Registration Dossier

Diss Factsheets

Administrative data

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
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:

The study was performed in rats according to OECD guideline 443 in compliance with GLP. The test substance was administered by the oral route. The basic configuration of EOGRTS was performed as based on the toxicological profile of the substance there are no concern-driven scientific triggers for the performance of the F2 generation (extension of Cohort 1B), developmental neurotoxicity (DNT; cohorts 2A and 2B) and/or developmental immunotoxicity (DIT; cohort 3) cohorts.

- Premating exposure duration for parental (P0) animals: A premating exposure period of 10 weeks is used to cover the full spermatogenesis and folliculogenesis before the mating.

- Basis for dose level selection: The highest dose level was selected with the aim to induce some systemic toxicity, but not death or severe suffering of the animals. Dose levels were selected based on the data from the previous studies in rats. A single oral dose at 1250 mg/kg caused mortality (3/5) and noted clinical signs. No signs were observed after a single oral dose at 625 mg/kg. In embryo-fetal development toxicity study in rats (study No. 239-0004-TX), test item-related changes (increases) on body weight gains, absolute weight gain during gestation, and food consumptions were observed at 470 and 940 mg/kg/day. Since systemic toxicity was observed at 470 mg/kg and higher, and the exposure duration in the EOGRTS is longer than in the PNDT, the dose levels were set at 75, 150 and 450 mg/kg bw/day for this study.

- Inclusion/exclusion of extension of Cohort 1B: According to column 2 (specific rules for adaptation from column 1) point 8.7.3 of the amended REACH Annex X, extension of cohort 1B to include the F2 generation shall be proposed by the registrant based on the following conditions being met (a and any of b(i), b(ii) or b(iii)). See also: Chapter R.7a: Endpoint sepcific guidance Version 5.0 - December 2016:
A. The substance has uses leading to significant exposure of consumers or professionals, taking into account, inter alia, consumer exposure from articles
No – The substance has no uses leading to significant exposure of consumers or professionals. The substance has only a limited professional use, which is not expected to affect many users. Furthermore, the substance is applied as building block in polyurethane foams and is chemically incorporated into the polymeric matrix of the article.
B (i). The substance displays genotoxic effects in somatic cell mutagenicity tests in vivo which could lead to classifying it as Mutagen Category 2, or
No – The substance is not classified as Mutagen Category 1A or 1B or 2.
B (ii). There are indications that the internal dose for the substance and/or any of its metabolites will reach a steady state in the test animals only after an extended exposure, or
No – The substance is not classified as a PBT or vPvB. The log Pow of Polyol IXOL B350 was determined to lie between -0.03 and 3.3 and does not trigger any bioaccumulation potential. The toxicokinetic behaviour of the substances gives no hints for very slow clearance, the NOAEC/LOAEC of subchronic studies are not more than 3 times lower than that the NOAEC/LOAEC from a subacute study. Therefore there are no indications that the internal dose for the substance and/or any of its metabolites will reach a steady state in the test animals only after an extended exposure.
B (iii) There are indications of one or more relevant modes of action related to endocrine disruption from available in vivo studies or non-animal approaches
No - There are no indications based on the available study results that endocrine disruption is a relevant mode of action for the substance.

Therefore, based on the above considerations, the registrant does not believe that there is a basis for extending cohort 1B to include the F2 generation.

- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B: The registrant does not believe there is a need to include cohorts 2A and 2B in the test design. This is based on:
• previous studies with the substance do not indicate neurotoxic effects such as changes in brain weight or in specific neural areas not secondary to body weight, changes in brain volume or specific neural areas or (histo)pathological findings in brain, spinal cord and/or nerves
• Neurobehavioural observations (arena and Functional Observational Battery testing) and motor activity assessment performed as part of the subchronic toxicity study, did not indicate any neurotoxic potential of the test material.

- Inclusion/exclusion of developmental immunotoxicity Cohort 3: The registrant does not believe there is a need to include cohort 3 in the test design. This is based on:
• the substance has not caused biologically significant changes in haematology/clinical chemistry and/or organ weight associated with immunotoxicity such as reduced leucocyte count in combination with reduced spleen weight in repeated dose studies
• the substance has not caused significant effects to immunology organs such as thymus atrophy in repeated dose studies

- Route of administration: The oral route has been selected as it is the most appropriate route of administration for substances to focus on the detection of hazardous properties on reproduction.

