Registration Dossier

Diss Factsheets

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

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
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
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:
entaerythritol; 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.
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 oral 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.
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
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/day. 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/day.
Animals were assigned to treatment groups on the last day of acclimatisation, according to the body-weight stratified random sampling method.
Positive control:
A positive control was not included.
Observations and examinations performed and frequency:
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. The following parameters were determined for 3 hour pooled samples: pH, protein, glucose, ketone, occult blood and sediment; the following parameters were determined for the 21 hour pooled samples: specific gravity, volume, sodium, potassium and chloride.
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 determination of the following parameters: red blood cell count, haematocrit, haemoglobin, mean cell volume, mean cell haemoglobin volume, mean cell haemoglobin concentration, reticulocyte count, platelet count, white blood cell count, coagulation time, prothrombin time, activated partial thromboplastin time, separate leukocyte group percentage. Blood chemistry parameters determined were: GOT, GPT, gamma-GTP, cholinesterase, glucose, total cholesterol, triglyceride, phospholipid, total bilirubin, urea nitrogen, creatinine, sodium, potassium, chloride, calcium, inorganic phosphorous, total protein, albumin, A/G ratio, protein fraction.
Sacrifice and pathology:
Males were sacrificed the day after the last dose was administered for necropsy. The organs and tissues were examined grossly for abnormalities, then the following organs were weighed: liver, kidneys, thymus, adrenal glands, testicle, and epididymis. The following organs were fixed in 10% neutral buffred formalin for histopathological examination: 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, testicle, epididymis, seminal vesicle (including the coagulating gland), and prostate gland, and the organs were stored. Additionally, the eyeball and Harderian gland were fixed in Davidson's solution and the testicle and epididymis in Bouin's solution. The liver, kidney, spleen, heart, lung, brain, pituitary gland, thymus, adrenal gland, thyroid gland, stomach (anterior stomach, glandular stomach), duodenum, jejunum, ileum, appendix, colon, rectum, testicle, epididymis, and prostate gland were embedded in paraffin and stained with Haematoxycillin and Eosin.

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.
Other examinations:
No other examinations were reported.
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.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
: intermittent soft stool and diarrhoea
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
MORTALITY

There was no mortality during the study.

CLINICAL SIGNS

Four males exhibited soft faeces in the 300 mg/kg bw/d group, and 1 male in this group had diarrhoea intermittently until administration Day 9. All males in the 1000 mg/kg bw/d group had soft faeces and diarrhoea intermittently from administration Day 2 until terminal sacrifice. In the 300 mg/kg bw/d 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 bw/d 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 bw/d group exhibited soft faeces intermittently, and 9 females had intermittent diarrhoea.

BODYWEIGHT, FOOD AND WATER CONSUMPTION

There were no effects of treatment on body weight gain in either males or females. There were no effects on food intake in males. In 300 mg/kg bw/d females, food intake was lower than the control from the day prior to the start of dosing; this difference was significant on administration Day 1, and gestational day 14. This finding was considered incidental and unrelated to treatment, as the differences in food intake were seen from the start of the study. Water consumption was increased in the 1000 mg/kg bw/d males compared to controls from Day 10 of administration, but the finding was not statistically significant. The increase in water consumption was thought to be a secondary effect of the intermittent diarrhoea observed in this group.

CLINICAL INVESTIGATIONS

There were no effects of treatment on urinalysis parameters (only determined in males).

Haematology (only determined in males) revealed a significant decrease in mean cell haemoglobin volume and mean cell haemoglobin concentration in the 300 and 1000 mg/kg bw/d males compared with controls. Findings of the clinical chemistry analysis (only determined in males) were a decrease in phospholipid and calcium in the 100 mg/kg bw/d males, and a decreaee in total cholesterol, phospholipid and calcium in the 300 mg/kg bw/d males. There was no dose-response and changes were reportedly within the normal range.

ORGAN WEIGHTS

There were no effects of treatment on organ weights in males or females.

PATHOLOGY

There were no abnormal findings at autopsy in male rats. In females, the reported findings were yellowish-white marks on the liver, fatty tissue adhesion on the spleen, liver and within the abdominal cavity, ileum diverticulum and ovarian cysts. These findings were seen in both treated and control rats, and were therefore considered unrelated to treatment. In one 1000 mg/kg bw/d female, there was placental separation on the left side of the uterus.
Histopathology revealed the following changes in both treated and control males: perilobular fatty change of the liver and hyaline droplet deposition and eosinophilic body deposition in tubular epithelium in mainly the proximal tubules of the kidney, urinary cast, ciliated epithelial cyst, cardiac focal infiltration of cells mainly composed of histiocytes , calcium deposition in lung blood vessel wall, ciliated epithelial cyst of the pituitary body, retention of homogeneous plasma-like substances in Rathke’s pouch, interstitial infiltration of cells mainly composed of lymphocytes in the prostate gland.
The following observations were noted in both treated and control females: 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 (abnormal body parts found during the autopsy were included in the histopathological examination). As histopathological findings occurred in both control and treated groups, and in the absence of a dose-relationship, they were therefore not considered to be treatment related.
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
Critical effects observed:
not specified

Treatment-related effects in this study were limited to clinical signs (diarrhoea, soft stool) in both sexes at 300 and 1000 mg/kg bw/d and increased water consumption in males at 1000 mg/kg bw/d.

Conclusions:
The NOAEL for this study is 100 mg/kg bw/d, based on clinical signs at higher dose levels.
Executive summary:

A combined repeat dose oral toxicity and reproductive/developmental toxicity study was conducted with pentaerythritol in male and female Crj: CD(SD) rats. Pentaerythritol was adminsitered orally by gavage at doses of 0 (vehicle control), 100, 300 and 1000 mg/kg bw/d. Males were exposed for 46 days (starting 14 days prior to mating), and females were exposed for 14 days prior to mating, during mating and gestation until lactation day 3. There were no mortalities during the study. The only treatment related effects were seen in the 300 mg/kg bw/d and 1000 mg/kg bw/d groups and consisted of intermittent soft faeces and diarrhoea. There was also a tendency towards increased water consumption in 1000 mg/kg bw/d males (only determined in males), the difference was not significant and could be attributed to the occurrence of intermittent diarrhoea in this group. The NOAEL for repeated dose toxicity was therefore 100 mg/kg bw/d.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report date:
1996

Materials and methods

Test guideline
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

Test material

Constituent 1
Chemical structure
Reference substance name:
Pentaerythritol
EC Number:
204-104-9
EC Name:
Pentaerythritol
Cas Number:
115-77-5
Molecular formula:
C5H12O4
IUPAC Name:
pentaerythritol
Details on test material:
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.
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.

Test animals

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.

Administration / exposure

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.
Doses / concentrationsopen allclose all
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.

Examinations

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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

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.

Effect levels (P0)

open allclose all
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

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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.

Effect levels (F1)

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

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.