaluate the treatment-related effects on neoplasm incidence.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Fischer 344/N

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Simonsen Laboratories (Gilroy, CA)
- Age at study initiation: 7 weeks
- Weight at study initiation: 126 - 159 g
- Housing: groups of 5 animals in polycarbonate cages with hardwood chips
- Diet: NIH-07 open-stock mash diet (Zeigler Bros., Inc., Gardners, PA), ad libitum
- Water: Worcester public water, ad libitum
- Acclimation period: 14 to 22 days; prior to study start, five rats from each study were randomly selected and killed for parasite evaluation and gross observation of disease.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 22
- Humidity (%): 47.1 ± 4.9
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Tricaprylin was dispensed into vials for gavage dosing on a weekly basis. After dispensing, the test item was stored at 4°C for no more than 3 weeks.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Accelerated bulk chemical stability studies were performed using gas chromatography. The test substance was found to be stable as a bulk chemical for 2 weeks when stored protected from light at temperatures up to 60°C. The stability of tricaprylin was monitored periodically by ultraviolet spectroscopy and gas chromatography. In addition, the peroxide concentration was determined prior to use. The acceptable peroxide concentration was set at 2 mEq/L. A bottle was discareded if it exceeded this specification. No significant degradation of the bulk chemical was observed throughout the study.

Duration of treatment / exposure:

104 weeks

Frequency of treatment:

daily, 5 days/week

Remarks:

Doses / Concentrations:
2.5, 5, 10 mL/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
2390, 4770, 9540 mg/kg bw/day
Basis:
other: actual ingested; based on a density of 0.9540 g/cm3 (Final Report on the Safety Assessment of Trilaurin, Triarachidin, Tribehenin, Tricaprin, Tricaprylin; International Journal of Toxicology, 2011)

No. of animals per sex per dose:

60 males/group; 10 from each group were sacrificed after 15 months

Control animals:

yes, concurrent no treatment

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, findings were recorded at least monthly

BODY WEIGHT: Yes
- Time schedule for examinations: at study initiation, than weekly for 13 weeks, and monthly thereafter

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at 15 months. Blood was collected from the posterior vena cava.
- How many animals: 10
- Parameters checked: haematocrit, haemoglobin, erythrocyte count, mean erythrocyte haemoglobin, mean erythrocyte haemoglobin concentration, mean erythrocyte haemoglobin volume, reticulocyte count, total and differential leukocyte count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at 15 months from the posterior vena cava
- How many animals: 10
- Parameters checked: potassium, total protein, albumin, cholesterol, alanine aminotransferase, creatine kinase, sorbitol dehydrogenase, bile acids

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, on all animals. Animals found in a moribund state, selected for the 15-month interim evaluations, or surviving to the end of the 2-year study were killed by the use of "Biotol", an ultra fast-acting barbiturate. All organs and tissues were examined for gross lesions.

HISTOPATHOLOGY: Yes, on all animals. All of the following tissues were preserved in 10% neutral puffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin for microscopic examination: adrenal gland, bone and marrow, brain, esophagus, gross lesions, heart, kidney, large intestine (cecum, colon, rectum), liver, lung, mammary gland, lymph nodes (mandibular and mesenteric), pancreas, parathyroid gland, pituitary gland, preputial gland, salivary gland, skin, small intestine (duodenum, jejunum, ileum), spleen, stomach, testis with epididymis and seminal vesicle, thymus, thyroid gland, tissue masses, trachea, and urinary bladder.

Other examinations:

Brain, kidney (right) and liver of each animal were weighed at the 15 month interim evaluation.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

9540 mg/kg bw/day: 30 animals died

Mortality:

mortality observed, treatment-related

Description (incidence):

9540 mg/kg bw/day: 30 animals died

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

9540 mg/kg bw/day: decreased body weights

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

9540 mg/kg bw/day: decreased food consumption (non-adverse)

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

9540 mg/kg bw/day: increase in the haematocrit, haemoglobin and erythrocyte levels

Clinical biochemistry findings:

no effects observed

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):

4770 mg/kg bw/day: significant decrease in the absolute kidney weight (non-adverse)

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

9540 mg/kg bw/day: decreased incidence in nephropathy and severity

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

dose-related increase in the incidences of pancreatic exocrine hyperplasia and adenoma; proliferative lesions of the forestomach

Details on results:

