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A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test was conducted with pentaerythritol in male and female Crj: CD(SD) rats. Pentaerythritol was adminstered orally by gavage, at doses of 0, 100, 300 or 1000 mg/kg bw/day. Males were exposed for 46 days, starting from 14 days prior to mating. Females were exposed for 14 days prior to mating, during the mating period and gestation until lactation day 3. The only treatment related findings of parental toxicity were the intermittent occurrence of soft faeces and diarrhoea in males and females of the 300 and 1000 mg/kg bw/day groups. There were no treatment related effects on reproduction. Therefore the NOAEL for repeated dose toxicity was established as 100 mg/kg bw/d, and the NOAEL for reproductive toxicity was established as 1000 mg/kg bw/day.

Oral administration of Pentaerythritol according to OECD TG 443 at dose levels up to and including 1000 mg/kg bw/day was well tolerated with no major treatment-related mortality, no adverse effects on general condition, body weight, food consumption, thyroid hormones, clinical pathology, organ weights, sperm parameters, spleen cell immunophenotyping or pathology on either the F0 or F1 generation. In addition the reproductive performance and fertility of the F0 animals and the survival/development of the F1 offspring up to weaning were unaffected by treatment at all dose levels. Based on the results of this study, it is concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for both systemic and reproductive toxicity in the Sprague Dawley rat is 1000 mg/kg bw/day - the highest tested dose in this study.

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Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 October to 20 December 1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
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)
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
Pentaerythritol; 2,2-bis(hydroxymethyl)-1-3-propanediol, abbreviated to BHP in study report. The test sample was provided by the Ministry of Health and Welfare Environmental Health Bureau, lot no. 50825, purity 92.7%, described as a water-soluble white granule, stored at room temperature avoiding direct sunlight. The test sample was confirmed for stability (until March 1994 for this lot) for a 7 month period.
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
The animals were 8 week old Crj: CD(SD) SPF rats, received from Charles River Laboratories Japan. Males weighed 259-298 g, and females weighed 161-193 g. The rats were acclimatised for 15 days, during which time the general appearance and body weights of the rats were monitored; animals showing satisafactory growth and development were selected for the experiment.
The animals were housed in a barrier-room, maintained at a temperature of 23±3°C, humidity of 55±10%, 10-15 air changes per hour and artificial light was provided on a 12 hour light/12 hour dark cycle. During breeding the rats were housed in wire-mesh floor cages, and from gestational day (GD) 17, females were housed in cages containing a stainless-steel inner tray and bedding (White Flake, Charles River). During acclimatisation, rats were housed in groups of up to 3, then single after assignment to treatment groups. During mating they were housed in pairs (1 male:1 female), and during gestation and laction females were housed singly. Individuals were identified by felt-tip pen marks on the tail, and tattoos behind each ear. Pups were identified using felt-tip pen markings on the dorsal region.
Pelleted diet (CRF-1, Oriental Yeast Co. Ltd.) and tap water (Sapporo) were provided ad libitum. Analysis and inspection of forage was performed by Japan Food Analysis Center Juridical Foundation and it was confirmed that the contained substance impurity was respectively within the permissible range of the company’s SOP. Water quality inspection of drinking water was performed by Kan-ei Industry Corporation and by Fukuda Hydrologic Center and it was confirmed that the water quality was within the permissible range of the company’s SOP.
Route of administration:
oral: gavage
Vehicle:
other: 0.5% CMC-Na solution
Details on exposure:
Doses were prepared every five days. The test substance was weighed and suspended with 0.5% CMC-Na solution (Japanese Pharmacopoeia CMC-Na : Lot No. 1X04, Maruishi Pharmaceutical Co., Ltd., Japanese Pharmacopoeia Purified Water: Lot No. 38A and Lot No. 39G, Yakuhan Pharmaceutical Co., Ltd.) to create 1, 3 , and 10w/v% solution.
Doses were adminstered by gavage, at a set volume of 10 ml/kg bw, based on body weight measurements obtained on the closest day to the adminstration date. Doses were administered between 10:00h and 13:00h. Average body weight at the start of administration was 398.6g (369 – 432g) for males, and 232.1g (207 – 254g) for females.
Details on mating procedure:
Males and females were housed in pairs (1 male:1 female) for a maximum of 14 days to allow mating. Successful mating was assessed by the presence of male sperm in the female vaginal mucous, and succesful gestation was assessed by the presence of an implantation site in the uterus. Females that did not become pregnant during the 14 day mating period were sacrificed.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The Mitsubishi Gas Chemical Company carried out the sample analysus using liquid chromatography: Shimadzu LC-GA; column Asahipak OPD-50 150 x 6.0 mmID; eluent water; flow rate 1.0 ml/min; detector RI. The anaylsis confirmed that the preparation solution of each concentration level of pentaerythritol matched the nominal concentration level (the analytical report states that the 10 and 20% solutions did not fully dissolve), and that 1 - 10% of the preparation solution had stability for 5 days.
Duration of treatment / exposure:
Males: 46 days starting 14 days prior to mating.
Females: 14 days prior mating, throughout mating and gestation until lactation day (LD) 3.
Frequency of treatment:
Daily
Details on study schedule:
Following the acclimatisation period, rats were treated for 14 days (age at start of treatment: 10 weeks), during the mating period (until evidence of mating, for a maximum of 14 days) and until LD3 (in females). Males were sacrificed after 46 days administration. Females and their litters were sacrificed on LD4. Females that failed to mate were sacrificed at the end of the 14 day mating period.
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12 rats/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
A 14 day range-finding study was conducted employing doses of 100, 300 and 1000 mg/kg bw/d. Soft faeces and diarrhoea were seen in males and females in the 1000 mg/kg/day group, but no other effects of toxicity were seen so doses for the main experiment were 100, 300 and 1000 mg/kg bw/d. Animals were assigned to treatment groups on the last day of acclimatisation, according to the body-weight stratified random sampling method. Oestrus cyclicity was observed in females during the acclimatisation period, and females which did showed abnormal cyclicity were not selected for use in the experiment.
Positive control:
A positive control was not examined and is not required.
Parental animals: Observations and examinations:
All animals were examined visually and handled daily during the test period to observe their behaviour and appearance. Male body weights were recorded on Day 1 of administration, Day 2, Day 5, Day 7, Day 10, Day 14 and every 7 days thereafter including on the day of sacrifice. Female bodyw eights were recorded on Day 1 of administration, Day 2, Day 5, Day 7, Day 10, Day 14, GD 0, GD 1, GD 3, GD 5, GD 7, GD 10, GD 14, GD 17, GD 20, LD 0, LD 1 and LD 4. Female body weights during the mating period were recorded on the same day as the male body weights. Females that failed to mate were also weighed immediately prior to sacrifice.
Food and water intake were measured on the same days as body weights were measured, excluding the mating period.

6 males from each group were housed in metabolic cages for 24 hours during the final week of administration (Day 43-44) for collected of non-fasted urine. Blood from male rats was collected from the femoral vein and abdominal aorta under ether anaesthesia on the day of sacrifice (the day after the last administration - Day 46) for haematology and blood chemistry. This part of the study is reported in greater detail in section 7.5.1.

Maternal behaviour was observed at delivery until sacrifice on LD 4.
Oestrous cyclicity (parental animals):
Oestrus cyclicity (vaginal pap smears prepared by Giemsa staining) was monitored daily during the acclimatisation period, for 10 days prior to mating, and during the mating period.
Sperm parameters (parental animals):
Sperm parameters were not measured.
Litter observations:
The total number of births, live pups, dead pups, gender and external appearance were recorded at the time of birth. The implantation site rate, the birth rate, the live birth rate, lactation rate at LD 4, sex ratio and gestation period were calculated.
Pup viability, mortality and general appearance were recorded daily from birth until terminal sacrifice. Newborns which were cannibalised or reported missing were counted in the mortality group.
Pup weights were recorded on LD 0, LD 1 and LD 4.
Postmortem examinations (parental animals):
Males were sacrificed the day after the last dose was administered for necropsy. This part of the study is reported in greater detail in section 7.5.1.

