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Repeated dose toxicity: oral

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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-conform OECD Guideline study.
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA, Health Effects Test Guidelines; OPPTS 870.3650: Combined Repeated Dose Toxicity Study With the Reproduction/Developmental Toxicity Screening Test
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
1,2,3-Propanetriol, oligomers, docosanoate
EC Number:
613-583-7
Cas Number:
64366-79-6
Molecular formula:
C22 H44 O2 . x (C3 H8 O3)x
IUPAC Name:
1,2,3-Propanetriol, oligomers, docosanoate
Test material form:
other: solid melt
Details on test material:
Purity: 100 area-%

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 10-11 weeks
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sulzfeld, Germany
- Weight at study initiation: Males: 321 - 322 g, Females 205 - 209 g
- Fasting period before study: no
- Housing: individually in Makrolon type M III cages;
- Exceptions:
- During overnight matings, male and female mating partners were housed together in Makrolon type M III cages
- Pregnant animals and their litters were housed together until PND 4 (end of lactation).
- For motor activity (MA) measurements the animals were housed individually in polycarbonate cages (floor area of about 800 cm2) and
small amounts of bedding material
- Diet, ad libitum: Ground Kliba maintenance diet mouse-rat “GLP” meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland
- Water, ad libitum: drinking water
- Acclimation period: approx. 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24°C
- Humidity: 30-70%
- Air changes (per hr): 15
- Photoperiod: 12-hour light/12-hour dark cycle

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was applied as a suspension. To prepare this suspension, the appropriate amounts of test substance were reduced to small pieces using a Dito Sama K 55E Cutter, then pestled and weighed out depending on the desired concentration. Then, drinking water containing 1% carboxymethylcellulose was filled up to the desired volume, subsequently released with a high speed homogenizer. During administration of the test substance, preparations were kept homogeneous by stirring with a magnetic stirrer. The test substance preparations were produced at least once a week.

VEHICLE
- Concentration in vehicle:
1.0 g/100 mL (100 mg/kg bw/d)*, 3.0 g/100 mL (300 mg/kg bw/d)*, 10.0 g/100 mL (1000 mg/kg bw/d)*
*) The dose refers to the body weight of the individual rats determined most recently.
- Amount of vehicle (if gavage): 10 mL/kg bw/d
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test substance in drinking water containing 1% carboxymethylcellulose for a period of 7 days at room temperature was proven before the start of the study. At the beginning of the administration period samples were taken from the lowest and highest concentration for homogeneity analysis (were also used for concentration control). From the mid concentration a concentration control analysis was carried out. The various analyses confirmed the stability of the test substance in the vehicle (drinking water containing 1% carboxymethylcellulose) over a period of 7 days at room temperature and confirmed the overall accuracy of the prepared concentrations.
Duration of treatment / exposure:
Males were exposed for 35 days (prior to mating, during mating, and up to termination) and females for 50 days (prior to mating, during mating, during post-coitum, and at least 4 days of lactation).
Frequency of treatment:
Daily
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the results of a dose range finding study.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity.
The littering and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams.
On weekdays (except public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings.

DETAILED CLINICAL OBSERVATIONS: Yes
Detailed clinical observations (DCO) were performed in all animals prior to the administration period and thereafter at weekly intervals. The animals were transferred to a standard arena (50 × 37.5 cm with sides of 25 cm high).
The following parameters were examined: abnormal behavior when handled, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, impairment of gait, lacrimation, palpebral closure, exophthalmus, feces (appearance/consistency), urine, pupil size

BODY WEIGHT: Yes
Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period body weight was determined on study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning).
The body weight change of the animals was calculated from these results.
The following exceptions are notable for the female animals:
- During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
- Females with litter were weighed on the day of parturition (PND 0) and on PND 4.
- Females without a litter and without positive evidence of sperm in the vaginal smear were weighed weekly. These body weight data were solely used for the calculations of the dose volume.

FOOD CONSUMPTION:
Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
- Food consumption was not determined during the mating period (male and female F0 animals)
- Food consumption of the F0 females with evidence of sperm was determined on GD 0-7, 7-14, 14-20.
- Food consumption of F0 females, which gave birth to a litter, was determined for PND 1-4.
Food consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel) and in males after the premating period.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: In the morning towards the end of the administration period
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group
- Parameters examined: see Table 1 in 'Any other information on materials and methods incl. tables'.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: In the morning towards the end of the administration period
- Animals fasted: Yes
- How many animals: first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group
- Parameters examined: see Table 2 in 'Any other information on materials and methods incl. tables'.

