1,2,3-Propanetriol, homopolymer, diisooctadecanoate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test”

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1,3-dihydroxypropan-2-yl 16-methylheptadecanoate; 2-hydroxy-3-[(16-methylheptadecanoyl)oxy]propyl 16-methylheptadecanoate; 2-hydroxy-3-{2-hydroxy-3-[(16-methylheptadecanoyl)oxy]propoxy}propyl 16-methylheptadecanoate; 3-(2,3-dihydroxypropoxy)-2-hydroxypropyl 16-methylheptadecanoate; 3-(2,3-dihydroxypropoxy)-2-hydroxypropyl hexadecanoate

EC Number:

613-333-7

Cas Number:

63705-03-3

Molecular formula:

Unspecified

IUPAC Name:

1,3-dihydroxypropan-2-yl 16-methylheptadecanoate; 2-hydroxy-3-[(16-methylheptadecanoyl)oxy]propyl 16-methylheptadecanoate; 2-hydroxy-3-{2-hydroxy-3-[(16-methylheptadecanoyl)oxy]propoxy}propyl 16-methylheptadecanoate; 3-(2,3-dihydroxypropoxy)-2-hydroxypropyl 16-methylheptadecanoate; 3-(2,3-dihydroxypropoxy)-2-hydroxypropyl hexadecanoate

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): 1,2,3-Propanetriol, homopolymer, diisooctadecanoate
- Physical state: yellowish/clear viscous liquid
- Analytical purity: 100% (reaction mixture)
- Batch No.: CD11110003
- Expiration date of the lot/batch: 31-Aug-2013
- Storage condition of test material: room temperature (15 - 25 °C)

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Age at study initiation: 11 weeks
- Weight at study initiation: 302 - 380 g (males),180 - 247 g (females)
- Housing: In groups of three to five in Makrolon type-4 cages with wire mesh tops up to the day of randomization and afterwards individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J. Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) with paper enrichment (Enviro-dri from Lillico, Biotechnology, Surrey, UK, batch/lot nos. 72 and 79 and ISO-BLOX from Harlan Laboratories B.V., Netherlands, batch/lot nos. T.6960C.CS-100099, 100267). During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus
cycles.
- Diet (ad libitum): pelleted standard Harlan Teklad 2018C (batch nos. 43/12 and 56/12) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland)
- Water (ad libitum): community tap-water
- Acclimation period: at least seven days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): Based on an observation on 01-Feb-2013, the automatic light cycle of 12 h on / 12 h off was not functional within a block of rooms including room numbers 015A and 011 B. Due to the breeding performance from November 2012 onwards during this and other studies conducted in rooms which belong to the affected block, it was considered probable that the animals had been maintained under constant lighting throughout the study period for the first delivery animals. From 01-Feb-2013 onwards, the automatic light cycle was restored.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: daily. The test substance was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle was added (w/v). Using an appropriate homogenizer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added. Separate formulations were prepared for each concentration.
Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

VEHICLE
- Amount of vehicle: 4 mL/kg bw (dose volume)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

On the first dose formulation day samples from the control group as well as three samples (top, middle and bottom) of about 1 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 1 g of each test item concentration were taken from the middle to confirm the stability (4 hours and 8 days at room temperature (20 ± 5°C)).
Towards the end of the study of the first and third delivery of animals, samples were taken from the middle to confirm concentration. Additional samples were taken on 21-Feb-2013 (first application of the second delivery of animals) from the middle to confirm concentration. The aliquots for analysis of dose formulations were frozen (-20 ± 5 °C) and delivered on dry ice or at room temperature to Dr. G. Heinemann (Harlan Laboratories Ltd., Zelgliweg 1, 4452 Itingen /Switzerland) and stored there at -20 ± 5 °C until analysis or were analyzed directly.

As the results obtained were out of the accepted limits, a new analytical method was implemented, which necessitated additional dose formulation samples mainly from the low dose levels (for details please refer the analytical report). For the implementation of the method additional samples were taken during the study, which were not reported.

The samples were analyzed by derivatisation with MSTFA/BSTFA and analysis by GC-FID or analyzed by HPLC using a method which was implemented within the present study. Analyzed samples were not discarded without written consent from the study director.

Duration of treatment / exposure:

Males: Minimum 28 days
Females: Approximately 6 weeks

Frequency of treatment:

once daily (within four hours)

Doses / concentrations

Remarks:

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

No. of animals per sex per dose:

at least 12 (see any other information on materials and methods)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on single dose toxicity data of the test compound and repeated dose toxicity data of chemically similar compounds, the results of a 14-day range-finding study the dose levels of 100, 300, and 1000 mg/kg bw per day were selected for the present study.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once prior to the first administration of the test item and weekly thereafter (in the gestation period on day 0, 6, 13 and 20 post coitum).
- detailed clinical observations were performed outside the home cage in a standard arena and the animals were observed for the following changes in: skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behavior were also reported.