Females that became pregnant and successfully littered were sacrificed on LD 4. Females that became pregnant but had not given birth by GD 25 were sacrificed on GD 26. If a female delivered an entirely still-born litter, she was sacrificed immediately after discovery of the dead pups. Females that did not become pregnant were sacrificed the day after the mating period ended.
At autopsy the organs and tissues were examined grossly. The number of uterine implantation sites and the number of corpora lutea in the ovaries were counted. Organ weights were recorded for the liver, kidneys, thymus, adrenal glands and ovaries. The following organs were fixed in 10% neutral buffered formalin: the liver, kidney, spleen, heart, lung, brain (cerebrum and cerebellulm), pituitary gland, adrenal gland, thyroid gland, parathyroid gland, thymus, messenteric lymph node, pancreas, tongue, lower jaw lymph node, submaxillary gland, sublingual gland, parotid gland, mammary gland, skin, eyeball, Harderian gland, breastbone and femur (including the bone marrow), spinal cord (neck region), skeletal muscle (lateral great muscle), thoracic aorta, larynx, trachea, bronchial tube, esophagus, stomach (anterior stomach, glandular stomach), duodenum, jejunum, ileum, appendix, colon, rectum, bladder, ovary, uterus, and vagina. Additionaly, the eye and Hrderian gland were fixed in Davidson's solution.
The following organs were embedded in paraffin and stained with Haematoxycillin and Eosin, or using the silver impregnation method: liver, kidney, spleen, heart, lung, brain, pituitary gland, thymus, adrenal gland, thyroid gland, stomach (anterior stomach, glandular stomach), duodenum, jejunum, ileum, appendix, colon, rectum, and ovary. The following organs were also stained from dams whose whole litter died: mammary gland and uterus.
Postmortem examinations (offspring):
Autopsies were performed on any pups found dead by fixing the entire body in 10% neutral buffered formalin. Pups that survived to LD 4 were observed for external appearance (including the mouth cavity) prior to sacrifice, after which the organs and tissues were examined grossly. The entire bodies were then fixed in 10% neutral buffered formalin.
Statistics:
Homogeneity of variance was determined using Bartlett's test, followed by ANOVA and Dunnett's test, or Kruskal-Wallis and Mann-Whitney U test as appropriate. Indices were analysed using Multi-Sample analsysis and the Two-Sample analysis, or Fisher's Exact Probability Test. Significance (between treated and control rats) was accepted at or below the 5% level.
Reproductive indices:
Copulation rate = (no. successfully mated rats / no. rats housed together) x100.
Conception rate = (no. impregnated rats / no. successfuly mated rats) x100.
Offspring viability indices:
Pup viability = ( no. viable pups LD 4 / no. confirmed births at time of delivery) x100.
Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Males:
Male data are presented in more detail in section 7.5.1. Four males exhibited soft faeces in the 300 mg/kg group, and 1 male in this group had diarrhoea intermittently until administration Day 9. All males in the 1000 mg/kg group had soft faeces and diarrhoea intermittently from administration Day 2 until terminal sacrifice. There were no effects of treatment on body weight gain, food intake, urinalysis parameters, organ weights or gross findings at necropsy. Water consumption was increased in the 1000 mg/kg group but the finding was not significantly different from control values. MCHC and MCHV were significantly decreased in the 300 and 1000 mg/kg groups compared with controls. There were some changes in blood chemistry parameters in the 100 and 300 mg/kg group, but there was no dose-response and changes were reportedly within the range of historical control data. Histopathological findings occurred in both control and treated groups, and were therefore not considered to be treatment related.

Females:
In the 300 mg/kg group 2 females exhibited soft faeces, and 1 female had diarrhoea on Day 5, and 1 on Day 9 of administration. All females in the 1000 mg/kg exhibited soft faeces and diarrhoea intermittently from administration Day 2 until the end of the mating period, and for 11 females the intermittent symptoms continued until the end of gestation. During lactation all females in the 1000 mg/kg group exhibited soft faeces intermittently, and 9 females had intermittent diarrhoea.
There were no treatment related effects on body weight gain. During the gestation period food intake was decreased in the 300 mg/kg group on GD 14 compared to controls, however this group also had reduced food intake compared to the controls on administration Day 1 (i.e. before the first dose was given). There were no differences between the groups during the lactation period.
There were no significant differences in organ weights between treated and control groups. Autopsy revealed yellowish-white marks on the liver, fatty tissue adhesion on spleen, liver, and within the abdominal cavity, ileum diverticulum, and ovarian cysts in both treated and control rats. Histopathological examination revealed focal necrosis of hepatocytes in the liver (included are the animal cases which showed abnormal findings during the autopsy), regeneration of tubular epithelium of the kidney (right), dilation of renal pelvis (right kidney), dilation of tubules (right kidney), urinary cast(right kidney), fibrous adhesion to liver and intra-abdominal adipose tissue and granuloma of the spleen (included are the abnormal body parts found during the autopsy), calcium deposition in lung blood vessel wall, ciliated epithelial cyst of the pituitary body, retention of homogeneous plasma-like substances in Rathke’s pouch, atrophy of the thymus, increase in RES cells in medulla, diverticulum of the ileum, and lutein cyst of the ovary (included are the abnormal body parts found during the autopsy). All histpathological findings were seen in both control and treated rats.

One female that failed to mate was found to have a continuation of dioestrus after the start of the mating period. This female was from the 300 mg/kg group, and all other rats demonstrated normal cycles therefore this effect was not considered to be related to treatment. 1 pair from the 100 mg/kg group and 1 pair from the 1000 mg/kg also failed to mate.

There was no significant differences in the number of corpora lutea, number of implantation sites, implantation index, total number of pups born, delivery index, number of live pups born, live birth index, gender ratio, number of dead pups born, gestation period, birthrate, and lactation index on Day 4 of lactation in comparison with the control group.

A litter to 1 female in the 1000 mg/kg all died within 1 day of birth; 18 pups were born in total, 5 of which were born alive. All the pups were cannibalised. The dam exhibited maternal licking behaviour and nest-building during delivery, however nursing behaviour was not observed following delivery. Predatory behaviour was observed during delivery. Autopsy of the dam did not reveal any significant findings, other than the placenta had separated from the uterus.
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: NOAEL for toxic effects
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: NOAEL for reproductive toxicity
Clinical signs:
effects observed, treatment-related
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Histopathological findings:
not examined
There were no effects of treatment on pup viability or pup body weights. There was finding of hypoplasia in the kidney (right) with partial dark red discolouration in one female pup in the 1000 mg/kg bw/d group and there was a finding of trauma (body part included in general appearance observation finding) in one female pup in the 300 mg/kg bw/d group.

Pups that died or were born dead exhibited trauma of the medial dorsal region, cervical region, cervical dorsal region, nasal region, left abdominal region and head in both control and treated groups. A dark red discolouration of the right side of the head was noted in some of the treated pups that died. The effects seen in pups at autopsy were not considered related to treatment, as they occurred in such low frequencies.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: NOAEL for developmental toxicity
Reproductive effects observed:
not specified

There was no effect on reproduction or development in this screening test, in the presence of slight parental toxicity.

Conclusions:
No evdience of reproductive or developmental toxicity was seen at the limit dose of 1000 mg/kg bw/d under the conditions of this study.
Executive summary:

A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test was conducted with Pentaerythritol in male and female Crj: CD(SD) rats. Pentaerythritol (in vehicle: 0.5% CMC-Na solution) was adminstered orally by gavage, at doses of 0 (vehicle only control), 100, 300 or 1000 mg/kg bw/d. Males were exposed for 46 days, starting from 14 days prior to mating. Females were exposed for 14 days prior to mating, during the mating period and gestation until lactation day 3. The only treatment related findings of parental toxicity were the intermittent occurrence of soft faeces and diarhhoea in males and females of the 300 and 1000 mg/kg bw/d groups. There were no treatment related effects on reproduction or the development of newborn pups (to lactation day 4). Therefore the NOAEL for repeated dose toxicity was established as 100 mg/kg bw/d, and the NOAEL for reproductive toxicity was established as 1000 mg/kg bw/d.

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 Nov 2019 - 01 Feb 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
Adopted 25 June 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS

- Premating exposure duration for parental (P0) animals: 10 weeks.
- Basis for dose level selection: Dose levels of 100, 300 and 1000 mg/kg/day were selected in conjunction with the Sponsor based on the findings from a preliminary reproductive study conducted at these laboratories. Based on the results of the preliminary study and in the absence of any overt toxicity, there was nothing to preclude the use of 1000 mg/kg/day as the high dose in this OECD443 study. Low and intermediate dose levels of 100 and 300 mg/kg/day were selected to provide an approximate 3-fold dose interval.
- Exclusion of extension of Cohort 1B: No information indicate the genotoxicity, endocrine disruption related effect of this substance. No information indicate the substance leads to significant exposure or specific metabolism pattern that would require extended exposure. Therefore, extension of cohort 1B to include the F2 generation was not performed.
- Exclusion of developmental neurotoxicity Cohorts 2A/2B and developmental immunotoxicity Cohort 3: No indication of the developmental neurotoxicity or immunotoxicity of this substance was seen from existing information. Cohort 2A/2B and cohort 3 are considered not needed at this moment.

- Route of administration
Based on the information provided in the technical dossier and/or in the chemical safety report, ECHA agreed that the oral route - which is the preferred one as indicated in ECHA Guidance on information requirements and chemical safety assessment (version 4.1, October 2015) Chapter R.7a, section R.7.5.4.3 - was the most appropriate route of administration. Additionally, this mimics the most likely route of exposure to humans. Hence, the test was performed by the oral route.
- Other considerations
The Sprague Dawley rat was chosen as the animal model for this study as it is a rodent species accepted by regulatory agencies for reproductive toxicity testing. The total number of animals used in this study was considered to be the minimum required to properly characterise the effects of the test item. This study was designed such that it did not require an unnecessary number of animals to accomplish its objectives.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier): Perstorp Holding AB (Sponsor)
- Lot/Batch number: 190100534 (exp. 08 Jan 2022)
- Purity: 99.2%


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient temperature (15 to 25°C)
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Homogeneity and stability of the dose formulation at 1 and 200 mg/mL was established for 24 hours at ambient temperature (15 to 25°C) and 15 days when stored refrigerated (2 to 8°C).

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Formulation fequency: weekly
Preparation: The required amount of test item was weighed, transferred to a mortar and ground using a pestle to a fine powder. Small amounts of vehicle were added and mixed to form a paste. Any agglomerates were broken down. The test item was fully washed from the container by rinsing, mixing and transferring the rinsings a minimum of three times or until visibly clean. Further amounts of vehicle were gradually added and mixed to produce a smooth, pourable suspension. The suspension was transferred to a measuring cylinder which had been wetted with vehicle. The mortar was rinsed with vehicle and this was added to the measuring cylinder. Vehicle was added to achieve the final volume and the suspension was transferred to a beaker and mixed using a high shear homogenizer. The suspension was stirred for a minimum of 20 minutes using a magnetic stirrer and then transferred to the final containers, via syringe whilst magnetically stirring. A series of formulations at the required concentrations were prepared by dilution of individual weighings of the test item.