URINALYSIS: Yes
- Time schedule for collection of urine: overnight towards the end of the administration period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: see Table 3 in 'Any other information on materials and methods incl. tables'.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Functional observational battery (FOB) was performed in the first five male animals per test group and the first five surviving females with litter (in order of delivery) of all test groups at the end of the administration period
- Dose groups that were examined: all

- Battery of functions tested:
Home cage observations:
Attention was paid to: Posture, Tremors, Convulsions, Abnormal movements, Gait abnormalities.
Open field observations:
Behavior when removed from cage, fur, skin, salivation, nose discharge, lacrimation, eyes/pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements/stereotypes, impairment of gait, activity/arousal level, feces excreted within two minutes (number/appearance/consistency), urine excreted within two minutes (amount/color), number of rearings within two minutes.
Sensorimotor tests/reflexes:
Approach response, touch response, vision ("visual placing response"), pupillary reflex, pinna reflex, audition ("startle response"), coordination of movements ("righting response"), behavior during "handling", vocalization, pain perception ("tail pinch"), grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test.

Motor activity (MA) measured on the same day as the FOB was performed in the first five parental males and the first five surviving females with litter (in order of delivery) per group. The animals were placed in new clean polycarbonate cages with a small amount of bedding for the duration of the measurement. Eighteen beams were allocated per cage. The numbers of beam interrupts was counted over 12 intervals of 5 minutes per interval. The sequence in which the animals were placed in the cages was selected at random. On account of the time needed to place the rats in the cages, the starting time was "staggered" for each animal. The measurement period began when the 1st beam was interrupted and finished exactly 1 hour later. No food or water was offered to the animals during these measurements and the measurement room was darkened after the transfer of the last animal.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs.
Organ weights: Weight assessment was carried out on all animals.
- The following weights were determined: Anesthetized animals, epididymides, testes.
- The following weights were determined in 5 animals/sex and test group (females with litters, same animals as used for clinical pathology examinations): Adrenal glands, brain, heart, kidneys, liver, spleen, thymus

HISTOPATHOLOGY: Yes
The following organs/ tissues were preserved in neutral-buffered 4% formaldehyde or in modified Davidson’s solution: Adrenal glands, All gross lesions, Aorta, Bone marrow (femur), Brain, Cecum, Cervix, Coagulating glands, Colon, Duodenum, Eyes with optic nerve, Esophagus, Extraorbital lacrimal gland, Epididymides (modified Davidson’s solution), Femur with knee joint, Heart, Ileum, Jejunum (with Peyer’s patches), Kidneys, Larynx, Liver, Lungs, Lymph nodes (axillary and mesenteric), Mammary gland (male and female), Nose (nasal cavity), Ovaries (modified Davidson’s solution), Oviducts, Pancreas, Parathyroid glands, Pharynx, Pituitary gland, Prostate gland, Rectum, Salivary glands, (mandibular and sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, Spinal cord (cervical, thoracic and lumbar cord), Spleen, Sternum with marrow, Stomach (forestomach and glandular stomach), Target organs, Testes (modified Davidson’s solution), Thymus, Thyroid glands, Trachea, Urinary bladder, Uterus, Vagina.
For further evaluation proceeding, see Table 4 in 'Any other information on materials and methods incl. tables'.
Other examinations:
The supplier assayed the food used in the study for chemical and microbiological contaminants.
The drinking water is regularly assayed for chemical contaminants.
The bedding and the enrichment are regularly assayed for contaminants (chlorinated hydrocarbons and heavy metals).
Statistics:
Please refer to 'Any other information on materials and methods incl. tables'.

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS AND MORTALITY (all groups)
Parental animals: No rat died prematurely in the present study. One female animal of test group 3 (1000 mg/kg bw/d) showed a hard abdomen on DCO day 49 – The histopathological finding was a severe dilation of the uterus. This finding was assessed as being spontaneous in nature and not related to the test substance dosing. In summary, no abnormal clinical signs were observed in males and females.
Reproductive performance: One sperm-positive F0 female of test group 0, one sperm-positive F0 female of test group 2 and one sperm-positive F0 female of the test group 3 did not deliver any F1 pups.

BODY WEIGHT AND WEIGHT GAIN
Mean body weights of the F0 males and females in all test groups were comparable to the concurrent control group throughout the entire study period. Body weight change was significantly decreased in males of test group 1 (100 mg/kg bw/d) on premating days 0 to 7 and 0 to 13 (up to -35.3%). In males of test group 2 (300 mg/kg bw/d) from premating days 7 to 13 and 0 to 13 (up to -45.6%) and in males of test group 3 (1000 mg/kg bw/d) from premating days 7 to 13 and 0 to 13 (up to -41.7%) the body weight change was also significantly decreased. Body weight change was significantly increased in females of test group 2 (300 mg/kg bw/d) from premating days 0 to 7. Because of no deviations in mean body weights of both sexes in all test groups and only slight reduced food consumption in test group 3 these findings were assessed as being incidental.

