2-({3-(octadecanoyloxy)-2,2-bis[[](octadecanoyloxy)methyl]propoxy}methyl)-2-[[](octadecanoyloxy)methyl]propane-1,3-diyl dioctadecanoate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

19 Feb 1998 - 30 March 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 1995

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom, UK

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, C5-9, hexaesters with dipentaerythritol

EC Number:

267-021-7

EC Name:

Fatty acids, C5-9, hexaesters with dipentaerythritol

Cas Number:

647028-25-9

Molecular formula:

Not available (UVCB)

IUPAC Name:

647028-25-9

Test animals

Species:

rat

Strain:

other: Sprague-Dawley Crl:CD®BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Limited, Margate, Kent, UK
- Age at study initiation: 5 to 6 weeks
- Weight at study initiation: 127 – 161 g (males) and 117 – 152 g (females)
- Housing: animals were housed in groups of 5 of the same sex per cage in polypropylene grid-floor cages
- Diet: Rat and Mouse SQC Expanded Diet No.1, pelleted, ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

arachis oil

Remarks:

BP

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared weekly and stored at approximately +4°C in the dark.

VEHICLE
- Concentration in vehicle: 75 mg/mL, 250 mg/mL, 500 mg/mL
- Amount of vehicle (if gavage): 2 mL/kg/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of test item in the test material formulations was determined by gas chromatography (GC) by means of an external standard technique. The prepared formulations were within ± 10% of the nominal concentration.

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily, 7 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: dose levels were chosen based on the results of a foregoing range-finding study, in which animals were orally exposed to 150, 500 and 1000 mg/kg bw/day by gavage for 14 days (Jones, L.J., 2000). No adverse effects were observed during the study period in any animal. Therefore, 150, 500 and 1000 mg/kg bw/day were selected as the dose levels for the main study.

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: immediately before dosing and one and five hours after dosing during the working week and immediately before dosing and one hour after dosing at weekends.

BODY WEIGHT: Yes
- Time schedule for examinations: individual body weights were recorded on day 0 (the day before the start of treatment) and on days 7, 14, 21, and 28. Body weights were also recorded at terminal of the study

FOOD CONSUMPTION AND EFFICIENCIES: Yes
- Cage group mean weekly food consumption and weekly food efficiencies were evaluated.

WATER CONSUMPTION: Yes
- Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at end of the study (Day 28)
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: all animals from each test and control group
- Parameters examined: haemoglobin, erythrocyte count, haematocrit, erythrocyte indices (mean corpuscular haemoglobin, mean corpuscular volume, mean corpuscular haemoglobin concentration (MCHC)), total leucocyte count, differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), platelet count (PLT), reticulocyte count.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at end of the study (Day 28)
- Animals fasted: No
- How many animals: all animals from each test and control group
- Parameters examined: urea, glucose, total protein, albumin, albumin/globulin ratio, calcium, inorganic phosphorus, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, creatinine, sodium, potassium, chloride, total cholesterol, total bilirubin

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: prior to the start of treatment and on days 2, 8, 15 and 23.
- Dose groups that were examined: all animals
- Battery of functions tested: sensory reactivity (grasp response, vocalisation, toe pinch, tail pinch, finger approach, touch escape, pupil reflex, startle reflex, blink reflex)/ grip strength (forelimb/hindlimb) / motor activity

BEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: prior to the start of treatment and on days 2, 8, 15 and 23.
- Dose groups that were examined: all animals
- Parameters observed: gait, tremors, twiches, convulsions, bizarre/abnormal/stereotypic behaviour, pilo-erection, exophthalmia, lachrymation, hyper/hypothermia, skin colour, respiration, palpebral closure, urination, defecation, transfer arousal, tail elevation.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
HISTOPATHOLOGY: Yes. Adrenals, aorta (thoracic), bone and bone marrow (femur including stifle joint), bone and bone marrow (sternum), muscle (skeletal), oesophagus, ovaries, pancreas, pituitary, brain (including cerebrum, cerebellum and pons), caecum, colon duodenum, epididymides, eyes, gross lesions, heart, ileum, jejunum, kidneys, liver, lungs (with bronchi), lymph nodes (cervical and mesenteric), prostate, rectum, salivary glands (submaxillary), sciatic nerve, seminal vesicles, skin (hind limb), spinal cord (cervical), spleen, stomach, testes, thymus, thyroid/parathyroid, trachea, urinary bladder, uterus.

Other examinations:

Organ weights: adrenals, kidneys, testes, brain, liver, thymus, epididymes, ovaries, heart, spleen

Statistics:

Haematological, blood chemical, organ weight (absolute and relative to terminal body weight), weekly body weight gain and quantitative functional performance and sensory reactivity data were considered for dose response relationships by linear regression analysis followed by one way analysis of variance (ANOVA) including Levene’s test for homogeneity of variance. Where variances were shown to be homogeneous pair wise comparisons were conducted by means of Dunnett’s test. Where Levene’s test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann Whitney “U” test.

The haematology variable basophils was not analysed since consistently greater than 30% of the data were recorded as the same value.