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The Sprague Dawley SD rat is the species and strain of choice because there is ample experience and background data on this species and strain. It is also the species and strain used in other toxicity studies of this substance which provide better results-comparison between studies.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Strain: Crl:CD (SD) rat
- Number of animals: 27 litters of four male siblings and 27 litters of four female siblings; no male/female sibling relationships
- Source: Charles River (UK) Ltd. Kent, UK
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (F0) 28-34 days, (F1) 21 days selection; 28 days allocation/formal start
- Weight at study initiation: (F0) Males: 54-102 g; Females: 48-104 g
- Fasting period before study: none
- Housing: cages compromised of polycarbonate body with stainless steel mesh lid; solid bottom cages used except during pairing when grid bottom cages were used; softwood based bark-free fiber bedding; number of animals per cage according to standards
- Diet (e.g. ad libitum): ad libitum SDS VRF1 Certified pelleted diet, no added antibiotic or other chemotherapeutic or prophylactic agent (restricted diet overnight before blood sampling for hematology or blood chemistry and during the period of urine collection).
- Water (e.g. ad libitum): potable water, ad libitum (except during urine collection)
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 40-70%
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours light: 12 hours dark

In-Life Dates:
13 November 2019 - 30 May 2020
Route of administration:
oral: gavage
Vehicle:
other: 1% (w/v) methylcellulose (aqueous)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The dosing formulations were prepared at least weekly, stored in a refrigerator set to maintain 2 to 8ºC, or transferred immediately to the animal unit, and dispensed daily.

VEHICLE
- Justification for use and choice of vehicle (if other than water):TG recomends "where possible, the use of an aqueous solution/suspension is considered first,followed by consideration of a solution/suspension in oil". Methylcellulose was deemed most appropriate for species selection and substance properties by the Sponsor after a formulation analysis study.
- Concentration in vehicle (nominal): 0, 10, 00, 100 mg/ml (dose = 0, 100, 300, 1000 mg/kg/day)
- Amount of vehicle (if gavage): 10 ml/kg

Details on mating procedure:
- M/F ratio per cage: 1/1(sibling pairing was not permitted)
- Length of cohabitation: max. 2 weeks (separation at detection of mating)
- Proof of pregnancy: The day of detection of an ejected copulatory plug in situ and/or of sperm in the lavage was designated GD 0.
- After successful mating each pregnant female was caged: Mated females were transferred to individual solid bottomed cages. From Day 20 after mating and throughout lactation, approximately two handfuls of paper shavings were provided to each cage as nesting material; this nesting material was changed at the same frequency as the cage bedding.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The samples were analyzed in accordance with the validated Covance Analytical Procedure (DFA/M080/19). The analytical method involved extraction and dilution in acetonitrile/water 10/90 v/v followed by reverse phase high performance liquid chromatographic analysis with ELSD detection. Sample concentrations were determined with reference to external standards prepared in the concentration range 25 μg/mL to 250 μg/mL.The formulations for Week 1 (F0 generation), Week 1 (F1 generation) and Last Week of F1 generation were sampled. For all groups, 4 × 1 mL (accurately weighed) was sampled from the middle of the formulation by Pharmacy personnel. Two samples from all groups were analyzed in accordance with the analytical procedure. The remaining samples were retained for contingency. Samples were disposed of once satisfactory results were achieved.

Homogeneity and stability of the dose formulation at 1 and 200 mg/mL was established for 24 hours at ambient temperature (15 to 25°C) and 15 days when stored refrigerated (2 to 8°C).The mean concentrations were within 10% of the nominal concentration, confirming the accuracy of formulation. The difference from mean remained within 2%, confirming precise analysis.

Stability analyses performed previously demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.

Samples of each formulation prepared for administration in Week 1 of each of the F0 and F1 generation and the last week of treatment for the F1 generation were analyzed for achieved concentration of the test item.
Duration of treatment / exposure:
The F0 animals were dosed for 10 weeks before pairing until termination after litters are weaned. The F1 animals were dosed from PND 21 (weaning) up to at least PND 90 (Cohort 1A 13 weeks, Cohort 2A 14 weeks)
Frequency of treatment:
Daily, approx. the same time each day
Details on study schedule:
- Age at mating of the mated animals in the study (F0): 14-15 weeks (after 10 weeks dosing)
-The F0 animals were dosed for 10 weeks before pairing until termination after litters are weaned. The F1 animals were dosed from PND 21 (weaning) up to at least PND 90 (Cohort 1A 13 weeks, Cohort 2A 14 weeks)
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
25 males + 25 females (F0), 20 males + 20 females (F1 cohort 1A), 20 males + 20 females (F1 cohort 1B)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels of 100, 300 and 1000 mg/kg/day were selected in conjunction with the Sponsor based on the findings from a preliminary reproductive study conducted at these laboratories (Covance study no. FW46HY). In the preliminary study dose levels of 300, 600 and 1000 mg/kg bw/day were well tolerated with no adverse effects of treatment on clinical condition of F0 or F1 animals, F0 body weight/food consumption, F1 offspring body weight and F1 selected body weight/food consumption. Possible effects of treatment were limited to a slight two day delay in attaining balano-preputial separation at 600 or 1000 mg/kg/day when compared with concurrent Controls but within the historical control range. Based on the results of the preliminary study and in the absence of any overt toxicity, there was nothing to preclude the use of 1000 mg/kg bw/day as the high dose in this OECD 443 study. Low and intermediate dose levels of 100 and 300 mg/kg bw/day were selected to provide an approximate 3-fold dose interval.

- Fasting period before blood sampling for clinical biochemistry: yes
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate. During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A detailed physical examination was performed on each animal to monitor general health according to the following schedule:
Physical examination Once each week
After mating of F0 females: Days 0, 5, 12, 18 and 20 after mating and Days 1, 7, 14 and 21 of lactation.
Particular attention was paid to possible signs of neurotoxicity such as convulsions, tremor and abnormalities of gait or behavior.

BODY WEIGHT: Yes
- Time schedule for examinations: :
F0 males Day that treatment commenced.
Each week.
Before necropsy.

F0 females Day that treatment commenced.
Each week until mating detected.
Days 0, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 after mating.
Days 1, 4, 7, 14, 21 and 28 post partum.
Before necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:
F0 males and females Weekly, from the day that treatment commenced until paired formating.
For females after mating food consumption was performed to match the body weight recording:
Days 0-1, 2-3, 4-5, 6-7, 8-9, 10-11, 12-13, 14-15, 16-17 and 18-20 after mating
Days 1-3, 4-6, 7-13, and 14-20 of lactation.
From these records the mean weekly or daily consumption per animal (g/animal/week or g/animal/day) was calculated for each relevant phase.

MORTALITY:
A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No (ad libitum)
Oestrous cyclicity (parental animals):
Dry and wet smears were taken as follows:
- Dry smears: For 15 days before pairing, using cotton swabs.

- Wet smears: After pairing until evidence of mating confirmed. For four days before scheduled termination (nominally Days 25 to 28 post partum).
Sperm parameters (parental animals):
Motility:
A sample of sperm was expressed from the left vas deferens (right side for F0 Group 4 Male 81) into prewarmed (target 37°C) medium M199, which contained 0.5% w/v bovine serum albumin (BSA Fraction V). A sample for assessment was taken into a 100 μm depth cannula by capillary action and, where possible, at least 200 sperm per animal analysed using the Hamilton Thorne IVOS II Computer Assisted Sperm Analyser (CASA).

Sperm morphology: Groups 1 and 4
200 µL aliquot of the sperm/medium mixture (described above) was diluted with 800 µL of 10% neutral buffered formalin. After staining with nigrosine and eosin an airdried smear was prepared. Slides were examined by light microscopy for the assessment of sperm morphology. At least 200 sperm were assessed for each male, where possible.

Sperm morphology: Groups 2 and 3
Fixed samples retained for possible future assessment.

Sperm count: Groups 1 and 4
The left cauda epididymis of each male was weighed and then the tunica was removed, then homogenised for at least 30 seconds in 10 mL of a mixture of 0.9% saline and 0.01% merthiolate (SM). An aliquot of this mixture was added to a pre-prepared IDENT stain tube before being assessed for sperm count using CASA.

Sperm count: Groups 2 and 3
Samples frozen for possible future assessment.

Homogenization-resistant spermatid counts: Groups 1 and 4
After removal of the tunica, the left testis of each male (right side for F0 Group 4 Male 81) were homogenised for at least 30 seconds in 25 mL of SM. An aliquot of this mixture was added to a pre-prepared IDENT stain tube before being assessed for homogenisation-resistant spermatid count using CASA. Homogenization-resistant spermatid counts: Groups 2 and 3 Samples frozen for possible future assessment

Homogenization-resistant spermatid counts: Groups 2 and 3
Samples frozen for possible future assessment
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes, litters culled to 10 (where possible 5 males and 5 females), When the total number of pups in a litter on PND 4 was ≤ 10 pups, no litter size adjustment occurred.

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring: Clinical observations, body weight, food consumption and macropathology examinations were performed on all animals for signs of toxicity, with special emphasis on the integrity and performance of the male and female reproductive system and the health (including thyroid hormone analysis), growth, development and function of the offspring. At weaning, two cohorts of selected males and females were assigned for further investigations, including sexual maturation, urinalysis, reproductive organ integrity and function.