CLINICAL SIGNS AND MORTALITY
Clinical findings including dyspnea, ataxia and lethargy were observed in 50 out of 60 animals in the high-dose group. However, most of the animals generally recovered prior to the next daily dosing and the incidence of clinical findings declined during the second half of the study.
The two-year survival rate was lower in test animals compared to the respective controls with statistical significance for the high-dose group (31/50, 30/50, 31/50, 23/53 for control, 2390, 4770, 9540 mg/kg bw/day, respectively (Table 1)). 23 rats of the high-dose group died or were killed between weeks 33 - 85 including 10 animals found dead and 13 animals killed moribund. In 20 of these animals, the cause of death or moribund condition could not be determined. 8 rats died between weeks 45 - 49 when the incidence of clinical findings (dyspnea, ataxia and lethargy) was highest. In addition, these animals revealed a significantly lower body weight than the mean group body weight (316 g vs 360 g) at 11 months. Only one of the moribund animals had a pulmonary mass that may explain the dyspnea. Up to 4 animals died or were killed in each subsequent month.

BODY WEIGHT AND WEIGHT GAIN
9540 mg/kg bw/day: Mean body weights were decreased in test animals compared to controls throughout the study. However, the difference was less than 5% after week 61.
No effects were observed in the low- and mid-dose group (Table 2).

FOOD CONSUMPTION
A dose-dependent decrease in food consumption was determined in test animals (Table 3) which resulted in a decreased protein consumption per day (protein consumption (g/rat/day): 3.88, 3.45, 3.09 and 2.70, for control, low-, mid- and high-dose animals, respectively). This effect is most probably due to the fact that rats exposed to tricaprylin received more than 10% of their caloric intake from the test item (10.4%, 19.8% and 35.4% for the low-, mid- and high-dose animals, respectively). Thus, the effect on food consumption is considered as non-adverse.

HAEMATOLOGY
Significant increases in the haematocrit, haemoglobin and erythocyte counts were determined for the high-dose group. No effects were observed in the low- and mid-dose group (Table 4).

ORGAN WEIGHTS
Test animals of the mid-dose group revealed a statistically significant lower absolute kidney weight. As the relative organ weight is not different from the control and the effect is not observed in the low- and high-dose group (Table 5), the effect is considered as incidental and not treatment-related.

HISTOPATHOLOGY: NON-NEOPLASTIC
A significant decrease in the incidence of nephropathy was determined in the high-dose group (control: 46/50, 2390 mg/kg bw/day: 42/50, 4770 mg/kg bw/day: 45/50, 9540 mg/kg bw/day: 27/49) with a dose-related decrease in severity (control: 2.0, 2390 mg/kg bw/day: 1.5, 4770 mg/kg bw/day: 1.7, 9540 mg/kg bw/day: 0.9, Table 6)). As the severity of nephropathy is normally related to the amount of protein in the diet, the decrease in severity induced by tricaprylin might be due to the lower food consumption in test animals (Table 3).
No further findings have been determined in control vs test animals.

HISTOPATHOLOGY: NEOPLASTIC
Tricaprylin induced dose-related increases in the tumor incidences of pancreatic exocrine hyperplasia reaching statistically significance for the mid-and high-dose group (control: 8/49, 2390 mg/kg bw/day: 9/49, 4770 mg/kg bw/day: 18/49, 9540 mg/kg bw day: 28/50), adenoma (control: 2/49, 2390 mg/kg bw/day: 6/49, 4770 mg/kg bw/day: 13/49, 9540 mg/kg bw/day: 18/50) and proliferative lesions in the forestomach (basal cell hyperplasia: control: 4/50, 2390 mg/kg bw/day: 7/50, 4770 mg/kg bw/day: 12/49, 9540 mg/kg bw/day: 21/52; squamous cell papilloma: control: 0/50, 2390 mg/kg bw/day: 0/50, 4770 mg/kg bw/day: 3/50, 9540 mg/kg bw/day: 10/53) reaching statistically significance for squamous cell papilloma in the high-dose group. In contrast, a decrease in the incidence of mononuclear cell leukemia was observed in high-dose animals (control: 23/50, 2390 mg/kg bw/day: 28/50, 4770 mg/kg bw/day: 22/50, 9540 mg/kg bw/day: 9/53). The incidence of pancreatic islet hyperplasia and adenoma or carcinoma decreased in a dose-dependent, but not significant manner (hyperplasia: control: 6/49, 2390 mg/kg bw/day: 5/48, 4770 mg/kg bw/day: 3/49, 9540 mg/kg bw/day: 1/49; adenoma/carcinoma: control: 5/49, 2390 mg/kg bw/day: 2/48, 4770 mg/kg bw/day: 3/49, 9540 mg/kg bw/day: 1/49) (Table 6).