BODY WEIGHT: Yes
- Time schedule for examinations: daily from treatment start to day of necropsy.

FOOD CONSUMPTION: Yes
- Males: Pre-pairing period days 1 - 4, 4 - 8, 8 - 11 and 11 - 14; after pairing period weekly.
- Females: Pre-pairing period days 1 - 4, 4 - 8, 8 - 11 and 11 - 14; gestation days 0 – 7, 7 14 and 14 – 21 and days 1 - 4 of the lactation
No food consumption was recorded during the pairing period.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were obtained on the day of the scheduled necropsy from 5 males randomly selected from each group. Blood samples from 5 lactating females from each group will be obtained on day 5 postpartum.
- Anaesthetic used for blood collection: Yes (light isoflurane anesthesia)
- Animals fasted: Yes, for approximately 18 hours
- How many animals: 5 males randomly selected from each group and from 5 lactating females from each group
- Parameters checked: erythrocyte count, haemoglobin, haematocrit, mean corpuscular volume, red cell volume distribution width, mean corpuscular haemoglobin, mean corpuscular hemoglobin concentration, haemoglobin concentration distribution width, leukocyte count (total), differential leukocyte count: platelet count, reticulocytes; prothrombin time (= thromboplastin time), activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: see haematology
- Animals fasted: Yes, for approximately 18 hours
- How many animals: see haematology
- Parameters checked:
Glucose
Urea
Creatinine
Bilirubin, total
Cholesterol, total
Triglycerides
Aspartate aminotransferase
Alanine aminotransferase
Alkaline phosphatase
Gamma-glutamyl-transferase
Bile acids
Sodium
Potassium
Chloride
Calcium
Phosphorus
Protein, total
Albumin
Globulin
Albumin/Globulin ratio

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: males towards the end of the first pairing period and females on day 3 post partum
- Dose groups that were examined: five P generation males and five P generation females randomly selected from each group
- Battery of functions tested:
• Cage-side observations: faeces-balls, urine and posture as well as resistance to removal.
• Hand-held observations: muscle tone, constituation, skin, pupile size, palpebral closure, lacrimation, salivation, reaction to handling and general abnormalities.
• Open field observations: level of ambulatory activity including rearing (one minute evaluation), unusual body movements (e.g. spasms, convulsions), gait evaluation, behavior, hair coat, respiration, quantity of faeces-balls and urine.
• Reflexes: blinking, palpebral closure, pinna reflex, extensor thrust response, paw pinch, responsiveness to sharp noise, righting reflex and hearing ability (Preyer’s reflex).
• Measurements / Counts: hind limb / fore limb grip strength, rectal temperature.
Additionally, locomotor activity was measured quantitatively for the same animals. Activity was measured with an Activity Monitor AMS-0151 (FMI, Germany). Activity of the animals (based on beam count) was recorded for 6-minute intervals over a period of 30 minutes. These data and the total activity over 30 minutes were reported.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
At the scheduled sacrifice, the testes and epididymides of all parental males were weighed separately. In addition, from 5 males and 5 females killed at the end of the study which were selected for hematology and clinical chemistry examination from each group, the following organs were trimmed from any adherent tissue, as appropriate, and their wet weight taken.
Adrenal glands (weighed as pairs)
Brain
Heart
Kidneys (weighed as pairs)
Uterus (including cervix)
Prostate
Liver
Thymus
Spleen
Thyroid (after fixation)
Ovaries (weighed as pairs)
Seminal vesicles (inclusive coagulating gland)

From all animals of the third delivery, following organs were weighed:
Testes
Epididymides
Heart
Liver
Kidneys (weighed as pairs)
Adrenal glands (weighed as pairs)

HISTOPATHOLOGY: Yes
The following tissues from all parental males were preserved in neutral phosphate buffered 4% formaldehyde solution:
Prostate
Seminal vesicles with coagulating gland
Testes (in Bouin’s fixative)
Epididymides (in Bouin’s fixative)
The following tissues from all parental females were preserved in neutral phosphate buffered 4%
formaldehyde solution:
Ovaries (with oviduct) Uterus (with vagina)

In addition, from all males and females the following tissues were preserved in neutral phosphate
buffered 4% formaldehyde solution:
Gross lesions
Brain
Spinal chord (cervical, thoracic, lumbar)
Small and large intestines (incl. Peyer’s patches)
Stomach (forestomach and glandular stomach)
Liver
Kidneys
Adrenals
Lymph nodes (axillary and mesenteric)
Urinary bladder
Aorta
Eyes with optic nerve and harderian gland
Lacrimal gland
Larynx
Nasal cavity
Esophagus
Heart
Thymus
Thyroids and parathyroids
Trachea and lungs (preserved by inflation
with fixative and then immersion)
Pituitary gland
Spleen
Peripheral nerve (sciatic)
Bone marrow (femur)
Femur with knee joint
Mammary gland (male and female)
Pancreas
Salivary glands – mandibular, sublingual
Skeletal muscle
Sternum with bone marrow
Pharynx