The numbers of live and dead pups born in each litter was recorded as soon as possible after completion of parturition on LD 0. The live pups were counted, sexed, weighed individually on PND 1 and examined daily for the presence of milk in the stomach and for any externally visible abnormalities daily up to and including PND 4. Additionally, on Day 1 of age, all offspring received a qualitative assessmentof body temperature, state of activity and reaction to handling and the ano-genital distance was mesured.

After being sexed dircetly before and after culling (PND 4), the pups were not sexed again until day 21 of age. From PND 5, the total live pups were counted daily and examined for ill-health, evidence of reaction to treatment again, and weighed on PND 7, 14 and 21. On day 13, male offsprings nipple/aerolae were counted.

- Selection and weaning of F1 animals for Cohort 1A and 1B
Two males and two females were selected from each selected litter and one male and one female from each litter, where available, were allocated to each of the two cohorts. If more were required, up to three males and three females were selected from each selected litter. Selected animals were microchipped on Day 18 to 21 of age and separated from littermates on Day 21 of age. Formal commencement of the F1 generation was on a nominal Day 28 of age (where possible 28 +/-2 days of age for selected F1 animals). Up to two male and two female F1 offspring per group were retained as spares, to provide potential replacement in the event of any mortality. These spares had body weights and clinical signs recorded weekly and were terminated after commencement of the F1 generation.

- Assessment of F1 Sexual Maturation (Cohorts 1A and 1B)
Commencing at PND 28, females were examined daily for vaginal opening. The day on which the vagina became open was recorded, along with the body weight on that day. For Cohort 1A: a wet smear was taken daily from the day of vaginal opening until first estrus was detected. Commencing at PND 38, males were examined daily for balano-preputial separation. The day on which separation occurred was recorded, along with the body weight on that day.

In-life procedures. observations and measurements F1 (Cohorts 1A and 1B)
- Mortality/Moribundity Checks
Observed approximately 24 hours after birth (Day 1 of age) and then daily for evidence of ill-health or reaction to treatment. A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary.

- Detailed Clinical Observations
Animals were subjected to detailed clinical observations weekly from weaning on PND 21, starting on a suitable day within one week of weaning of the majority of litters. The animals were removed from the cage for examination. The examinations included, but were not limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture, response to handling, as well as the presence of clonic or tonic movements, stereotypy or bizarre behaviour were also examined. Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.

- Pre and Postdose Observations
Detailed observations were performed to establish and confirm a pattern of signs in association with dosing according to the following schedule:
Week 1 - Daily.
Weeks 2 to 4 - twice weekly (middle and end of the week)
Week 5 onward - once each week.
Detailed observations were recorded at the following times in relation to dose administration:
- Prior to dosing
- One to two hours after completion of dosing
- As late as possible in the working day

- Body weights
Recorded on Days 1, 4, 7, 14 and 21 of age; Selected F1 generation: Days 23, 25, 27* and 29* of age. (* Only applicable before formal commencement of the F1 generation at nominal 4 Weeks of age (Day 28 of age ± 2 days)). Plus before necropsy.


- Food consumption
From nominal Week 4 of age, twice during Week 1 of the F1 generation and weekly thereafter.

- Oestrus Cycle Monitoring
Sexual maturation was assessed by daily examination from Day 28 of age until vaginal opening occurred. Body weight was recorded on the day of vaginal opening.
For Cohort 1A: a wet smear was taken daily from the day of vaginal opening until first estrus was detected.

- Haematology (Cohort 1A; 10 rats/sex/group):
At day of necropsy
Analysed 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), Activated partial thromboplastin time, Fibrinogen and prothrombin time

- Clinical Chemistry (Cohort 1A; 10 rats/sex/group):
At day of necropsy
Analysed parameters: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Gamma-glutamyltransferase, Creatine kinase, Total bilirubin, Urea, Creatinine, Calcium, Phosphate, Total protein, Albumin, Globulin, Albumin/globulin ratio, Glucose, Cholesterol, Triglycerides
Sodium, Potassium, Chloride, Sample quality

- Urinalysis (Cohort 1A; 10 rats/sex/group):
Last week of dosing
Analysed parameters: Colour, Appearance/Clarity, Specific gravity, Volume, pH, Protein, Glucose, Bilirubin, Ketones, Blood, sodum, potassium, and chloride. microscopic examination of the urine sediment was performed. An aliquot of the urine sample was centrifuged, stained with Kova stain and the resulting deposit spread on a microscope slide. The number of elements seen in nine high or low power fields (HPF or LPF) was recorded in the raw data and entered onto the database and the number seen /HPF or /LPF was derived from these data as described below.
-Epithelial cells (Epi)
-Leucocytes (WBC)
- Erythrocytes (RBC)
- Casts
- Other abnormal components (A)
The slide was also examined for abnormalities in spermatozoa and crystals


- Thyroid stimulating hormone, TSH and T4 (Conditions, blood sample site, and anaesthetic caried based on age)
F1 - culled offspring Ten litters per group - pooled litter sample on Day 4 of age.* Plus ten male and ten female animals per group on Day 22 of age from as many litters as possible)
F1 adults- Cohort 1A - Ten male and 14 female animals per group at termination
Postmortem examinations (parental animals):
SACRIFICE
F0 animals:
- Male animals: After weaning of the F1 animals, after confirmation that no further mating was required
- Maternal animals: Day 28 post partum
- Females failing to mate: Following completion of pairing females terminated after showing an estrus smear
- Method:
F0 and F1 adults with terminal blood samples: Exsanguination under terminal anesthesia.
F0 and F1 adults with no terminal blood samples: Carbon dioxide asphyxiation with subsequent exsanguination.

GROSS NECROPSY
- F0
All animals, including surplus offspring culled on Day 4 of age were subject to a complete macroscopic examination. Any abnormality in the appearance or size of any organ and tissue was recorded and the required tissue samples preserved in appropriate fixative. Decedents offspring <=21 days of age, (found dead or welfare kill), where possible, were examined and carcass retained.

ORGAN WEIGHTS
F0:
adrenals, brain, L+R epididymides, heart (including auricular and ventricular regions), kidneys, liver (section from two lobes), optic nerves, pituitary, prostate- dorsolateral and ventral combined, seminal vesciles (with coagulating gland), spleen, L+ R testes, thymus, thyroid with parathyroids (weighed after partial fixation), uterus with cervix and oviducts. Paired organs were weighed together. Organ to body weight percentages (using the terminal body weight) were calculated.

MICROSCOPIC EVALUATION
F0:
abnormalities, adrenals, brain (cerebellum, cerebrum, midbrain), cecum, colon, duodenum, epididymides, esophagus, eyes, femurs, heart, ileum, jejunum, kidneys, liver (section from two lobes), lungs (section from two major lobes including bronchi), optic nerves, ovarie, pancreas, pituitary, prostate (dorsolateral and ventral combined), rectum, scieatic nerve, seminal vesicles, skeletal muscles, skin with mammary glands (inguinal area), spinal cord (transverse and longitudinal sections at the cervial thoracic and lumbar levels), spleen, sternum, stomach, testes, thymus, throid with parathyroids, trachea, urinary bladder, uterus with cervix and oviducts, vagina, and vas deferens
Postmortem examinations (offspring):
SACRIFICE
F1 animals:
Unselected offspring On Day 4 and Day 22 of age.
Cohort 1A animals At approximately 13 weeks of age.
Cohort 1B animals At approximately 14 weeks of age.
- Method:
F1 adults: Carbon dioxide asphyxiation with subsequent exsanguination.
F1 offspring 14 days and older: Carbon dioxide asphyxiation with subsequent exsanguination
F1 offspring less than 14 days of age: Subcutaneous injection of sodium pentobarbitone.

GROSS NECROPSY
F1 animals:
All animals, including surplus offspring culled on Day 4 of age were subject to a complete macroscopic examination. Any abnormality in the appearance or size of any organ and tissue was recorded and the required tissue samples preserved in appropriate fixative. Decedents offspring <=21 days of age, (found dead or welfare kill), where possible, were examined and carcass retained.

ORGAN WEIGHTS
- F1 Cohort 1A
adrenals, brain, L+R epididymides, heart (including auricular and ventricular regions), kidneys, liver (section from two lobes), lymph nodes (mesenteric, left axillary), oovaries (L+R), pituitary, prostate- dorsolateral and ventral combined, seminal vesciles (with coagulating gland), spleen, L+ R testes, thymus, thyroid with parathyroids (weighed after partial fixation), uterus with cervix and oviducts. Paired organs were weighed together. Organ to body weight percentages (using the terminal body weight) were calculated.
- F1 Cohort 1B
epididymides, ovaries (L+R), pituitary, prostate (dorsolateral and ventral combined), seminal vesciles (with coagulation gland), testes, uterus with cervix and oviducts
- Unselected F1 offspring on Day 22 of age
brain (cerebellum, cerebrum, midbrain), spleen, thymus


MICROSCOPIC EVALUATION
- F1 Cohort 1A
abnormalities, adrenals, brain (cerebellum, cerebrum, midbrain), cecum, colon, duodenum, epididymides, esophagus, eyes, femurs, heart, ileum, jejunum, kidneys, liver (section from two lobes), lungs (section from two major lobes including bronchi), optic nerves, ovarie, pancreas, pituitary, prostate (dorsolateral and ventral combined), rectum, scieatic nerve, seminal vesicles, skeletal muscles, skin with mammary glands (inguinal area), spinal cord (transverse and longitudinal sections at the cervial thoracic and lumbar levels), spleen, sternum, stomach, testes, thymus, throid with parathyroids, trachea, urinary bladder, uterus with cervix and oviducts, vagina, and vas deferens

- F1 Cohort 1B
Abnormalities
Statistics:
All statistical analyses were carried out separately for males and females. Data relating to food consumption were analyzed on a cage basis. For all other adult parameters, the analyses were carried out using the individual animal as the basic experimental unit. For litter/fetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population. The following data types were analyzed at each timepoint separately:
-Body weight, using absolute weights and gains over appropriate study periods
-Food consumption, over appropriate study periods
-Hematology
-Blood chemistry
-Urinalysis
-Estrous cycles
-Pre-coital interval
-Gestation length and gestation index
-Stage of estrous cycle at termination
-Litter (implantations, litter size, sex ratio - percentage male, post implantation survival index, live birth index and viability index), for before cull study periods
-Ano-genital distance, adjusted for Day 1 offspring body weight
-Sexual maturation, age and body weight at completion
-Organ weights, absolute and relative to body weight
-Corpora lutea and ovarian primordial follicle count
The following comparisons were performed:
-Group 1 vs 2, 3 and 4
-Group 1 vs 4 for ovarian follicle counts and corpora lutea data

Significant differences between the groups compared were expressed at the 5% (p<0.05) or1% (p<0.01) level.