Dose descriptor:

LOAEL

Effect level:

9 540 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: systemic toxicity (clinical signs, mortality, body weight ); corresponding to 10 mL/kg bw /day (calculation based on a density of 0.9540 g/cm³)

Dose descriptor:

NOAEL

Effect level:

4 770 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: systemic toxicity (clinical signs, mortality, body weight ); corresponding to 5 mL/kg bw /day (calculation based on a density of 0.9540 g/cm³)

Critical effects observed:

not specified

Table 1: Mortality data after tricaprylin exposure

	control	2390 mg/kg bw/day	4770 mg/kg bw/day	9540 mg/kg bw/day
number of animals included in the study	60	60	60	60
sacrificed for 15-month interim evaluation^a	10	10	10	7
natural deaths	4	7	4	13
moribund kills	15	13	15	17
animals surviving until study termination	31^d	30	31^d	23^d
mean survival (days)^b	651	639	642	553
survival analysis^c	P = 0.004	P = 0.944	P = 0.969	P = 0.014

a: censored from survival analyses

b: mean of all death including uncensored, censored and terminal sacrifice

c: The result of the life table trend test is shown in the control column whereas the results of life table pairwise comparisons with the controls are listed in the dosage columns.

d: Includes 2 rats for the control and 1 rat for the mid- and high-dose groups that died during the last week of the study.

 

Table 2: Effects on body weight after tricaprylin exposure

	body weight (g)
weeks on study	control	2390 mg/kg bw/day	4770 mg/kg bw/day	9540 mg/kg bw/day
1	146	144	145	145
2	172	171	169	168
3	199	198	193	190
4	219	215	215	207
5	243	241	239	231
7	263	259	255	247
8	266	265	257	254
9	283	282	277	266
10	300	295	285	278
12	306	301	294	283
13	306	301	296	284
14	317	311	303	289
17	332	329	319	303
21	351	350	339	319
25	363	367	354	330
29	376	372	357	335
33	381	379	371	345
37	386	387	376	353
41	388	389	377	347
45	389	389	382	358
49	388	395	388	368
53	401	408	396	380
57	404	411	394	380
61	403	416	400	390
65	408	419	399	392
69^a	406	421	400	392
73	408	422	403	395
77	414	423	406	403
81	413	419	405	404
85	405	412	396	399
89	402	412	390	395
93	400	413	393	397
97	393	406	386	391
101	387	404	379	379
104	386	391	371	366
mean (week 1–13)	246	243	239	232
mean (week 14–52)	367	367	357	335
mean (week 53–104)	402	413	394	390

a: interim evaluation occurred during week 67

 

Table 3: Effects on food consumption after tricaprylin exposure

	food consumption per animal per day (g)
weeks on study	control	2390 mg/kg bw/day	4770 mg/kg bw/day	9540 mg/kg bw/day
2	14.2	13.7	12.5	11.4
5	16.9	15.9	16.0	14.4
9	17.1	16.5	16.4	13.6
13	13.0	12.1	9.9	8.9
17	20.2	16.9	16.0	13.5
21	15.8	14.9	12.5	10.0
25	18.3	18.3	16.5	13.5
29	17.8	16.2	14.0	12.2
33	17.8	16.3	16.0	13.7
37	18.3	15.6	14.2	12.1
41	15.2	12.8	11.5	8.8
45	16.7	14.9	12.5	10.8
49	20.3	17.7	15.8	13.3
53	18.2	16.2	14.2	12.1
57	20.4	17.5	14.1	12.7
61	17.9	16.5	14.2	12.5
65	16.8	13.7	11.9	10.6
69	18.6	17.3	16.5	15.6
73	16.8	14.1	12.9	10.2
77	17.7	14.3	12.9	11.2
81	17.7	14.5	13.2	10.7
85	15.9	14.3	12.2	10.3
89	13.9	12.6	10.6	9.5
93	16.6	14.4	15.9	15.1
97	16.5	12.3	12.3	12.9
101	13.7	12.3	9.4	8.7
104	13.5	10.6	9.2	7.6
mean	17.0	15.1	13.6	11.8

 

Table 4: Haematology data after tricaprylin exposure at the 15-month interim evaluation

	control	2390 mg/kg bw/day	4770 mg/kg bw/day	9540 mg/kg bw/day
number of animals	9	10	8	7
haematocrit (%)	45.6 ± 1.1	50.7 ± 2.7	49.2 ± 2.3	51.7 ± 2.0*
haemoglobin (g/dL)	15.6 ± 0.4	17.3 ± 0.8	16.7 ± 0.6	17.5 ± 0.6*
erythrocytes (10^6/ µL)	8.77 ± 0.31	9.72 ± 0.41	9.30 ± 0.34	9.91 ± 0.32*