All organ and tissue samples to be examined by the study pathologist were processed, embedded and cut at an approximate thickness of 2 - 4 micrometers and stained with hematoxylin and eosin. Additionally, the testis was stained by PAS-hematoxylin.
Testes, epididymides, prostate, seminal vesicles, ovaries, oviduct, vagina and uterus from all animals of all three animal deliveries of the control and high-dose group were examined. The same applied to all occurring gross lesions. The remaining organs/tissues of 5 randomly selected males and females of the control and high-dose group, respectively, were examined histopathologically. From the 3th delivery of animals no additional organs/tissues of 5 randomly selected males and females of the control and high dose group were examined.
Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.
Histological examination of ovaries was carried out on the females that did not give birth. In addition, microscopic examination of the reproductive organs of males that did not successfully mate with the allocated female was performed.

Statistics:

The following statistical methods were used to analyze food consumption, body weights and reproduction data:
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables were assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test will be applied if the variables could be dichotomized without loss of information

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

see details on results

Mortality:

mortality observed, treatment-related

Description (incidence):

see details on results

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

see details on results

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

see details on results

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):

see details on results

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

see details on results

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

see details on results

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

see details on results

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

see details on results

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

see details on results

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
At a dose level of 1000 mg/kg bw/day, one male (no. 171, third animal delivery) died spontaneously on day 1 of the after pairing period. No clinical signs were observed and a normal body weight gain was recorded until day 13 of the paring period. One day before he died he showed a body weight loss of 3%. During necropsy, a dark red discolored thymus was recorded.

BODY WEIGHT AND WEIGHT GAIN
Males:
Mean body weight gain was statistically significantly lower at 1000 mg/kg bw/day during the pre-pairing period, resulting in slightly reduced mean body weights (-3% at the end of this period compared to the controls). This was a result of the minor body weight loss (1%) between days 1 to 2. During the pairing period, body weight gain at the high dose level was similar to the control value. Due to this fact, the reduction in mean body weight gain and body weights were considered to be not adverse. The overall differences in mean body weight gain at the dose levels of 0, 100, 300 and
1000 mg/kg bw/day were: +13%, +12%, +11% and +11% during the pre-pairing period, +5%, +5%, +3% and +4% during the pairing period and +7%, +9%, +7% and +8% during the after pairing period (percentages refer to the body weight gain within the period).

Females:
No effect of the test item on mean body weights and mean body weight gain was noted for females at any dose level. Towards the end of the gestation period, body weights and body weight gain were reduced in females at 1000 mg/kg bw/day (statistically significantly different body weight gains from day 17 post coitum onwards). These differences were considered to be a result of the increased post implantation loss and low number of pups, which were noted for dams at this dose level. The overall differences in mean body weight gain at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day were: +8%, +9%, +8% and +7% during the pre-pairing period, +63%, +57%, +57% and +51% during the gestation period and +4%, +7%, +4% and +3% during days 1 to 4 of the lactation period (percentages refer to the body weight gain within the period).

FOOD CONSUMPTION AND COMPOUND INTAKE
Males:
At 1000 mg/kg bw/day, mean food consumption was statistically significantly reduced during the first 11 days of treatment in males of the first and second delivery (-9.3% on day 11 of prepairing when compared with controls). Thereafter, the food consumption remained slightly lower than in controls, but without reaching statistical significance. Food consumption was also lower in males of the third delivery, but not to a statistically significant degree. Since this reduction in food consumption occurred only transiently, the reduction was considered to be not adverse.

Females:
There were no effects on mean food consumption at any dose level and in any study phase.

HAEMATOLOGY
The assessment of the hematology data did not reveal any test item-related effects in males and females at any dose level.

CLINICAL CHEMISTRY
The assessment of the clinical biochemistry data did not reveal any test item-related effects in males and females at any dose level.
Slightly higher urea levels were noted in males at 300 mg/kg bw/day and in females at 1000 mg/kg bw/day. Additionally, lower alkaline phosphatase levels were noted in females at 300 mg/kg bw/day, higher creatine levels in males and lower glucose levels in females at 1000 mg/kg bw/day, respectively. However, in the absence of dose-dependency and/or since values were additionally within the historical control range, a test item-related effect can be excluded.

