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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 2016 to October 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
GLP compliance:
yes (incl. QA statement)
Limit test:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Pentaerythritol
EC Number:
204-104-9
EC Name:
Pentaerythritol
Cas Number:
115-77-5
Molecular formula:
C5H12O4
IUPAC Name:
pentaerythritol
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The Han Wistar rat was chosen as the animal model for this study as it is a rodent species and strain accepted by regulatory agencies for toxicity testing.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Source: Charles River UK Limited
Age of animals at initiation of dosing: 7-8 weeks
Weight of animals at initiation of dosing: 202 to 277 g (males) and 139 to 199g (females)
Housing: Prior to stratification, animals were housed up to 5 per cage per sex. Following stratification, animals were housed 2 per cage by sex.
Caging: polycarbonate cages with stainless steel grip tops and and solid bottoms
Bedding: sterilised wood shavings with no known contaminants
Temperature: 20-21°C
Relative humidity: 40-70%
Photoperiod: 12 hour light/dark cycle
Air changes: At least 10 changes/hour
Diet: SDS Rat and Mouse (modified) No. 1 Diet SQC Expanded; ad libitum
Water: Public supply; ad libitum
Enrichment: Chew devices and devices for hiding in provided

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The dose volume was 10 mL/kg. The dose volume was based on the most recent body weight measurement. Doses were given using a syringe with attached gavage cannula.
Vehicle:
corn oil
Details on oral exposure:
The oral route of administration was selected for this study as this was the preferred route of exposure specified by the regulatory requirements.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples were collected for analysis on Day 1, during Week 3, 6 and 12 of dosing. The dosing solutions for all groups (including the vehicle contol) were analysed for acheived concentration and the test item formulations were analysed for homogeneity.

Duplicate top, middle and bottom (duplicate middle only for control) sets of samples (0.1 mL) for each sampling time point were sent to the analytical laboratory for analysis. Triplicate top, middle and bottom (duplicate middle only for control) samples (0.1mL) were maintained at the Test Facility as backup samples.

Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% of theoretical concentration. Each individual sample concentration result was considered acceptable if it was within or equal to ± 15%. Homogeneity results were considered acceptable if the relative standard deviation (RSD) of the mean value at each sampling location was ≤10%. After acceptance of the analytical results, backup samples were discarded.

Stability analyses performed previously demonstrated that the test item was stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in this study.
Duration of treatment / exposure:
91 days
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Vehicle control
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
The dose levels were justified based on previous repeated dose studies. A 14-day range-finding study reported effects limited to soft faeces/diarrhoea in females at 1000 mg/kg bw/d. An OECD 422 screening study reported intermittent soft faeces /diarrhoea at 300 and 1000 mg/kg bw/d and a tendency to increased water consumption in males at 1000 mg/kg bw/d. A 28-day study reported no effects at 1000 mg/kg bw/d.
Positive control:
Not required for this study type

Examinations

Observations and examinations performed and frequency:
Animals were observed, twice daily (once at the start and once towards the end of the working day throughout the study), for general health/mortality and moribundity. Animals were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.

All animals were removed from the cage for detailed clinical observations and examined weekly commencing in Week -1.

All the animals were examined for reaction to treatment regularly throughout the day. The onset, intensity and duration of these signs were recorded (if appropriate), particular attention being paid to the animals during and for the first hour after dosing.

All animals were individually weighed twice during pretreatment then daily from Day 1 to completion of the study. A weight was recorded on the days of scheduled necropsy.

Food consumption was quantitatively measured twice during pretreatment then weekly from Day 1 to completion of the study.

The eyes were examined once during pretreatment for all animals, and control and high dose animals in Week 13, using an indirect ophthalmoscope after the application of a mydriatic agent (1% Tropicamide, Mydriacyl®).

Detailed functional observations were performed once during pretreatment and once during Week 12. These examinations were conducted by a technician not involved in the dosing procedures or in the collection of body weight and food consumption data, and were performed at an approximately standardised time of day. Before the independent technician entered the animal room to perform the examinations, the cage card showing treatment group was removed from each cage, leaving the second pre-prepared card as the functional observation animal identifier. In each cage all animals had their tail marked with their functional observation battery number to allow the independent technician to identify each animal.

Homecage observations:
Posture/condition on first approach (animal undisturbed), checking for: Prostration, Stereotypy / bizarre behaviour, Tremors (head, limbs, whole body), Convulsions, Ease of removal from the cage.

Body temperature:
The rectal temperature was recorded in degrees Celsius (ºC), using a probe inserted approximately 2cm past the anal sphincter.

Handling observations:
Condition of the eyes, checking for:Pupillary function, Miosis / Mydriasis, Enophthalmos/ Exophthalmos, Lacrimation and Evaluation of diameter of the pupil.
Condition of the coat
Body tone
Pinna response
Presence of salivation
Overall ease of handling
Respiration rate and pattern

Air righting:
Holding the animal in a supine position, it was dropped from approximately 30 cm and the air righting response was rated.