*: significantly different from control by Dunn´s or Shirley´s test (P ≤ 0.05)

 

Table 5: Organ weights of the kidney (right) after tricaprylin exposure at the 15-month interim evaluation

	control	2390 mg/kg bw/day	4770 mg/kg bw/day	9540 mg/kg bw/day
number of animals	10	10	10	7
body weight at necropsy	430 ± 15	419 ± 11	402 ± 15	406 ± 12
absolute	1.349 ± 0.04	1.23 ± 0.036	1.194 ± 0.041*	1.328 ± 0.053
relative	3.15 ± 0.07	2.95 ± 0.07	2.98 ± 0.07	3.27 ± 0.08

*: significantly different from the control by William´s or Dunnett´s test (P ≤ 0.05)

Organ weights are given in grams; organ-weight-to body-weight ratios are given as mg organ/g bw

 

Table 6: Histopathological findings after tricaprylin exposure

		control	2390 mg/kg bw/day	4770 mg/kg bw/day	9540 mg/kg bw/day
non-neoplastic	nephropathy incidence	46/50	42/50	45/50	27/49
	nephropathy severity	2.0 ± 0.12	1.5 ± 0.13	1.7 ± 0.11	0.9 ± 0.13
neoplastic	exocrine pancreas hyperplasia	8/49	9/49	18/49	28/50
	exocrine pancreas adenoma	2/49	6/49	13/49	18/50
	forestomach: basal cell hyperplasia	4/50	7/50	12/49	21/52
	forestomach: squamous cell papilloma	0/50	0/50	3/50	10/53
	mononuclear cell leukemia	23/50	28/50	22/50	9/53
	pancreatic islet hyperplasia	6/49	5/48	3/49	1/49
	pancreatic islet adenoma/carcinoma	5/49	2/48	3/49	1/49

For the interpretation of carcinogenic effects, please see Section 7.7, study entry "NTP, 1994, 2y, rat, gavage, RL2".  

Reference 8

Endpoint:

sub-chronic toxicity: oral

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted similarly to guideline .

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

: no ophthalmoscopy, no neurology

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Simonsen Laboratories, Gilroy, CA, USA
- Age at study initiation: 6 weeks
- Weight at study initiation: 126 - 132 g (males), 107- 110 g (females)
- Housing: rats: 5 per cage
- Diet (Control feed (NIH 07) or diet formulations of castor oil): ad libitum; feeders were changed twice per week throughout the study.
- Water (automatic watering system): ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 68-76
- Humidity (%): 42 - 72
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulated diets were prepared by blending the appropriate amount of castor oil with a small quantity of feed to prepare a premix. The premix then was layered between the required amount of feed in a twin-shell blender and blended for 15 minutes to achieve a uniform mix. The homogeneity of castor oil in feed at 10% (100 mg/g) was determined by gravimetric analysis, and blends at 0.5% (5 mg/g) were determined by HPLC analysis. These concentrations of chemical in feed were found to be homogeneously distributed by this mixing procedure. The stability of the 0.5% dose level was determined using HPLC; it was found to be stable for at least 21 days when stored in the dark at 5°C and for 3 days when stored open to air and light in a rodent cage. During the studies, formulated diets were stored for no longer than 3 weeks at 5°C; feed hoppers in the animal cages were changed twice weekly.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The results of the analyses for all dose mixtures given to the animals ranged from 97% to 106% of the target concentrations.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

daily ad libitum feeding

Remarks:

Doses / Concentrations:
0.62, 1.25, 2.50, 5.00, 10.0 % (w/w)
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
404, 809, 1583, 3067 and 5835 mg/kg bw/day
Basis:
other: actual ingested: male rats

Remarks:

Doses / Concentrations:
401, 797, 1569, 3045, 5725 mg/kg bw/day
Basis:
other: actual ingested: female rats

No. of animals per sex per dose:

10; 10 additional rats/sex were included at each dose level for evaluation of hematological and clinical chemistry parameters at days 5 and 21.

Control animals:

yes, plain diet

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS/ CLINICAL OBSERVATIONS: Yes
- Time schedule: twice a day

BODY WEIGHT: Yes
- Time schedule for examinations: initially and 1 x wk thereafter.