Probability values (p) are presented as follows:

p< 0.001***
p < 0.01**
p< 0.05*
p ≥0.05 (not significant)

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

1000 and 500 mg/kg/day: statistically significant reduction in body weight in males during week 1 (non adverse)

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

no effects observed

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):

500 and 1000 mg/kg/day: statistical significant increase in platelet count and reduction in mean corpuscular haemoglobin concentration respectively (non adverse)

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg/day: increase in total and asymptotic motor activity in males. Females from the same treatment group showed a decrease in asymptotic activity (non adverse)

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

500 mg/kg/day: statistically significant reduction in absolute epididymides weight (non adverse)

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

150 mg/kg/day (males and females) and 1000 mg/kg/day (one female): dark foci on the lungs (non adverse)

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg/day: 3 males showed globular accumulations of eosinophilic material in the proximal tubular epithelium (non adverse)

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
No mortality occurred during the study period. No clinical signs of toxicity were observed in test or control animals throughout the study. However, noisy respiration was observed in one treated male animal (1000 mg/kg/day group) and one treated female animal (500 mg/kg/day group) one hour after dosing on day 2. These isolated and transient observations showed no convincing dose-relationship and were considered to be of no toxicological importance.
One treated female (1000 mg/kg/day) showed fur loss from day 21 onwards and a control female developed a scab on the head from day 25. Since such findings are occasionally reported in group housed rats, they are considered to be incidental.

BODY WEIGHT AND WEIGHT GAIN
No adverse effect on body weight was noted. Nevertheless a statistically significant reduction in body weight gain was apparent for 1000 and 500 mg/kg/day males during week 1 of the study. The dose relationship was not credible and the intergroup differences were considered to be a result of slightly higher than usual control group body weights gains.

HAEMATOLOGY
No treatment-related changed in the haematological parameters were measured.
Males treated with 1000 mg/kg/day showed a statistically significant reduction (p< 0.05) in mean corpuscular haemoglobin concentration when compared to controls. Under these conditions and in absence of any other haematological changes, the intergroup difference was considered to be accidental.
A statistically significant (p < 0.05) increase in platelet count was detected but this was confined to 500 mg/kg/day males and was considered to be incidental (see Table 2 under "Any other information on results incl. tables").

FUNCTIONAL PERFORMANCE TESTS
Males treated with 1000 mg/kg/day showed a statistically significant increase in total and asymptotic motor activity while females from the same treatment group displayed a statistically significant decrease in asymptotic activity. In the absence of any other evidence to suggest neurotoxicity or other toxicologically significant effects of treatment, these isolated intergroup differences were considered to be accidental and of no toxicological significance (see Table 3 under "Any other information on results incl. tables").

ORGAN WEIGHTS
No treatment-related effect on body weight was noted. Males treated with 500 mg/kg/day showed a statistically significant reduction in absolute epididymides weight when compared with control but, in the absence of a convincing dose-response relationship, the intergroup difference was considered to be incidental and of no toxicological importance (see Table 4 under "Any other information on results incl. tables").

GROSS PATHOLOGY
Necropsy revealed no substance-related findings. One male and female animal treated with 150 mg/kg/day and a female animal treated with 1000 mg/kg/day displayed dark foci on the lungs. These findings showed non dose-related response and where considered to be of non toxicological importance.

HISTOPATHOLOGY: NON-NEOPLASTIC
Three males treated with 1000 mg/kg/day demonstrated globular accumulations of eosinophilic material in the proximal tubular epithelium which should be regarded as a possible effect of treatment. The authors considered this finding consistent with the appearance of hydrocarbon nephropathy, which results from the excessive accumulation of α2-microglobulin in renal proximal tubular epithelium of adult male rats, which does not represent a hazard to human health.

Effect levels

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

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Table 1. Group mean weekly body weight gains (males).

Dose Level	Increase in bodyweight (g) during Week         1            2            3          4
Group 1 0 mg/kg bw	62 ± 5	53 ± 4	51 ± 8	35 ± 5
Group 2 150 mg/kg bw	56 ± 5	54 ± 4	53 ± 8	38 ± 6
Group 3 500 mg/kg bw	50 ± 6**	51 ± 3	47 ± 9	38 ± 10
Group 4 1000 mg/kg bw	53 ± 5*	51 ± 5	51 ± 9	35 ± 6

*: significant different from control group p < 0.05

**: significant different from control group p < 0.01

Table 2. Group mean haematological values (males).

Dose Level	MCHC (g/d)	PLT (10^9/L)
Group 1 0 mg/kg bw	34.6 ± 0.4	792 ± 67
Group 2 150 mg/kg bw	34.3 ± 0.5	928 ± 115
Group 3 500 mg/kg bw	34.2 ± 0.3	944 ± 68*
Group 4 1000 mg/kg bw	33.7 ± 0.7*	920 ± 76

*: significant different from control group p < 0.05

Table 3. Group mean functional performance test values.

Dose Level (mg/kg bw/day)	Motor activity overall/ Number of animals (% mobile)	Motor activity final 20% of trial/ Number of animals (% mobile)	Motor activity final 20% of trial/ Number of animals (% activity)
control males	5.0 ± 3.8	0.0 ± 0.0	15.1± 25.4
control females	15.4 ± 6.9	9.2± 6.9	49.5± 6.9
150 males	5.0 ± 4.3	0.4± 0.9	21.1± 43.7
150 females	21.5 ± 4.6	14.9± 11.5	49.4± 28.7
500 males	5.8 ± 6.1	0.4± 0.5	11.8± 13.0
500 females	21.7 ± 7.5	47.2 ± 27.3	47.2± 27.3
1000 males	18.3 ± 5.9**	9.9 ± 8.5*	55.4± 32.2
1000 females	17.2 ± 6.0	0.3± 0.3	5.1± 7.6*

*: significant different from control group p < 0.05

**: significant different from control group p < 0.01

Table 4. Group mean absolute organ weights.

Dose Level	Epididymides (g)
Group 1 0 mg/kg bw	1.2251 ± 0.0555
Group 2 150 mg/kg bw	1.0521 ± 0.1711
Group 3 500 mg/kg bw	0.9431 ± 1.031**
Group 4 1000 mg/kg bw	1.0538 ± 0.1125

**: significant different from control group p < 0.01