Extensor thrust
The animal was grasped at the thorax gently from behind and raised off the surface in a vertical position. With their free hand the observer gently but briskly pressed the tips of two fingers (or one finger and thumb) into the middle of the plantar surface (i.e. footpads) of each hind limb (one digit into each footpad). As the rodent extended the hind limbs, the presence/strength of the extensor thrust reflex was evaluated via digital palpation. Several presses were sometimes required to elicit the extensor response.

Observations in a standardised arena (2 minute observation period):
Rearing
Grooming
Urination and defecation
Arousal (level of alertness)
Posture
Tremor (head, limbs, whole body)
Convulsions
Vocalisation
Piloerection
Palpebral closure
Gait abnormalities.
Stereotypy (excessive repetition of behaviours) and/or unusual behaviours.

Functional tests:
Detailed functional observations were performed once during pretreatment and once during Week 12.
The following additional functional tests were performed. Again, these assessments were performed at an approximately standardised time of day: Reaction to sudden sound (click above the head) and reaction to touch on the rump with a blunt probe.
Grip strength:
This was measured using a Dual/Single Channel Grip Strength Meter (Linton Instruments) to which was attached a wire screen assembly. Once the animal had gripped the screen, the body was pulled until its grasp was broken; the strain gauge recorded the force required. The procedure was repeated 3 times for the forelimbs and 3 times for the hindlimbs, and the mean fore and hind grip strengths calculated.
Pain perception:
This was assessed by measurement of the tail flick response, using a technique based on the method devised by D`Amour and Smith (1941)(ii). The apparatus used shone a calibrated infra-red heat source onto the tail and automatically measured the reaction time of the animal (accurate to 0.1 s). It was ensured that no visible injury to the tail was caused in this test.
Landing Foot Splay:
Tempera paint was applied to the hind feet of each animal. The animal was then held in a horizontal, prone position with the nose ca 30 cm above a bench surface covered with absorbent paper. When the animal was calm, it was dropped. The distance between the prints of the central footpads was measured and the average measurement recorded. The procedure was repeated twice. If the rat did not land
properly on its feet, this was recorded.
Motor activity:
Each animal was placed in an individual cage held within a SmartFrame utilising infra-red pyroelectric detectors. Movement was detected in 2 dimensions anywhere in the cage, and was differentiated into basic and fine movements, and X and Y ambulation. Each animal was monitored for one session of 1 h, activity counts being recorded over successive period of 5 minutes each.
Other physical/functional abnormalities:
Any other abnormality not already recorded in the above screening battery.

Clinical pathology:
Blood was collected from the orbital sinus under isoflurane anaesthesia using a capillary tube. Urine was collected over 6 hours with absence of food but presence of water. After collection, samples were transferred to the appropriate laboratory for processing. Prior to blood sampling, animals were not deprived of food. Samples were collected from all animals during week 13 for urinalysis and from all animals prior to necropsy for haematology, coagulation and clinical chemistry.

For haematology, blood samples (0.5 mL) were collected, transferred into tubes containing K2EDTA and analysed for the following parameters:
Red blood cell count
Haemoglobin concentration
Haematocrit
Mean corpuscular volume
Red Blood Cell Distribution Width
Mean corpuscular haemoglobin
concentration
Mean corpuscular haemoglobin
Reticulocyte count (absolute)
Platelet count
White blood cell count
Neutrophil count (absolute)
Lymphocyte count (absolute)
Monocyte count (absolute)
Eosinophil count (absolute)
Basophil count (absolute)
Large unstained cells (absolute)
A blood smear was prepared from each haematology sample. Blood smears were labelled, stained, and stored. Blood smears were evaluated as required to confirm analyser results as per Test Facility SOPs.

For coagulation: Blood samples (0.9 mL) were collected, transferred into tubes containing 3.8% (w/v) trisodium citrate and processed for plasma, which was analysed for the parameters as follows:
Activated partial thromboplastin time
Fibrinogen
Prothrombin time
Sample Quality

For clinical biochemistry: Blood samples (1.0 mL) were collected, transferred into tubes containing lithium heparin and processed for plasma, which was analysed for the parameters as follows:
Alanine aminotransferase
Aspartate aminotransferase
Alkaline phosphatase
Gamma-glutamyltransferase
Creatine Kinase
Total bilirubina
Urea
Creatinine
Calcium
Phosphate
Total protein
Albumin
Globulin
Albumin/globulin ratio
Glucose
Cholesterol
Triglycerides
Sodium
Potassium
Chloride
Sample Quality

Urine samples were analysed for the following parameters:
Colour
Appearance/Clarity
Specific gravity
Volume
pH
Protein
Glucose
Bilirubin
Ketones
Blood
Sacrifice and pathology:
All animals were euthanised by exposure to carbon dioxide, weighed and exsanguinated. When possible, the animals were euthanised in a rotating order across dose groups such that similar numbers of animals from each group, including controls were necropsied at similar times throughout the day. Animals were not fasted before their scheduled necropsy. All animals were subjected to a complete necropsy examination, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues.
Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available.