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


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: from additional rats at days 5 and 21, and at the end of study
- Anaesthetic used for blood collection: Yes, CO2
- How many animals: 10 additional animals
- Parameters checked: red blood cell (RBC) count, red blood cell morphologic assessment, hematocrit (HCT), hemoglobin concentration (HGB), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), white blood cell count (WBC), white blood cell differential count, reticulocyte count (absolute), and platelet counts (absolute).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: from additional rats at days 5 and 21, and at the end of study
- How many animals: 10 additional animals
- Parameters checked: alkaline phosphatase activity (ALP), albumin (ALB), urea nitrogen (UN), creatinine (CREA), alanine aminotransferase activity
(ALT), total bile acids (TBA), sorbitol dehydrogenase activity (SDH), total protein (TP), and creatinine kinase (CK).


URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Complete histopathology examinations were conducted on all rats from the control and 10% dose groups. Livers were examined from male rats in all other dose groups; histologic sections of gross lesions were examined from all rats. Organ weights were determined to the nearest milligram for the liver, right kidney, right testicle, heart, thymus, and lungs. All tissues were preserved in 10% neutral buffered formalin.

The following tissues were routinely processed for preparation of histologic sections and microscopic examination: adrenal glands, brain, cecum, colon, duodenum, epididymis/seminal vesicles/prostate/testes or ovaries/uterus, esophagus, eyes (if grossly abnormal), femur (including marrow), heart, ileum, jejunum, kidneys, liver, lungs and mainstem bronchi, mammary gland, mandibular and mesenteric lymph nodes, nasal cavity and turbinates, pancreas, parathyroid glands, pituitary gland, preputial or clitoral glands, rectum, salivary glands, skin, spinal cord and sciatic nerve (if neurologic signs present), spleen, forestomach and glandular stomach, thymus, thyroid gland, trachea, urinary bladder, zymbal glands, and all gross lesions and tissue masses including regional lymph nodes. A complete histopathologic examination was conducted on all rats from the control and 10% dose groups. Liver was examined from male rats in all other dose groups, and histologic sections of gross lesions were examined from all rats.

Statistics:

Body weight and organ weight data were statistically analyzed within each sex by one-way Analysis of Variance tests, followed by Dunnett's t-test if pair-wise comparisons were indicated (p < 0.05)(Dunnett, 1955).

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 examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

no adverse biologically significant effects

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

no adverse biologically significant effects

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):

no adverse biologically significant effects

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
No effects

BODY WEIGHT AND WEIGHT GAIN
Group mean body weights of rats receiving diets containing castor oil did not differ significantly from controls. Mean body weights of exposed female rats were slightly lower than the mean body weights of controls but the differences were not dose-related.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
No significant differences in average food consumption among each sex were observed, although food consumption of male and female rats receiving diets containing 10% castor oil was slightly lower than that of controls.

HAEMATOLOGY
Hematological effects of the castor oil diets among male rats included a slight decrease in MCHC at day 21 in those receiving the 10% diet; a statistically significant decrease in MCV among the 10% group; a decrease in MCH among the 5% and 10% groups; and an increase in platelets among the 1.25%, 5%, and 10% groups. The only change observed among female rats was a statistically significant decrease in reticulocyte counts at day 5 in groups receiving the 0.62% or 10% diets. None of these changes was considered biologically significant.

CLINICAL CHEMISTRY
A treatment- and dose-related increase in the activity of serum alkaline phosphatase was observed in male and female rats at days 5 and 21, and at study termination. Total bile acids were increased among males receiving the higher dietary levels at days 5 and 21 but were not increased at study termination. Other minor changes included increases in albumin observed at study termination in males receiving 5% diets and at day 5 in females receiving 10% diets, and an increase in urea nitrogen at study termination in males that received 0.62% diets and a decrease at day 5 in females that received castor oil at 10% in the diet.

ORGAN WEIGHTS
Absolute liver weights and the liver-to-body-weight ratio were increased in male rats that received diets containing 10% castor oil. Heart-to-body-weight ratios were increased in groups of male rats receiving 0.62%, 2.5%, and 10% diets; however, absolute heart weights were not increased, and the differences in body weight ratios were small and not considered treatment related. Using light microscopy, it was determined there were no morphologic changes associated with the slight differences in organ weights between groups.

In male rats, there was a slight decrease in epididymal weight (6-7%) which occurred in the middle- and high-dose groups, but this was not dose-related. There were no effects on any other male rat reproductive endpoint, or on any female rat reproductive endpoint. Although there was some variation in epididymal weights, their small magnitude and the absence of changes in other endpoints suggested that there was little or no evidence of any reproductive toxicity associated with castor oil exposure. Histopathologic examination revealed an absence of compound-related lesions in any organ or tissue of rats exposed to castor oil in the diet.

Dose descriptor:

NOAEL

Effect level:

10 other: % w/w

Based on:

test mat.