Test material

Constituent 1
Chemical structure
Reference substance name:
2-Butyne-1,4-diol, polymer with 2-(chloromethyl)oxirane, brominated, dehydrochlorinated, methoxylated
EC Number:
614-503-3
Cas Number:
68441-62-3
Molecular formula:
(C3H7O2)xC4H4O2Br2(C4H9O2)y with x + y = 2.5
IUPAC Name:
2-Butyne-1,4-diol, polymer with 2-(chloromethyl)oxirane, brominated, dehydrochlorinated, methoxylated
Test material form:
liquid: viscous
Details on test material:
- Name of test material (as cited in study report): Polyol IXOL B350
- Substance type: Halogenated polyetherpolyol
- Physical state: viscous, dark brown liquid
- Analytical purity: treated as 100% pure
- Lot/batch No.: 3436146
- Expiration date of the lot/batch: 2020-10-01
- Storage condition of test material: Room Temperature

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: BioLASCO Taiwan Co., Ltd.
- Age at study initiation: Females and males, approx. 9-10 weeks
- Weight at study initiation: 219-272 g (F0 females) and 315-359 g (F0 males),
- Housing: During the pre-pairing period, the animals (F0) were individually housed in solid bottom cages, with bedding. During the mating period (F0), the rats were housed on the basis of one male to one female. The mated female (F0) was separated from the male and housed individually in a maternity cage where provided with appropriate nesting materials. After parturition, litters were housed with their mothers until weaning. After weaning (PND 21), F1 animals were group housed (up to 3 animals of the same sex and same dosing group together).
- Diet: rodent feed, ad libitum (except during fast period)
- Water: reverse-osmosis purified and chlorinated water, ad libitum
- Acclimation period: The pretest period (from arrival to the study to first day of dosing) was at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): targeted rage between 20 and 25°C, actual range 21.5 to 25.7°C. The temperature was occasionally out of the target range, but each of these excursions lasted less than 2°C.
- Humidity (%): targeted rage between 40 and 70%, actual range 38.8 to 104.2%. The humidity was occasionally out of the target range, but each of these excursions lasted less than 3 hours.
- Air changes (per hr): 10 to 20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 1% (w/v) Gum tragacanth powder in purified water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared at least once weekly. The dose formulations intended for dosing were stirred constantly at room temperature from at least 30 minutes before dosing to completion of the dosing. Homogeneity was determined in this study for the low- and high-dose formulations following the first and last preparation for F0 and F1 animals. No correction was made for the purity of the test substance. Solutions were stored at ambient temperature.

VEHICLE
- Justification for use and choice of vehicle: 1% (w/v) Gum tragacanth powder in purified water. This vehicle was selected based on trial formulations performed at WuXi AppTec.
- Amount of vehicle (if gavage): 10 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 14 days
- Proof of pregnancy: sperm in vaginal smear referred to as Day 0 of pregnancy (GD 0)
- After successful mating each pregnant female was caged: individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
CONCENTRATION VERIFICATION:
The concentration of Polyol IXOL B350 was determined in all dose formulations and the control according to a validated method (WuXi AppTec Study No.: 239-0002-AC). For concentration analysis, triplicate samples (one for analysis, the other 2 for backup) were collected from the middle portion of the mid-dose formulations and control formulations. For the low- and high-dose formulations, mean results of the homogeneity results were used as concentration verification and no additional samples were collected.
The concentration of Polyol IXOL B350 in the dosing formulations were within 99 to 104% of nominal values. The control samples were tested and there was no detectable peak observed at the retention time of Polyol IXOL 350 (< LOQ).

HOMOGENEITY:
Homogeneity was determined for the low- and high-dose formulations following the first preparation (for F0 animals and F1 animals) and the last preparation of dose formulations. For homogeneity analysis, triplicate samples (one for analysis, the other 2 for backup) were collected from the top, middle, and bottom layers of the low-and high-concentrations formulations.
The homogeneity of the dose formulations at 7.5 and 45 mg/mL were performed. The concentrations of the top, middle and bottom samples were within 99% to 103% of nominal values. The relative standard deviations (RSD) of top, middle, and bottom samples were within 0.1% to 1%.