The brain, epididymis (paired organ), adrenal gland (paired organ), pituitary gland, prostate gland, thyroid gland (paired organ weighed after fixation), heart, kidney (paired organ), liver, lung, ovary (paired organ), spleen, testis (paired organ), thymus and uterus were weighed at necropsy for all scheduled euthanasia animals. Paired organs were weighed together. Organs were weighed before fixation unless stated. Organ to body weight percentage (using the terminal body weight) were calculated.

Representative samples of the tissueslisted below were collected from all animals and preserved in 10% neutral buffered formalin, unless otherwise indicated:
Artery, aorta
Bone marrow smear (air dried and fixed in methanol)
Bone marrow, femur
Bone marrow, sternum
Bone, femur
Bone, sternum
Brain
Cervix
Epididymis
Eye (preserved in Davidson's fixative)
Gland, adrenal
Gland, harderian
Gland, lacrimal
Gland, mammary
Gland, parathyroid
Gland, pituitary
Gland, prostate
Gland, salivary (mandibular)
Gland, seminal vesicle
Gland, thyroid
Gut-associated lymphoid tissue (Peyer’s patches)
Heart
Kidney
Large intestine, caecum
Large intestine, colon
Large intestine, rectum
Lesions/masses
Liver
Lung
Lymph node, mandibular
Lymph node, mesenteric
Muscle, skeletal
Nerve, optic (preserved in Davidson's fixative)
Nerve, sciatic
Oesophagus
Ovary
Oviduct
Pancreas
Skin
Small intestine, duodenum
Small intestine, ileum
Small intestine, jejunum
Spinal cord
Spleen
Stomach
Testis (preserved in Modified Davidson's fixative)
Thymus
Tongue
Trachea
Ureter
Urinary bladder
Uterus
Vagina
The tissue samples, with the exception of bone marrow smears) were embedded in paraffin, sectioned, mounted on glass slides, and stained with haematoxylin and eosin.Histopathological evaluation was performed by a veterinary pathologist with training and experience in laboratory animal pathology.
Two bone marrow smears were taken from the femur. Both bone marrow smears were air dried, fixed in methanol, stained with May-Grunwald-Giemsa stain and coverslipped. Bone marrow smears were not evaluated.
Other examinations:
None
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 0.1%, 1%, and 5% levels. Numerical data collected on scheduled occasions for the listed variables were analysed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible.
Values were also expressed as a percentage of predose or control values when deemed appropriate. Inferential statistics were performed when possible,
(but excluded semi-quantitative data, and any group with less than 3 observations) for the following variables:
Body Weight
Food Consumption
Haematology Variables
Coagulation Variables
Clinical Chemistry Variables
Urinalysis Variables
Organ Weights
FOB Quantitative Variables
Body Weight Change
Organ Weight relative to Body Weight

Pairwise comprisons were made for each test article treated group versus the control group.

Levene’s test was used to assess the homogeneity of group variances. Datasets with at least 3 groups were compared using an overall one-way ANOVA F-test if Levene’s test was not significant or the Kruskal-Wallis test if it was. If the overall F-test or Kruskal-Wallis test was found to be significant, then the above pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively. Datasets with 2 groups (the designated control group and 1 other group) were compared using a t-test if Levene’s test was not significant or Wilcoxon Rank-Sum test if it was.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Ploughing behaviour (pushing nose into shavings) and salivation were observed at all dose levels throughout the study, including the control group. Although also observed in the control group, the number of occasions of ploughing and salivation were observed was generally higher in the groups receiving Pentaerythritol. Ploughing was seen on 7/18, 119/52, 86/58 or 126/109 occasions in males/females, respectively and salivation was seen on 4/29, 73/36, 38/31, or 55/57 occasions in males/females, respectively at 0, 100, 300 or 1000 mg/kg/day. These observations were considered to be a palatability effect of the test item and vehicle and of no toxicological significance. Isolated instances of decreased activity, erect fur and pedalling behaviours were observed, however, there were no patterns, trends or dose response relationships and therefore these were considered of no toxicological significance.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths throughout the course of this study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Bodyweights and body weight gain in treated groups were generally comparable to controls throughout the study.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was unaffected by treatment throughout the course of this study.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no ophthalmoscopy findings considered to be related to Pentaerythritol.
Haematological findings:
no effects observed
Description (incidence and severity):
There were no changes in any haematology or coagulation parameters that were considered to be related to Pentaerythritol.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no changes in any clinical chemistry parameters that were considered to be related to Pentaerythritol. Any observed changes were within normal background ranges and therefore considered unrelated to administration of Pentaerythritol.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no changes in any urinalysis parameters that were considered to be related to Pentaerythritol.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no changes in any function observation parameter that were considered to be related to Pentaerythritol.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No treatment-related organ and body weight changes were noted. Minor, organ and body weight differences observed were considered incidental and unrelated to administration of Pentaerythritol.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment-related gross findings were noted. The gross findings observed were considered incidental, of nature commonly observed in this strain and age of rats, and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to administration of Pentaerythritol.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment-related microscopic findings were noted. The microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of Pentaerythritol.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
No neoplastic findings were observed in any group
Other effects:
not examined
Details on results:
There were no effects of treatment in this study with the exception of post-dose salivation and ploughing