FOOD CONSUMPTION
Food consumption of the male animals in test group 3 (1000 mg/kg bw/d) was slightly decreased in the premating period from study days 7-13 (-5.1%). Because of a single incidence this finding was assessed as being incidental.

HAEMATOLOGY
No treatment-related changes among hematological parameters were observed. In males of test group 1 (100 mg/kg bw/d) hematokrit values were lower compared to controls, but the means were not dose-dependently changed. In males of test group 3 (1000 mg/kg bw/d) absolute monocyte counts were increased, but the mean was within the historical control range (absolute monocyte counts 0.06-0.16 Giga/L). Therefore, both mentioned alterations were regarded as incidental and not treatment-related.

CLINICAL CHEMISTRY
No treatment-related changes among clinical chemistry parameters were observed. In males of test group 1 (100 mg/kg bw/d), potassium levels were decreased, but the means were not dose-dependently changed. In females of test group 2 (300 mg/kg bw/d) sodium and chloride values were higher compared to controls and additionally in females of test group 3 (1000 mg/kg bw/d) sodium levels were increased, but all mentioned values were within historical control ranges (sodium 139.6-148.8 mmol/L, chloride 100.2-107.2 mmol/L). Therefore, all mentioned alterations were regarded as incidental and not treatment-related.

URINALYSIS
No treatment-related changes among urinalysis parameters were observed. In males of test group 3 (1000 mg/kg bw/d) specific gravity of the urine was increased and urine volume was decreased (the latter value not statistically significantly altered). These alterations were regarded as adaptive reaction of the kidneys towards less fluid intake and therefore were regarded as not adverse.

NEUROBEHAVIOUR
Home cage observations: No test substance-related effects were observed.
Open field observations: No test substance-related effects were observed.
Sensorimotor tests/reflexes: No test substance-related effects were observed.
Quantitative Parameters: No test substance-related effects were observed.
Motor activity measurement: There were no significant deviations concerning the overall motor activity (summation of all intervals) in male and female animals.

ORGAN WEIGHTS
All mean absolute and relative weight parameters did not show significant differences when compared to the control group 0 and are therefore considered to be within the normal range.

GROSS PATHOLOGY / HISTOPATHOLOGY
All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
The three female animals (one each from test groups 0, 2, and 3), which were not pregnant as well as the male mating partners did not show relevant gross lesions. One male with impaired fertility of the control group showed a moderate multifocal lymphoid infiltration in the epididymis. The infiltration was minimal and does not explain the infertility. Two of the female animals that were not pregnant (one each from test groups 0 and 3) and the male mating partner (from test group 3) did not show relevant histopathological findings (the female and male animals with impaired fertility from test group 2 were not investigated histopathologically).

Effect levels

open allclose all
Dose descriptor:
NOAEL
Remarks:
general, systemic toxicity
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
1 000 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:
Under the conditions of this reproduction/developmental toxicity screening test the NOAEL (no observed adverse effect level) for reproductive performance and fertility was 1000 mg/kg bw/d. The NOAEL for developmental toxicity in the F1 progeny was found to be 1000 mg/kg bw/d. The NOAEL for general, systemic toxicity was 1000 mg/kg bw/d.
Executive summary:

The study was performed according to OECD guideline 422 in compliance with GLP.

Polyglycerintribehenat was administered orally via gavage to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels of 0 mg/kg bw/d (test group 0), 100 mg/kg bw/d (test group 1), 300 mg/kg bw/d (test group 2) and 1000 mg/kg bw/d (test group 3).

Regarding clinical examinations, no signs of general systemic toxicity were observed in male or female parental animals of all test groups (100, 300 and 1000 mg/kg bw/d) during the entire study.

Regarding fertility and reproductive performance, no signs of toxicity were observed in male or female parental animals of all test groups (100, 300 and 1000 mg/kg bw/d) during the entire study. 

Regarding clinical pathology, no treatment-related, adverse effects were observed up to a dose of the compound of 1000 mg/kg bw/d.

Regarding pathology, there were neither treatment-related organ weight changes, nor macroscopic or histopathologic findings.

All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

The treatment of male and female Wistar rats with dose levels of 100, 300 and 1000 mg/kg bw/d of the test substance did not lead to adverse findings. Especially no effects of the test substance on the reproduction tract were observed. All other findings were considered to be incidental or spontaneous in origin and without any relation to treatment.

Conclusion: Under the conditions of this reproduction/developmental toxicity screening test the NOAEL for reproductive performance and fertility was 1000 mg/kg bw/d. The NOAEL for developmental toxicity in the F1 progeny was found to be1000 mg/kg bw/d. The NOAEL for general, systemic toxicity was 1000 mg/kg bw/d.