STABILITY:
At the first preparation (for F0 animals), three sets of samples (1 mL each) for stability analysis were collected from the middle portion of the test item formulations at the low concentration (7.5 mg/mL). The stability of samples in vehicle was determined after storage of at least 8 days at room temperature under normal laboratory light conditions. POLYOL IXOL B350 in dosing formulation at 7.5 mg/mL was stable for at least 8 days at room temperature.
Duration of treatment / exposure:
Parental animals:
Males: F0 males were treated 10 weeks prior to mating and throughout mating to termination (until weaning of all F1 animals).
Females: F0 females were treated 10 weeks prior to mating, throughout mating, pregnancy and lactation until termination after the weaning of their litters. If LD (Lactation Day) 0 date was confirmed before daily dosing, the dam was not dosed on LD 0.

F1 Offspring:
F1 Cohort 1A animals were treated from weaning (PND 21) for 10 weeks. F1 Cohort 1B males were treated from weaning (PND 21) for 11 weeks.
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
75 mg/kg bw/day
Dose / conc.:
150 mg/kg bw/day
Dose / conc.:
450 mg/kg bw/day
No. of animals per sex per dose:
Parent generation: 28/sex/group
Cohort 1A: One male and/or one female per litter were selected (20/sex/group)
Cohort 1B: One male and/or one female pup per litter were selected (20/sex/group)
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS:
Viability (morbidity and mortality) checks were performed twice daily, except on animal receipt and the day of necropsy, where animal will be examined at least once. Cage side observation were conducted once daily at least from Day -1 (for F0 animals), and at least once daily during the dosing period (at approximately 2 to 3 hours post dose) for all F0 study animals. Cage side observation were not conducted if a detailed observation is scheduled at the same time slot. Pregnant rats were examined for parturition at least 3 times daily for signs of parturition from GD 21 until deliver but not over GD 24.

DETAILED CLINICAL OBSERVATIONS:
Detailed clinical observations were conducted at least once prior to randomization for all F0 animals, at least once on the first dosing day and at least once weekly thereafter during the dosing period at approximately 2 to 3 hours post dose for F0 and F1 study animals. During gestation and lactation period, detailed observations were conducted at least once on GD 0, 3, 6, 9, 12, 15, 18, 20 and LD 1, 4, 7, 10, 13, 16, 19, 21 (at approximately 2 to 3 hours post dose) for F0 dams. Detailed observations were also conducted at least once on the day of scheduled necropsies.

BODY WEIGHT:
Each F0 animal was weighed at least once pretest for randomization, once on Day 1 prior to dosing and at least once weekly throughout the treatment phase for study animals. During the F0 gestation and lactation period, body weight of study animals (dams) were weighed at least once on GD 0, 3, 6, 9, 12, 15, 18, 20 and LD 1, 4, 7, 10, 13, 16, 19, 21. Body weight of study animals was also collected on the day of scheduled necropsies.

FOOD CONSUMPTION:
Quantitative food consumption was measured (weighed) once for F0 study during pretest and at least once weekly throughout the study (except the week during mating). During the F0 gestation and lactation period, food consumption on GD 0-3, 3-6, 6-9, 9-12, 12-15, 15-18 and 18-20 and LD 1-4, 4-7, 7-10, 10-13, 13-16, 16-19, 19-21 was measured for dams. For the F0 males, in the last week before scheduled males necropsy initiation, the food end of each cage was measured on the day prior to the initiation of scheduled necropsy period of males.

URINALYSIS:
- Time schedule: For F0 females and F0 males prior to necropsy
- Number of animals used: 10 animals/sex/group
- Metabolism cages used: Yes, one rat per cage, urine was collected overnight
- Fasted: Animals were fasted overnight
- Parameters checked: volume, color and clarity, specific gravity, pH, glucose, microscopic examination of sediment, bilirubin, ketones, occult blood, protein, urobilinogen.

HAEMATOLOGY:
- Time schedule: For F0 females and F0 males prior to necropsy
- Number of animals used: 10 animals/sex/group
- Fasted: Animals were fasted overnight
- Anesthesia used: isoflurane
- Parameters examined: See Table 1 in ‘Any other information on materials and methods incl. tables’.

CLINICAL CHEMISTRY:
- Time schedule: For F0 females and F0 males prior to necropsy
- Number of animals used: 10 animals/sex/group
- Fasted: Animals were fasted overnight
- Anesthesia used: isoflurane
- Parameters examined: See Table 2 in ‘Any other information on materials and methods incl. tables’.