Remarks:

corresponding to doses of 5835 and 5725 mg/kg bw/day in males and females, respectively, as calculated from the reported food consumption and body weights

Sex:

male/female

Basis for effect level:

other: no overall effects

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The available studies on oral and dermal repeated dose toxicity support the view that Fatty Acid Glycerides are inherently harmless, non-toxic substances:

 

Subchronic repeated dose toxicity oral:

 

A 90 day oral feeding study with Castor oil (CAS No. 8001 -79 -4) was performed equivalent to OECD Guideline 408 in F344/N rats and B6C3F1 mice (Irwin, NTP report 1992). The test substance was mixed at concentrations of 0, 0.62, 1.25, 2.50, 5.00, 10.0 % (w/w) to the diet and the animals were fed ad libitum for 13 weeks. 10 animals per sex and per dose were used. The highest dose was equivalent to approx. 5.7 g/kg/day for rats and approx. 15 g/kg/day for mice. Exposure to castor oil at dietary concentrations as high as 10% in 13-week studies did not affect survival or body weight gains of rats or mice. There were no biologically significant effects noted in hematologic analyses in rats. Mild increases in total bile acids and in serum alkaline phosphatase were noted at various times during the studies in rats receiving the higher dietary concentrations of castor oil. Liver weights were increased in male rats receiving the 10% dietary concentration and in male and female mice receiving diets containing 5% or 10% castor oil. However, there were no histopathologic lesions associated with these liver changes, nor were there any compound-related morphologic changes in any organ in rats or mice. No significant changes were noted in a screening for male reproductive endpoints, including sperm count and motility, and no changes were observed in the length of oestrous cycles of rats or mice given diets containing castor oil. Thus, no significant adverse effects of castor oil administration were noted in these studies. A NOAEL of 5000 mg/kg bw/day for rats and a NOAEL of 15000 mg/kg bw/day for mice could be identified.

 

A 90 day oral feeding study with medium chain triglycerides was performed similar to OECD Guideline 409 in adult beagle dogs (Matulka, 2009). The medium chain triglycerides (MCTs) were comprised of a glycerol backbone esterified to medium chain length (8¿12 carbon) fatty acids (FA) and, were all saturated FA. All dogs received on 91 consecutive days approximately 200 g of conventional feed with 0%, 5%, 10%, or 15% MCT for a three hour feeding regimen (4 animals per dose). Based on examination of clinical signs, body weight measurements, food consumption level, physical examinations, haematology and serum chemistry, ophthalmic examinations, and urinalysis the NOAEL for medium chain triglycerides was found to be greater than 15 % , which was calculated to be approximately 3750 mg/kg/day.

   

A 90d oral feeding study (Klimmer, 1971) was performed in young Wistar rats with mixed decanoyl and octanoyl glycerides (CAS No. 73398 -61 -5). The test substance was mixed to the diet at concentrations of 10,000 and 50,000 ppm. 20 animals per dose were fed ad libitum. No treatment-related abnormalities were observed concerning clinical signs, body weight increase, food consumption, haematology, clinical chemistry, urine analysis and gross pathology. Thus, the NOAEL was found to be 5000 mg/kg bw/day assuming 1 ppm in food being equivalent to 0.1 mg/kg bw/ day for young rats.

 

A 56 day oral feeding study with medium- and long-chain triglycerides was performed in Wistar rats (Matsuo, 2004). The effects of structured medium- and long-chain triacylglycerols (MLCT) in diets containing 50, 100, 150 or 200 g test substance/kg bw on body fat accumulation were compared with those of long-chain triacylglycerols (LCT). The diets were fed to groups of 6 young adult male Wistar rats for 56 days. Based on clinical observations, body weight, food efficiency, liver weights and clinical serum chemistry the NOAEL for rats was found to be 20 g/kg bw.

 

Subacute repeated dose toxicity oral:

 

A 28 day oral gavage study was performed according to 79/831/EWG, Annex V, Part B in male and female Sprague-Dawley rats (10 animals per dose) with C16 -18 and C18 mono- and dihydroxy fatty acid glycerides (CAS No. 91845 -19-1) at concentrations of 0, 100, 500 and 1000 mg/kg bw (Potokar, 1985). The test substance was administered once a day on 5 days per week using peanut oil as a vehicle. A post-exposure recovery satellite group of 5 male and 5 female animals was observed for an additional period of 33 days. Histopathologic evaluation revealed dilatation of lymphatic vessels in the small intestine stroma and the occurrence of foreign body giant cells and vacuolization in Peyer Plaques without signs of inflammation. These findings were observed at 500 and 1000 mg/kg/d and were considered to be adaptive responses, not adverse effects. No other treatment-related abnormalities were observed. The NOAEL was found to be 1000 mg/kg bw/day in male and female rats.