NEUROBEHAVIOUR
No clinical signs were noted in males during the course of this study.
Incidentally, one control female (no. 45) showed a slightly decreased activity, ruffled fur and vaginal bleeding on day 25 post coitum. Necropsy of this female revealed masses in the uterus and vagina. Furthermore, one female (no. 66) treated at 100 mg/kg bw/day that did not give birth around day 21 post coitum either showed a wound on the anterior dorsum during the last three days prior to necropsy on day 25 post coitum. During necropsy reddish discolored ovaries and enlarged axillary lymph nodes were noted. Both females were pregnant.
These isolated findings were not related to treatment with the test item.

No findings were noted at detailed weekly clinical observation during the whole course of the
study.

There were no treatment-related changes noted at functional observational battery. One male at 100 mg/kg bw/day had increased faeces-balls and one female at 300 mg/kg bw/day showed decreased rearings. These isolated findings showing no dose relationship were deemed unrelated to treatment with the test item. A statistically significantly lower mean body temperature was noted in males of the first and second delivery at 1000 mg/kg bw/day (37.8 °C compared to 38.5 °C in the control group). No similar effect was noted for males treated at 100 and 300 mg/kg bw/day or in females. Since the value of 37.8°C was borderline to the range of the historical control data (38.0°C-38.6°C), and no change was noted in females, this finding was considered to be incidental.

ORGAN WEIGHTS
Treatment with the test item at 1000 mg/kg bw/day resulted in statistically significantly higher absolute and relative liver and kidney weights in animals of both genders, which was confirmed in animals of the third delivery except for kidney weights in females. Since there was no evidence for an impairment of organ function by clinical pathology and histopathology, these findings were not considered to be adverse. Additionally, in females, absolute and relative heart weights were also increased. This finding was without histopathological correlate, and was therefore considered to be incidental.
There were no effects of treatment at 100 and 300 mg/kg bw/day.

GROSS PATHOLOGY
All findings were typical of this strain and age of rat and were considered to be incidental.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no treatment-related findings.
In particular, qualitative examination of the stages of spermatogenesis in the testis did not reveal any treatment-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle. No treatment-related microscopic abnormalities were observed
in the evaluation of the ovarian follicles and corpora lutea of the ovaries or the evaluation of the uterus.
All findings were typical of this strain and age of rat and were considered to be incidental.

Effect levels

1

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

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

Based on:

test mat.

Sex:

male/female

2

Dose descriptor:

NOEL

Remarks:

systemic

Effect level:

>= 300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Based on these results, the NOAEL for general toxicity was considered to be 1000 mg/kg bw/day. The NOEL for general toxicity was established at 300 mg/kg bw/day.

Executive summary:

A study investigating the toxicological effects of the test item 1,2,3 -Propanetriol, homopolymer, diidoctadecanoate to rats according to OECD guideline 422 resulting from repeated oral-gavage administration is performed. 1,2,3 -Propanetriol, homopolymer, diidoctadecanoate was administered in corn oil as vehivle at dosages of 100, 300, 1000 mg/kg bw/day, and control received the vehicle control only.

To meet guideline requirements concerning number of pregnant females per group, male and female rats were added in a second delivery for breeding due to reduced fertility observed in all groups of the first delivery. It is believed, that a disturbance of the light / dark cycle was the reason for the reduced mating rate of the females of the first delivery. The additional animals ran through the whole study as the animals from the first delivery with the exception that the males went to necropsy on day 24 of the after pairing period and not on day 16 of the pairing period as the males of the first delivery (logistical reasons). Therefore, 1,2,3-Propanetriol, homopolymer, diisooctadecanoate was administered to male rats for up to 28 days (first delivery) and up to 41 days (second delivery) and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

Because an impact of the light/dark cycle disturbance during the study on these results (a prolonged duration of gestation and an increased post-implantation loss was observed at the high dose) could not be excluded, the study was repeated with a third animal delivery of control and high-dose (1000 mg/kg bw/day) groups under proper light conditions. Test item was administered

to 12 male rats per group for up to 33 days and to 12 female rats per group for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

All animals survived until scheduled necropsy. There were no treatment-related clinical signs or effects on fuctional observation battery or locomotor activity. No test-titem related findings were noted during the clinical laboratory investigations.

Liver and kidney weights were slightly increased at 1000 mg/kg bw/day in both sexes of the first and second delivery, which was confirmed in animals of the third delivery except for kidney weights in females. These changes were considered compound-related, but not adverse, since there was no evidence for an impairment of organ function by clinical pathology and histopathology. There were no treatment-related necropsy or microscopic findings.

Overall at 1000 mg/kg bw/day, food consumption and body weight gain was transiently reduced in males at the beginning of the treatment period which was considered to be not adverse.

Based on these results, the NOAEL (No Observed Adverse Effect Level) for general parental toxicity was considered to be 1000 mg/kg body weight/day. The NOEL (No Observed Effect Level) for general parental toxicity was established at 300 mg/kg bw/day.