Parametric/Non-Parametric
Tests include Bartlett's test, Willams' test, t-tests, Dunnett's tests, Shirley's test Kruskal-Wallis' tests, Fisher's exacttest, Wilcoxon rank sum tests, Cochran-Armitage tests, Chi-square tests, one- and two-tailed linear-by-linear tests
Reproductive indices:
Post implantation survival, live birth, viability, and lactation indices were calculate for F0.

Post implantation survival index (%) = (Total number of offspring born)/(Total number of uterine implantation sites) x 100
Post-implantation survival index was expressed as 100% where the number of offspring exceeded the number of implantation sites recorded.


Live birth index (%) = (Numer of offspring on Day 1 after littering)/(Total number of offspring born) x 100
Offspring viability indices:
Viability index (%) = (Number of live offspring on Day 4 before culling) / (Number of live offspring on Day 1 after littering) x 100

Lactation index (%) = (Number of live offspring on Day 21 after littering) / (Number of live offspring on Day 4 (after culling)) x 100
Clinical signs:
no effects observed
Description (incidence and severity):
There were no signs at routine physical examination that could be attributed to treatment and no signs were observed in association with dose administration.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No Pentaerythritol-related mortality occurred.

There were three F0 animals that died or were killed for welfare reasons due to dosing trauma.
- F0 Control female 204 was found dead on Day 64;
- F0 Female 226 at 100 mg/kg bw/day was euthanized on Day 49;
- F0 Female 251 at 300 mg/kg bw/day was found dead on Day 65; F1 Male 537 at 300 mg/kg bw/day was euthanized on Day 33;

Histopathological examination revealed a number of findings that were indicative of gavage injury; these included inflammation or inflammatory cell infiltrate seen submucosally in the esophagus, pleura inflammation seen in the lungs and or around the heart and thymus, foreign material in the thorax and esophageal perforation.

In addition there were three F0 decedents
- F0 Male 50 at 100 mg/kg bw/day was euthanized for welfare reasons on Day 77;
- F0 Male 91 at 1000 mg/kg bw/day was found dead on Day 90;
- F0 Control Female 212 was euthanized for welfare reasons on Day 22;

Histopathological examination did not determine a cause of death for these animals. As these deaths were isolated and were not restricted to groups that received Pentaerythritol they are considered to be unrelated to treatment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Bodyweight gain for F0 males during treatment and F0 females before pairing was unaffected by administration of Pentaerythritol at dose levels up to and including 1000 mg/kg bw/day.

During gestation body weight gain from GD0-20 was slightly but significantly low for females receiving 1000 mg/kg bw/day (p<0.05); gestation body weight gain at 100 or 300 mg/kg bw/day was unaffected by treatment.

Body weight gain of females during lactation was unaffected by administration of Pentaerythritol at all dose levels.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption for males and females before pairing and for females during gestation and lactation was unaffected by treatment at dose levels up to and including 1000 mg/kg bw/day.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Haematological investigations did not reveal any differences that could be attributed to treatment with Pentaerythritol; differences in clotting times were minor and lacked dose response.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Plasma glucose concentrations at 1000 mg/kg bw/day were slightly low for both males and females with the difference attaining statistical significance for males (p<0.01). Phosphorous and calcium levels for females at 1000 mg/kg bw/day were slightly low when compared with Controls (p<0.05 and p<0.01, respectively). There were no other differences that were considered to be related to administration of Pentaerythritol.
Endocrine findings:
effects observed, non-treatment-related
Description (incidence and severity):
There was no treatment related effect observed on serum TSH concentrations in male orfemale rats after dosing with Pentaerythritol up to a dose of 1000 mg/kg bw/day administered via oral gavage.

The mean serum T4 concentrations for F0 adult animals at scheduled termination showed inter-/intra-group variation when compared to their respective Control groups; however there were no differences that could be attributed to administration of Pentaerythritol at dose levels up to and including 1000 mg/kg bw/day.


Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Incidences of increased agression or vocalization observed but deemed to not be dose related.
Immunological findings:
effects observed, non-treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No Pentaerythritol-related microscopic observations were noted in the F0 adult animals at scheduled termination.
Histopathological findings: neoplastic:
not specified
Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day there was a slight increase in the incidence of females exhibiting 4/5 day cycles and an increase in the incidence of acyclic females at 300 and 1000 mg/kg bw/day; a dose response was not apparent. Following weaning on Day 21 post partum and scheduled termination on Day 28 post partum all females showed estrus.
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
No adverse effects on sperm motility or motion parameters were observed following treatment with Pentaerythritol up to 1000 mg/kg bw/day. At 1000 mg/kg bw/day, morphological examination revealed increases in head abnormalities when compared with both the concurrent Control and historical control data; these included decapitate and flat heads with incidence of flat heads attaining statistical significance.
Reproductive performance:
no effects observed
Description (incidence and severity):
Precoital interval:
The majority of females mated within 4 days of pairing and no adverse effect of treatment was inferred.

Mating performance & Fertility:
Mating performance and fertility were unaffected by treatment.

Gestation Length & Gestation Index:
The majority of animals showed a gestation length within the normal range of 22 to 23.5 days; one Control female and two females at 1000 mg/kg bw/day had a gestation length slightly greater than 23.5 days. The gestation index was unaffected by administration of Pentaerythritol.
Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day
Sex:
male/female
Basis for effect level:
reproductive function (sperm measures)
reproductive performance
Key result
Critical effects observed:
no
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no signs at routine physical examination that could be attributed to treatment and no signs were observed in association with dose administration.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
No Pentaerythritol-related mortality occurred.

There were two F1 animals that died or were killed for welfare reasons due to dosing trauma.
- F1 Male 537 at 300 mg/kg/day was euthanized on Day 33;
- F1 Male 542 at 1000 mg/kg/day was euthanized on Day 2.

Histopathological examination revealed a number of findings that were indicative of gavage injury; these included inflammation or inflammatory cell infiltrate seen submucosally in the esophagus, pleura inflammation seen in the lungs and or around the heart and thymus, foreign material in the thorax and esophageal perforation.

In addition there were two F1 decedents.
- F1 Control Male 494 was euthanized for welfare reasons on Day 17;
- F1 Male 522 at 300 mg/kg bw/day was found dead on Day 14.

Histopathological examination did not determine a cause of death for these animals. As these deaths were isolated and were not restricted to groups that received Pentaerythritol they are considered to be unrelated to treatment.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The mean body weight of selected animals on Day 21 of age was essentially similar across the groups. On Day 1 of the F1 generation at 28±2 days of age the mean body weight of male animals at 1000 mg/kg bw/day was slightly but significantly high when compared with Controls (p<0.05); this was not evident for females at this dose level. Overall the body weight gain for selected F1 animals up to scheduled termination was unaffected by administration of Pentaerythritol at dose levels up to and including 1000 mg/kg bw/day.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was unaffected by administration of Pentaerythritol at dose levels up to and including 1000 mg/kg bw/day.
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):
The large unstained cell count for females that received Pentaerythritol was slightly but significantly high when compared with Controls (p<0.05); a dose response was not apparent, and this difference was not evident in male animals. Mean cell hemoglobin concentration was slightly high for females at 1000 mg/kg bw/day when compared with Controls (p<0.05); this difference was not evident in male animals.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Blood chemistry did not reveal any differences and was considered unaffected by administration of Pentaerythritol.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Urinary pH was low (p<0.05) and specific gravity was high (p<0.01) for males that received 1000 mg/kg bw/day; this was not apparent for females at this dose level.
Sexual maturation:
no effects observed
Description (incidence and severity):
The age and body weight of males on completion of balano preputial separation showed no adverse effects of treatment with Pentaerythritol at all dose levels.