 

A 28-day oral gavage screening study (van Otterdijk, 2010) was performed with Glycerides, C8-18 and C18-unsatd. mono- and di-, acetates (CAS 91052-13-0) according to OECD guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction /Developmental Toxicity Screening Test) in Crl:WI(Han) (outbred, SPF-Quality) Wistar Han rats. Doses of 0, 100, 300 and 1000 mg/kg bw/d were given to groups of four Main groups of 10 male and 5 female rats. Additionally, 5 Recovery group males and females in the control and high dose group were allowed 14 days of recovery. Additionally, 10 females were added to each group for the assessment of reproduction and developmental toxicity. Recovery animals were exposed for at least 28 days from start of treatment up to termination or start of recovery. Females used for the assessment of reproduction/developmental toxicity were exposed for 41-49 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation.

No treatment related abnormalities were found based on clinical signs, functional observations, body weights, food consumption, clinical pathology, macroscopy, organ weights, and histopathology examinations. Thus a 28 -day oral repeated dose NOAEL of 1000 mg/kg bw/d (highest dose tested) was found in Wistar rats.

 

Structural analogue read-across from Glycerides, C8-18 and C18-unsatd. mono- and di-, acetates (CAS No. 91052-13-0) for systemic mammalian toxicity endpoints was judged to be justified for the following reasons: This read across substance is also a glyceride, containing mainly C12-14 fatty acid and actetate moiety as well as glycerol. It¿s an organic liquid with a pour point of -8 °C and a melting point of 357.85 °C and a vapour pressure < 0.01 Pa at room temperature. In contrast to the glycerides of the fatty acid glyceride category, it has a higher water solubility of 8.75 mg/L, which might influence its environmental distribution, but not the mammalian metabolism upon systemic uptake. Therefore it is expected to feed into the same mammalian physiological pathways as the members of the fatty acid glyceride category, like citric acid cycle, sugar synthesis and lipid synthesis. These processes are described in detail within the category justification.

 

A 30 day oral gavage study was performed with Glycerides, mixed decanoyl and octanoyl (CAS No. 73398 -61 -5) in male Wistar rats (Klimmer, 1971). Groups of 10 animals received daily doses of up to approx. 10 g/kg bw/d. No treatment related abnormalities were observed based on clinical signs, body weight increase, food consumption, urine analysis and gross pathology at any dose level. No other parameters were analyzed. Based on the study results, a subacute NOAEL of 10000 mg/kg bw/d was found for male Wistar rats.

 

A 28 day safety evaluation of a medium- and long-chain triacylglycerol (MLCT) oil - placebo-controlled, double blind - human clinical trial study was performed utilizing 10 test subjects per group (Matulka, 2006). The test subjects were healthy men and women at the age range o 21¿39 years. The study food (bread) containing either LCT (i.e., mixed rapeseed and soybean oils (7:3) as a control) or MLCT was consumed during the morning, afternoon, and evening meals. The amount of oil consumed via the bread source was calculated at 42 g per day. Food consumption was targeted at an average daily intake of 2100¿2600 kcal and 70¿80 g of fat. Monitoring of body weight, blood analysis and urinalysis revealed no significant differences between or within LCT and MLCT groups. No adverse effects of MLCTs were observed in humans when ingested at a dose of 42 g/day for 4 weeks.

 

Repeated dose toxicity dermal:

A subchronic dermal repeated dose toxicity study was performed with glycerides, mixed decanoyl and octanoyl (CAS No. 73398 -61 -5) in female rats (reviewed by Elder, 1980). A perfumed skin softener formulation containing 4% Caprylic/Capric Triglyceride was applied to the shaved skin of 15 female rats at a dose of 2 mL/kg five days per week for 13 weeks, equivalent to 75 mg/kg bw/d. This treatment had no effects on the body weight, clinical appearance or behaviour. All blood-cell and serum chemistry parameters measured one week before termination of the study were within normal limits and comparable to those seen in an equal group at controls. At necropsy, organ weights and gross finding revealed no effects of the test substance, and no histopathological changes were observed. There were no localized effects on the skin. Based on this study results the subchronic dermal NOAEL for female rats was found to be 75 mg/kg bw/d (highest dose tested).