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
Remarks on result:
other: There were no toxicologically relevant effects of treatment at the highest dose level

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

All study samples analysed had mean concentrations within or equal to the acceptance criteria of ±10% (individual values within or equal to ±15%) of their theoretical concentrations, except for Day 1, Groups 2-4 (16.0%, 12.7% and 13.0% respectively), and Week 3, Group 3 (-10.7%). Day 1, Group 4 was rerun and found to confirm these results (13.6%). Day 1, Groups 2-3 analytical samples were also reran for confirmation but these results cannot be reported due to there being no proven stability in solution at these concentrations. The backup samples for Day 1 were not successfully analysed due to a sample preparation error. No residues from Day 1 dose aliquots were available to be analysed as they had been discarded. For these reasons it cannot therefore be confirmed whether the Day 1 samples are actually out of specification or whether the initial results are due to an analytical issue so an extra analytical timepoint at Week 3 was added by the Study Director. As part of the investigation for Week 3, Group 3, the backup samples were analysed in triplicate, and the results (2.0%) were within the acceptance criteria. The investigation demonstrated that Week 3, Group 3 was likely within specification and that all formulations were prepared correctly, therefore this was considered to have had no impact on the outcome or integrity of the study.

For homogeneity, the RSD of concentrations for all samples in each group was within the acceptance criteria of <10%.

Clinical signs

Dose level (mg/kg bw/day)

Male

Female

0

100

300

1000

0

100

300

1000

Decreased activity

0/10

1/10 (1)

2/10 (2)

0/10

0/10

0/10

0/10

0/10

Erect fur

0/10

4/10 (4)

5/10 (5)

10/10 (12)

0/10

0/10

0/10

0/10

Scab

0/10

0/10

0/10

0/10

0/10

0/10

0/10

1/10 (7)

Thin fur

1/10 (2)

0/10

0/10

0/10

1/10 (9)

0/10

0/10

0/10

Pedaling

0/10

1/10 (1)

0/10

0/10

1/10 (1)

0/10

0/10

1/10 (1)

Salivation

3/10 (4)

10/10 (73)

5/10 (38)

10/10 (55)

6/10 (29)

6/10 (36)

3/10 (31)

10/10 (57)

Ploughing

5/10 (7)

10/10 (119)

10/10 (86)

10/10 (126)

9/10 (18)

10/10 (52)

10/10 (58)

10/10 (109)

Numbers of observations are shown in parentheses.

Applicant's summary and conclusion

Conclusions:
Administration of Pentaerythritol by once daily oral gavage was well tolerated in rats at dose levels of up to 1000 mg/kg bw/d, with only instances of ploughing and salivation noted. No target organ effects were observed at any dose level. Based on these results, the NOAEL was considered to be 1000 mg/kg bw/d.
Executive summary:

A 90-day study was conducted in groups of Han Wistar rats (10/sex) according to OECD guideline 408. The animals were dosed orally by gavage with pentaerythritol at dose levels of 0, 100, 300 and 1000 mg/kg bw/d. Ten rats of each sex were tested in each each group. The animals were examined for clinical signs, body weight, body weight gain, food consumption, ophthalmology, functional observations, clinical pathology parameters which comprised haematology, coagulation, clinical chemistry and urinalysis, gross necropsy, organ weights and histopathological analysis. No mortality was observed. Ploughing and salivation were observed at all dose levels with a generally dose related incidence. There were no treatment-related changes in food consumption, ophthalmology, functional observations, clinical pathology parameters, body weights and organ weights. No gross necropsy or histopathological findings were related to treatment. Pentaerythritol was therefore well tolerated in the rat at levels up to 1000 mg/kg bw/d when administered for 90 days. A NOAEL of 1000 mg/kg bw/d can be determined for this study in the absence of any toxicologically significant effects at any dose level. The increased incidences of post-dose salivation and ploughing seen in this study are considered to be a reaction to an unpalatable test materisl and are not considered to be an adverse effect of treatment.