For females receiving 1000 mg/kg bw/day the mean day of age on completion of vaginal opening was 38 compared with 36 in the Control group (p<0.05) and the mean bodyweight was 145 g compared with a Control value of 129 g (p<0.01). Review of historical control data showed that the mean values at 1000 mg/kg bw/day were essentially within the 5-95% limits and that the individual values for the day of completion were within the concurrent Control range; therefore no effect of treatment was inferred.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
The ano-genital distance for male and female offspring on Day 1 of age showed no adverse effect of treatment.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
No nipples were observed for male offspring examined on Day 13 of age.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Mean absolute and body weight relative spleen weights were low for both males and females treated at 1000 mg/kg bw/day in Cohort 1A; with body weight relative differences attaining statistical significance (p<0.05). No Pentaerythritol-related differences in mean organ weight was evident for animals in Cohort 1B.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Macroscopic examination of F1 males and females from Cohorts 1A and 1B at schedule termination did not reveal any findings that could be attributed to treatment with Pentaerythritol
Histopathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No Pentaerythritol-related microscopic findings were observed in the F1 males and females from Cohorts 1A and 1B.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Endocrine:
There was no treatment related effect evident on serum TSH concentrations in F1 animals after dosing with Pentaerythritol at doses up to and including 1000 mg/kg bw/day. Group 2 (100 mg/kg bw/day) male F1 offspring on Day 22 of age appeared to have high mean TSH levels when compared with Controls but the male Control mean was low when compared with the female Control mean and in the absence of a similar effect in female

Estrous cycles:
The interval between completion of vaginal opening and the first estrus smear did not show any adverse effect of treatment. Assessment of estrous cycles from nominal Day 75 of age revealed that the majority of females showed 4 and 4/5 day cycles. Two females at 1000 mg/kg bw/day showed 5-day cycles, one Control, two females at 100 mg/kg bw/day and one female at 300 mg/kg bw/day showed irregular cycles and one female at 300 mg/kg bw/day was acyclic. The majority of females showed an estrus smear during the four day assessment period before termination, with the exception of three F1B females receiving 300 mg/kg bw/day.

Ovarian Follicle counts & Corpea Lutea:
Ovarian follicle and corpora lutea counts were unaffected by treatment.

Sperm Assessment:
No adverse effects on sperm motility or motion parameters were observed following treatment with Pentaerythritol at dose levels up to and including 1000 mg/kg bw/day. At 1000 mg/kg bw/day, there was a slight decrease in cauda epididymal sperm numbers with increases in spermatid numbers when compared with the concurrent Controls; this can be associated with a blockage between the testis and epididymis, however in the absence of an adverse effect on sperm motility, the significance, if any, of these findings are unclear. At 1000 mg/kg bw/day, morphological examination revealed statistically significantly high incidences of midpiece abnormality, bent/kinked, when compared with Controls but within the historical control range.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Development of the F1 offspring up to weaning were unaffected by treatment at all dose levels.
Developmental immunotoxicity:
effects observed, non-treatment-related
Description (incidence and severity):
Minor differences were observed in the immunophenotyping parameters between treated groups for both males and females, but these differences were not attributed to administration of Pentaerythritol as they were within the ranges observed in the concurrent Control group
Key result
Dose descriptor:
NOAEL
Generation:
F1 (cohort 1A)
Effect level:
> 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse treatment related effects were seen
Key result
Dose descriptor:
NOAEL
Generation:
F1 (cohort 1B)
Effect level:
> 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse treatment related effects were seen
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day
Treatment related:
no
Conclusions:
Oral administration of Pentaerythritol at dose levels up to and including 1000 mg/kg bw/day was well tolerated with no treatment-related mortality, no adverse effects on general condition, body weight, food consumption, thyroid hormones, seminology, organ weights or pathology on either the F0 or F1 generation. Reproductive performance for the F0 adults and spleen cell immunophenotyping and ovarian follicle counts for the F1 animals were unaffected by treatment at dose levels up to and including 1000 mg/kg bw/day. The clinical condition of offspring, litter size, offspring survival and development were unaffected by parental treatment at dose levels up to and including 1000 mg/kg bw/day. Based on the results of this study, it is concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for both systemic and reproductive toxicity in the Sprague Dawley rat is 1000 mg/kg bw/day - the highest tested dose in this study.
Executive summary:

In an extended one-generation reproductive toxicity study (OECD TG 443) in rats, including Cohorts 1A and 1B, Pentaerythritol was administered to groups of 25 animals per sex per dose level by gavage at dose levels of 100, 300, or 1000 mg/kg bw/day (F0) at a volume dose of 10 mL/kg bw/day. Additionally, Pentaerythritol was administered to 40 animals per sex per dose level by gavage at dose levels 100, 300, or 1000mg/kg bw/day (F1, Cohorts 1A and 1B) at a volume dose of 104 mL/kg bw/day. A similarly constituted Control group received the vehicle (F0 & F1), 1% w/w methylcellulose (aqueous), at the same volume dose as the treated groups throughout the same periods.

 

Males were treated for ten weeks before pairing, up to necropsy after litters were weaned. Females were treated for ten weeks before pairing, throughout pairing up to necropsy on Day 28 of lactation. All F1 animals were then dosed directly from postnatal day (PND) 21 to at least PND 90. At weaning, two cohorts of selected males and females were assigned for further investigations, including sexual maturation, urinalysis, reproductive organ integrity and function (Cohorts 1A and 1B). 

 

Throughout the study, 10 animals were either found dead or were killed for reasons of animal welfare, none were deemed treatment-related. Various effects were seen throughout the study (gestation body weight, plasma glucose concentrations, sperm morphology, haemoglobin, urinary pH and specific gravity, spleen weights), but differences and/or effects were concluded to be non-treatment-related since they were either minor, lacked a dose response or were inconsistent between the sexes.

 

Overall, oral administration of Pentaerythritol at dose levels up to and including 1000 mg/kg bw/day was well tolerated with no treatment-related mortality, no adverse effects on general condition, body weight, food consumption, thyroid hormones, seminology, organ weights or pathology on either the F0 or F1 generation. Reproductive performance for the F0 adults and spleen cell immunophenotyping and ovarian follicle counts for the F1 animals were unaffected by treatment at dose levels up to and including 1000 mg/kg/day. The clinical condition of offspring, litter size, offspring survival and development were unaffected by parental treatment at dose levels up to and including 1000 mg/kg bw/day.

 

Based on the results of this study, it is concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for both systemic and reproductive toxicity in the Sprague Dawley rat is 1000 mg/kg bw/day - the highest tested dose in this study.

 

This study is acceptable and satisfies the guideline requirements for an extended one-generation reproductive study (OECD 443) in the rat.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
A guideline-compliant screening study is available for the submission substance. Sufficient to address requirements.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Description of key information

A GLP study conducted according to OECD TG 414 (prenatal developmental toxicity study) in the rat is available. Four groups of 24 females Sprague-Dawley rats were dosed orally by gavage on Days 6-19 of gestation (where Day 0 was the day of detection of mating), at dose levels of 0, 100, 300 or 1000 mg/kg bw/day in corn oil. At dose levels up to and including 1000 mg/kg bw/day were no test item-related effects on clinical observations, body weight gain, food consumption, gross pathology findings, pregnancy performance parameters, embryofetal survival or fetal weights, fetal abnormalities and variants. In conclusion, under the conditions of this study, the maternal and embryofetal no observed effect level (NOEL) was considered to be 1000 mg/kg bw/day - the highest tested dose.

Based on the results from an OECD TG 414 study in rabbits, administration of 100, 300 or 1000 mg/kg bw/day in 1% (w/v) methylcellulose (aqueous) by oral gavage administration, from Day 6 to 28 after mating did not adversely affect maternal performance or fetal development and growth. The maternal and fetal No Observed Adverse Effect Level (NOAEL) is considered to be 1000 mg/kg bw/day - the highest dose tested.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 June to 29 September 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
- Name of test material (as cited in study report): Pentaerythritol
- Physical state: crystalline solid
- Analytical purity: 98.8%
- Lot/batch No.: 4410102701
- Expiration date of the lot/batch: 18 December 2016
- Storage condition of test material: ambient
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent, UK
- Strain: Crl:CD(SD) Sprague-Dawleys
- Age at study initiation: 9-10 weeks
- Weight at study initiation: 211 - 325 g
- Housing: Animals were housed up to 2 per cage in appropriately sized suspended polycarbonate/polypropylene cages with stainless steel grid tops and solid bottoms. Bedding material was sterilised white wood shavings which was provided with a certificate of analysis for significant contaminants. An analytical certificate for each batch of bedding used are retained at Charles River, Edinburgh. For psychological/environmental enrichment, animals were provided with items such as a device for hiding in and an object for chewing (certificates of analysis are retained at the test facility).
- Diet: SDS VRF-1 breeder diet ad libitum
- Water: Water from the public supply ad libitum
- Acclimation period: up to 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-25°C (target 19-23°C)
- Humidity (%): 39% - 78% (target 40% to 70%)
- Air changes (per hr): at least 10/hour (100% fresh air)
- Photoperiod (hrs dark / hrs light): 12 h cycle

IN-LIFE DATES: From: 19 June 2015 To: 16 July 2015
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The control item, corn oil, was prepared weekly, stored in a refrigerator set to maintain 4°C and dispensed daily for administration to Group 1 control animals. The control item was removed from the refrigerator and stirred for at least 30 minutes before dosing. The control item was also stirred continuously during dosing. Test item dosing formulations were prepared based on a method established at the Test Facility at appropriate concentrations to meet dosage level requirements. The dosing formulations were prepared weekly, stored in a refrigerator set to maintain 4°C, and dispensed daily. The dosing formulations were removed from the refrigerator and stirred for at least 30 minutes before dosing. The dosing formulations were also stirred continuously during dosing. Any residual volumes were discarded.