 

An additional study with a tanning butter formulation containing 22% Caprylic/Capric Triglyceride (CAS No. 73398 -61 -5) was applied to the clipped backs of three male and three female New Zealand stain albino rabbits at a dose of 2000 mg/kg five times per week for 28 days (reviewed by Elder, 1980). Throughout the test, no effects attributable to the treatment were noted on body weight, physical appearance and behaviour. Blood samples taken 23 days after initiation of the test showed no effects on haematocrit, haemoglobin concentration, cell counts, urea nitrogen, alkaline phosphatase or glutamic pyruvic transaminase activites, or glucose concentration. At the end of the test, no systemic, gross or histopathologic changes referable to the test material were observed. On the treated area of the skin there was slight to moderate erythema and slight peeling and cracking regardless of whether the skin was abraded or left intact. Based on the study results a sub-acute (28-day) dermal NOAEL of 2000 mg/kg bw/d was found for male and female rabbits.

 

Repeated dose toxicity inhalation:

Repeated dose toxicity inhalation studies were not available.

For the following reasons the inhalation route was judged to be of minor relevance:

- For liquid fatty acid glycerides with a vapour pressure at room temperature < 0.01 Pa exposure to vapour can be excluded.

- For waxy solid fatty acid glycerides inhalation exposure seems very unlikely.

- Fatty acid glycerides with melting temperatures below 55ºC are commonly presented in pellet, flake, block or paste form.

- Fatty acid glycerides with melting temperatures above 55ºC may be turned into powder using conventional spray-cooling technology

- The particle size of powdered fatty acid glycerides is not a function of chemical composition, but is determined by the spray-cooling conditions.

- For the majority of industrial applications powders with mean particle size (D(0.5)) above 150µm are preferred.

- Powders with mean particle size (D(0.5)) down to 40µm can be produced for special applications (mainly for use as food additives).

- For standard and coarse powders with mean particle size above 150µm the proportion of respirable dust (powder with particle size below 10µm is negligible.

- For fine powders the proportion of respirable dust is below 5.5% and is not considered to be significant.

- Fatty acid glycerides are not corrosive and are readily hydrolysed in the body by ubiquitously expressed lipases. The likelihood of persistence in the lungs is considered to be negligible.

- The potential risk to human health due to respirable dust in fine powders produced for special applications is very low.

   

Additional toxicological information (see 7.12):

 

In 1975, the FDA - Select Committee on GRAS Substances (SCOGS) summarized their opinion on the GRAS status of Glycerin and Glycerides (FDA, 1975): There was no evidence in the available information on mono- and diglycerides of fat-formaing fatty acids that demonstrates or suggests reasonable grounds to suspect a hazard to the public when they are used at levels that are now current or that might reasonably be expected in the future. Further, there was no evidence in the available information on acetooleins and acetostearins that demonstrates or suggests reasonable grounds to suspect a hazard to the public when they are used at levels that are now current or that might reasonably be expected in the future.

 

According to Annex I of theEU Directive No 95/2/ECon food additives other than colours and sweeteners, Mono- and diglycerides of fatty acids (E471) is listed as "Food Additive generally permitted for use in foodstuffs not referred to in article 2 (3)". Substances on this list may be added to all foodstuffs with the exception of those referred to in Article 2 (3) following thequantum satisprinciple. According to Annex II of this directive the maximum level of E 471 in non-emulsified oils and fats of animal or vegetable origin (except virgin oils and olive oils) is 10 g/L.

According to Annex VI of this directive the maximum level for the use of E 471 as "Food additive permitted in infant formulae for infants in good health" is 4g/L.

 

Pursuant to 21 CFR § 170.3, medium- and long-chain triacylglycerol (MLCT)-Oil has been determined generally recognized as safe (GRAS) by scientific procedures for its intended conditions of use (FDA, 2006). The safety of MLCT-Oil is supported by preclinical and clinical studies, and the fact that medium-chain triacylglycerols have been administered to patients withmalabsorption syndromes, added to infant formulations, and consumed in the diet from natural sources, while long-chain triacylglycerols contained in vegetable oils have been commonly consumed in the human diet. MLCT-Oil (containing approximately 12% MCFA) manufactured via transesterification from LCT (from vegetable oils such as rapeseed oil) and MCT (from coconut and/or palm kernel oil) to produce a novel food product, was granted ¿Food for Specified Health Use¿ (FOSHU) status by the Ministry of Health, Labor, and Welfare inon December 6, 2002. FOSHU is defined as a food that has beneficial, effective ingredients added to help in the maintenance of a healthy body condition.

 

 

Justification for classification or non-classification

According to DSD (67/548/EEC) or CLP (1272/2008/EC) classification criteria for repeated dose toxicity, no classification is required.