- Concentration in vehicle: 0, 25, 75 and 250 mg/mL
- Dose volume: 4 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were collected from all groups for analysis of concentration on Day 1 and and Week 2. Samples were collected from the test substance groups for analysis of homogeneity of Day 1 and Week 2. Samples were analysed by gas chromatography with flame ionisation using a validated method. Stability analyses were performed for a previous study conducted at the test facility - stability in the vehicle was demonstrated.
Details on mating procedure:
Time-mated females were delivered to the test facility.
Duration of treatment / exposure:
Days 6-19 of gestation
Frequency of treatment:
Once daily
Duration of test:
15 days (treatment on Days 6-19 gestation; scheduled euthanasia on Day 20 of gestation)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
24 females per group
Control animals:
yes, concurrent vehicle
Details on study design:
The oral gavage route of administration was selected for this study as this is a regulatory requirement. Dose levels were selected in agreement with the Sponsor following review of all available toxicological data where dose levels up to 1000 mg/kg/day were well tolerated with only minimal effects of toxicity.
Maternal examinations:
Animals were observed for general health/mortality and moribundity twice daily, once in the morning and once as late as possible each day. Detailed clinical observations were made daily from the start of dosing. Prior to dosing and regularly throughout the day, all animals were examined for reaction to treatment. 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. Body weights were recorded once during pretreatment (Day 5 of gestation) and daily (Day 6 - 20 of gestation) during the dosing period. Food consumption was quantitatively measured daily from Day 6 of gestation. Scheduled euthanasia took place on Day 20 of gestation. All adult 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. Representative samples of abnormal tissues were collected and preserved in 10% neutral buffered formalin or other appropriate fixative.
Ovaries and uterine content:
The reproductive tract was dissected from the abdominal cavity. The gravid uterus was weighed. The uterus was opened and the contents were examined. The fetuses were removed from the uterus. The ovaries and uterus were examined for number and distribution of Corpora Lutea, Implantation Sites, Placentae (size, colour or shape) any abnormalities were recorded, Live and Dead Fetuses and Early and Late Embryonic Deaths.
Fetal examinations:
Fetuses were examined for external abnormalities. Late resorptions and dead fetuses were examined for external abnormalities to the extent possible. Each implant was classified as being live, or a dead fetus (dead full term fetus that shows no sign of maceration), or a late embryonic death (macerated tissue identifiable as an embryo fetus, with recognisable external features such as tail, limbs, mouth and nares present; attached to distinct identifiable placentae), or an early embryonic death (discrete, formless, discoloured tissue mass attached to the internal uterine wall; may be of varying size). The body weight of each fetus was recorded. Fetuses were individually identified within litters.

Visceral examination: Half of the viable fetuses from each uterus were fixed in methylated ethyl alcohol, examined internally for sex and eviscerated following initial fixation, the viscera was not examined from fetuses prior to disposal. The remaining half of viable fetuses from each uterus were fixed in Bouin's fluid.

The fetuses fixed in Bouin's fluid were examined for soft tissue abnormalities and sex using a freehand sectioning technique derived from that of Wilson.

Skeletal examination: The eviscerated carcasses were then macerated in potassium hydroxide, the skeletons stained with Alizarin Red S, and then the fetuses cleared with aqueous glycerol solutions. These preparations were examined for the presence of skeletal abnormalities and for the extent of ossification.
Statistics:
Means and standard deviations were calculated for body weight, food consumption and pregnancy data. To assist interpretations, tests were applied to determine the statistical significance of observed differences between Control and groups receiving test item. Unless otherwise stated, all statistical tests were two-sided and performed at the 5% significance level using in house software. Pairwise comparisons were only performed against the control group. Body weight and food consumption, data were analysed for homogeneity of variance using the ‘F-Max' test. If the group variances appeared homogeneous, a parametric ANOVA was used and pairwise comparisons were made using Fisher’s F protected LSD method via Student's t test ie pairwise comparisons were made only if the overall F-test was significant. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilise the variances. If the variances remained heterogeneous, then a Kruskal-Wallis non-parametric ANOVA was used and pairwise comparisons were made using chi squared protection (via z tests, the non-parametric equivalent of Student's t test).
Indices:
Not applicable.
Historical control data:
Not reported.
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
There were no mortalities and no clinical signs of toxicity. There were no treatment-related effects on body weight or body weight gain. Food consumption was unaffected by treatment. No abnormalities were detected at necropsy.
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
At dose levels up to and including 1000 mg/kg/day there was no effect on pregnancy frequency, gravid uterine weight, mean fetal weight, number of corpora lutea, number of implants or embryofetal survival including pre-implantation loss. The type and distribution of all fetal abnormalities and variations, including those indicating the extent of skeletal ossification were similar in all groups and did not indicate any association with test item administration.
Dose descriptor:
NOEL
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks on result:
not determinable due to adverse toxic effects at highest dose / concentration tested
Abnormalities:
not specified
Developmental effects observed:
not specified

Dose formulation analyses

All study samples analyzed had mean concentrations within the acceptance criteria of ± 10% of their theoretical concentrations, except for Day 1, Group 2 (-16.0%); therefore, as part of the investigation, the backup samples were analyzed in triplicate, and the results (-8.4%) were within the acceptance criteria. The investigation demonstrated that Day 1, Group 2 was likely to have been within specification. For homogeneity, the RSD of concentrations for all samples in each group was within the acceptance criteria of ≤ 10%, except for Week 2, Group 2 (12.7%); therefore as part of the investigation, the backup samples were analyzed in triplicate, and the result (6.2%) was within the acceptance criteria. The investigation demonstrated that Week 2, Group 2 was likely to have been within specification. The dose formulations were within specification. Homogeneity testing showed that the formulation technique used produced homogeneous preparations.

Conclusions:
Under the conditions of this study, the maternal and embryofetal no observed effect levels (NOEL) were considered to be 1000 mg/kg/day.
Executive summary:

The potential toxicity of pentaerythritol when administered orally during the period of organogenesis and foetal period to pregnant rats was investigated according to OECD 414. Four groups of 24 females Sprague-Dawley rats were dosed orally by gavage on Days 6-19

of gestation (where Day 0 was the day of detection of mating), at dose levels of 0, 100, 300 or 1000 mg/kg bw/day in corn oil.

The following parameters and end points were evaluated: clinical signs, body weights, food consumption, gross necropsy findings, gravid uterine weight, and examination of pregnancies and fetal examinations (external abnormalities, fetal weights, visceral and

skeletal evaluations). Animals were killed on Day 20 of gestation. At dose levels up to and including 1000 mg/kg/day were no test item-related effects on clinical observations, body weight gain, food consumption, gross pathology findings, pregnancy performance parameters, embryofetal survival or fetal weights, fetal abnormalities and variants. In conclusion, under the conditions of this study, the maternal and embryofetal no observed effect levels (NOEL) were considered to be 1000 mg/kg/day.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 September 2019 - 09 November 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
OECD 414 Prenatal Developmental Toxicity Study (2018)
Deviations:
yes
Remarks:
Please refer to any other information section for details.
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source: Sponsor
- Llot/batch number of test material: 190100534
- Purity: 99.2%

STABILITY
-Storage: Refrigerated (2 to 8°C)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS (UK)
- Age at study initiation: 16 to 22 weeks old
- Weight at study initiation: 2.49 to 4.25 kg
- Housing: Suspended cages fitted with perforated floor panels and mounted in batteries. Undertrays lined with absorbent paper were changed at least three times a week.
- Diet (e.g. ad libitum): Restricted (200 g/animal/day).
- Water (e.g. ad libitum):ad libitum
- Acclimation period:Five days before commencement of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15-21°C
- Humidity (%): 45-70%
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod (hrs dark / hrs light):14 hours light : 10 hours dark
Route of administration:
oral: gavage
Vehicle:
other: 1% (w/v) methylcellulose
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical method involved extraction and dilution in acetonitrile/water 10/90 v/v followed by reverse phase high performance liquid chromatographic analysis with evaporative light scattering detection. Sample concentrations were determined with reference to external standards prepared in the concentration range 25 μg/mL to 250 μg/mL.
Duration of treatment / exposure:
Females were treated once daily at approximately the same time each day from Day 6 to Day 28 (inclusive) after mating.
Frequency of treatment:
Females were treated once daily at approximately the same time each day from Day 6 to Day 28 (inclusive) after mating.
Duration of test:
Day 6 to Day 28 of gestation
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
24/dose
Control animals:
yes, concurrent vehicle
Details on study design:
Dose levels of 100, 300 and 1000 mg/kg bw/day were selected based on findings from a preliminary embryo-fetal development study in the New Zealand White Rabbit. In the preliminary study, oral administration of Pentaerythritol to pregnant New Zealand White rabbits during organogenesis and the fetal growth phase at dose levels up to and including 1000 mg/kg bw/day was well tolerated with no adverse effects on maternal clinical condition, body weight, food consumption or macropathology and there were no adverse effects on embryo-fetal survival/development that could be attributed to maternal administration of the test item. Based on these findings there was nothing to preclude the use of 1000 mg/kg bw/day as the high dose in this OECD 414 study. Low and intermediate dose levels of 100 and 300 mg/kg bw/day were selected providing approximate three-fold dose intervals allowing assessment of any dose response.
Maternal examinations:
A detailed physical examination was performed on each animal on arrival (Day 1 after mating) and Days 6, 12, 18, 24 and 29 after mating to monitor general health. The weight of each adult was recorded on arrival (Day 1 after mating), on Day 3 and Days 6 to 29 after mating. The weight of food supplied to each animal, that remaining and an estimate of any spilled
was recorded daily from Day 2 after mating. All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded.
Ovaries and uterine content:
Gravid uterine weight (including cervix and ovaries) was recorded for all animals. For each ovary/uterine horn the number of Corpora lutea, Implantation sites, Intrauterine deaths (classified as early or late, resorptions), Fetuses (live and dead) was recorded. The absence or number of uterine implantation sites was
confirmed.
Fetal examinations:
The examination of all viable fetuses and placentae included the following: individually weighed and identified within the litter using a coding system based on their position in the uterus. Examined externally with abnormalities recorded, sampled as appropriate and retained in appropriate fixative. All fetuses were subject to a gross internal examination of the viscera of the neck, thorax and abdominal cavities and the sex of each fetus was also recorded. Serial sections of the fetal heads were examined for soft tissue abnormalities. Alizarin Red stained fetuses and torsos were assessed for skeletal development and abnormalities.
Statistics:
For adult parameters, the analyses were performed using the individual animal as the basic experimental unit. For litter/fetal findings, the litter was considered the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.
Historical control data:
The New Zealand White strain was used because historical control data for this strain was available in the test laboratory in which the study was conducted.
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
At 1000 mg/kg bw/day, minor mean body weight loss of 20 g was recorded between Days 6-10 of gestation followed by marginally low weight gain to Day 13. This resulted in overall body weight stasis between Days 6 and 13 of gestation compared with a mean weight gain of 60 g in Control animals. Thereafter, mean body weight gain improved in these females throughout the remainder of the treatment period, with body weight gain comparable with, or exceeding Control animals. Body weight gain was unaffected in females treated at 100 or 300 mg/kg bw/day.

There was no effect of treatment on mean gravid uterine weight or on maternal body weight change when adjusted for gravid uterine weight.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
At 1000 mg/kg bw/day, mean food consumption was statistically significantly lower than that of Control animals from Day 6 to Day 12. Thereafter, food intake increased with mean food consumption comparable with, or exceeding Control animals. Food consumption at 100 or 300 mg/kg bw/day was unaffected by administration of the substance.
Food efficiency:
not examined
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:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Pale spleens were observed for one female at 300 mg/kg bw/day and for one female at 1000 mg/kg bw/day. Pale liver lobes was observed for one female at 1000 mg/kg bw/day and the placentae for one female at 1000 mg/kg bw/day were also pale. These findings were isolated and showed no correlation with treatment and are therefore considered to be a consequence of natural biological variation and unrelated to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
not specified
Other effects:
not specified
Details on maternal toxic effects:
Administration of Pentaerythritol by oral gavage at dose levels of 100, 300 or 1000 mg/kg bw/day was well tolerated without adverse effect on general condition, reproductive performance, including the number of implantation sites, resorptions (early or late), the extent of pre or post-implantation loss, numbers of live young, sex ratio and placental, litter and fetal weights of the maternal animals.

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: Highest dose tested without adverse effects.
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
At 300 and 1000 mg/kg bw/day, there was an increase in incidence of full supernumerary 13th rib with associated 20 thoracolumbar vertebrae and unilateral shift of pelvic girdle compared to concurrent Control animals, with the incidence of unilateral shift of pelvic girdle being outside of the historical control data range. This indicated a shift in rib/vertebral configuration which as variants, are not considered adverse, but are considered to be associated to treatment with Pentaerythritol.

At 100, 300 and 1000 mg/kg bw/day, there was an increase in the incidence of incompletely ossified epiphyses and metacarpals compared with concurrent Control animals and at 300 mg/kg/day, an increase in the incidence of short supernumerary cervical rib compared with concurrent Control animals was also evident, However, these findings were within the historical control data range and therefore considered not to be related to treatment with Pentaerythritol.
Visceral malformations:
no effects observed
Other effects:
not specified
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effect occurring at the highest dose tested.
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Summary of Cesarean Section Data

   Group  Control  100 mg/kg bw/day  300 mg/kg bw/day

 1000 mg/kg bw/day

 Number of females pregnant at cesarean section  n  24/24  18/21  20/22  22/23
 Corpora Lutea  Mean  11.0  10.3  11.0  10.2
   SD  2.82  2.47  2.17  2.15
 Implantations  Mean  9.2  7.6  9.2  8.3
   SD  2.35  2.75  2.25  3.42
 Pre-implantation loss (%)  Mean  16.1  26.6  15.2  20.5
   SD  13.25  20.92  15.99  26.73
 Post-implantation loss (%)  Mean  10.8  11.4  10.0  8.5
   SD  16.70  14.86  10.13  9.29
 Resoprtions - early  Mean  0.8  0.6  0.7  0.7
   SD  1.72  0.81  0.89  0.71
 Resoprtions - late  Mean  0.3  0.2  0.3  0.2
   SD  0.64  0.4  0.48  0.42
 Resoprtions - total  Mean  1.1  0.8  1.0  0.9
   SD  2.01  1.00  0.98  0.97
 Live young - male  Mean  4.2  3.4  3.5  3.7
   SD  2.08  2.09  1.26  2.03
 Live young - female  Mean  3.8  3.4  4.7  3.7
   SD  1.77  1.50  1.52  2.29
 Total live young  Mean  8.0  6.8  8.2  7.4
   SD  2.31  2.86  1.93  2.89
 %   33   38    41   34
  Sex ratio (% male)   Mean   51.8 48.7     43.5   51.0
 SD  20.03 14.72   13.62  25.78
 Body weight - day 6 (kg)   Mean 3.35   3.28  3.49  3.24
   SD  0.421 0.368   0.400  0.414
 Body weight - day 29 (kg)  Mean  3.69  3.63  3.85  3.56
   SD  0.410  0.345  0.408 0.374 
 Body weight change day 6 -29 (kg)  Mean  0.33  0.34  0.36  0.32
 SD  0.148  0.119  0.121  0.126
  Gravid uterine weight (kg)  Mean  0.485  0.424  0.508  0.427
   SD  0.1133  0.1434  0.1088  0.1224
 Number mated  n  24  24  24  24
 Number not pregnant  n  0 2  1
 Number pregnant  n  24 21  22  23
 Number of total litter resorptions  n  0  0  0
 Number of early deliveries/abortions  n  0  0  0  0
 Number of litters with dead foetuses  n  0  0  0  1
 Number of litters with resoprtions  n  13  10  14  12
 Number of litters subject to foetal pathology  n  24  21  22  23

Summary of Litter Data

   Group  Control  100 mg/kg bw/day  300 mg/kg bw/day  1000 mg/kg bw/day
 Male foetal weight (g)  Mean  40.9  41.2  40.6  40.7
   SD  4.50  5.27  4.49  7.96
 Female foetal weight (g)  Mean  38.6  41.6  40.1  40.1
   SD  4.97  5.46  4.44  6.00
 Overall foetal weight (g)  Mean  39.6  41.5  40.4  40.6
   SD  4.22  4.81  4.31  7.01

Please refer to the attached documents for data on foetal examinations and historical control data.

Conclusions:
Based on the results from the OECD TG 414 study in rabbits, dose levels up to and including 1000 mg/kg bw/day did not adversely affect maternal performance or fetal development and growth. Therefore, the maternal and fetal No Observed Adverse Effect Level (NOAEL) is considered to be 1000 mg/kg bw/day - the highest dose tested.
Executive summary:

The influence of Pentaerythritol on embryo-fetal survival and development when administered during the organogenesis and fetal growth phases of pregnancy (from Day 6 to Day 28 after mating) in the New Zealand White Rabbit was evaluated in the OECD TG 414 study. Three groups of 24 females were administered Pentaerythritol at doses of 100, 300 or 1000 mg/kg bw/day by oral gavage administration, from Day 6 to 28 after mating. A similarly constituted Control group received the vehicle, 1% (w/v) methylcellulose (aqueous), at the same dose volume as the treated groups. Animals were killed on Day 29 after mating for reproductive assessment and fetal examination. No signs were observed at routine physical examination of maternal animals, in association with dose administration or at macroscopic examination that were attributable to treatment with Pentaerythritol. Reproductive performance, including the number of implantation sites, resorptions (early or late), the extent of pre or post-implantation loss, numbers of live young, sex ratio and placental, litter and fetal weights was unaffected by maternal treatment with Pentaerythritol. There was no evidence that treatment with Pentaerythritol induced major fetal abnormalities. There was some skeletal malformations observed in the treated groups, but these were not considered to be adverse and/or were within historical control range. Based on the results from the OECD TG 414 study in rabbits, dose levels up to and including 1000 mg/kg bw/day did not adversely affect maternal performance or fetal development and growth. Therefore, the maternal and fetal No Observed Adverse Effect Level (NOAEL) is considered to be 1000 mg/kg bw/day - the highest dose tested.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
A GLP and guideline-compliant study is available in two species (rat and rabbit) for pentaerythritol. Sufficient to address requirements.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

No evidence of reproductive or developmental toxicity potential was seen in a combined screening assay performed with pentaerythritol at dose levels of up to and including the limit dose of 1000 mg/kg bw/day.

No evidence of maternal toxicity or developmental toxicity potential was observed in a prenatal developmental toxicity study conducted with pentaerythritol in the rat or the rabbit, at dose levels up to and including 1000 mg/kg bw/day.

In an extended one generation reproductive toxicty study, 100, 300, and 1000 mg Pentaerythritol/kg bw/day was adminitered via oral gavage. The substance was well tolerated with no treatment-related mortality, no adverse effects on general condition, body weight, food consumption, thyroid hormones, clinical pathology, organ weights, sperm parameters, spleen cell immunophenotyping or pathology on either the F0 or F1 generation. In addition the reproductive performance and fertility of the F0 animals and the survival/development of the F1 offspring up to weaning were unaffected by treatment at all dose levels. Based on the results of this study, it is concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for both systemic and reproductive toxicity in the Sprague Dawley rat is 1000 mg/kg bw/day.

Given the lack of adverse findings from rat reproductive toxicity studies (OECD 422 and OECD 443), developmental toxicity study in the rat and in the rabbit (OECD 414), classification of the test item Pentaerythritol is not justified for reproductive and developmental toxicity according to EC Regulation 1272/2008.

Additional information