OTHER: T4 AND TSH HORMONE DETERMINATIONS:
- Time schedule: For F0 females and F0 males prior to necropsy
- Number of animals used: 10 animals/sex/group
- Fasted: Animals were fasted overnight
- Anesthesia used: isoflurane
- Analysis for T4 and TSH hormones were performed with commercially available ELISA kits. The ELISA was performed according to a validated method based on the manufacturer’s protocol.
Oestrous cyclicity (parental animals):
Vaginal smears were prepared daily and used to determine the stages of estrus cycle during from 2 weeks of treatment prior to mating of the F0 study females and continued through mating period. In addition, vaginal smears were prepared for F0 study females at the day of sacrifice (unscheduled or scheduled). Vaginal smears were not prepared for females found with a retained vaginal plug which were considered on estrus.
Sperm parameters (parental animals):
Parameters examined in male parental generation:
The sperm motility, morphology, and count was evaluated for all F0 males of each group at scheduled necropsy. The sperm sample was collected from the left cauda epididymis which was weighed in advance (right may be used if there is an observation on left that would interfere with the sperm analysis). The epididymal sperm concentration was calculated as follows:
Sperm concentration = sperm count per cauda/weight of cauda epididymis (g)
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible). If there are 10 or less pups in a litter, no culling was performed. If there are 5 or less pups of either sex in a litter, none of these pups will be culled. If there are less than 5 pups of either sex but more than 10 pups in a litter, the sex with more pups will be culled to give a total of 10 pups.The other culled pups were euthanized on the day of culling.

PARAMETERS EXAMINED
The following parameters were examined in pups that were not selected for the cohorts:
- Litter evaluation: After littering was finished, the entire litter was observed for maternal and pup nesting behavior, the viability and a qualitative assessment of body temperature (normal or cold to touch) of each pup on PND 0. Each pup was identified and evaluated for sex, body weight and external alterations on PND 1. The pup with external malformation was euthanized and saved in 10% neutral buffered formalin for possible further evaluation. Pups viability and count was conducted at least once daily at lactation (PND 0 to PND 21).
- Body weight: The F1 animals were weighed individually on PND 1, 4, 7, 10, 13, 16, 19, 21
- Anogenital distance: At day lactation day 4 the anogenital distance (AGD) was measured of each pup before culling of the litter.
- Nipple retention in male pups: On postnatal day 11/12/13 all surviving male pups were quantitatively examined (number of nipples/areolae per pup will be counted) for the presence of nipples and/or areolae.
- Reflexological and Sensory Development: During lactation, all selected F1 animals were examined for reflexological and sensory functions, including: surface righting reflex, air righting reflex, cliff avoidance, negative geotaxis, and auricular startle response.
- T4 and TSH hormone determinations: On PND 4, at least 10 surplus pups/sex/group, 1 male and/or 1 female surplus pups/litter/group (if applicable) were randomly selected for blood collection for T4 analysis. As much blood as possible (approximately 0.3~0.5 mL) was collected by cardiac puncture as a terminal procedure for each pup (the pup was anaesthetized by intraperitoneal injection of pentobarbital sodium with dosage of 3 mg/pup prior to blood collection). If the blood volume was less than 0.2 mL, and there was another animals in the same group with low blood volume (less than 0.2 mL), they were pooled, if no other animals in the same group with low blood volume (less than 0.2 mL), attempt was made to obtain serum from these samples.
On PND 22, at least 10 surplus pups/sex/group, 1 male and/or 1 female surplus pups/litter/group (if applicable) were randomly selected for blood collection for TSH and T4 analysis. As much blood as possible (approximately 1.5 mL) was collected from the abdominal aorta (vena cava may be used if required) at necropsy as a terminal procedure after anesthesia.
- Selection of the Cohorts: Pups were selected randomly for all available litters on PND 21, with the exception those obvious runts (animals with a body weight more than two standard deviations below the mean pup weight of the respective litter). If there were 2 or less pups of either sex in a litter, none of these pups were culled. Ten culled males and female pups (from different litters) on PND 21 at control group were selected for spare F1 animals. These spare F1 animals were used for possible replacement within 5 days after the initiation of F1 dosing.

The following parameters were examined in pups that were selected for the cohorts (1A and 1B).
- General clinical observations: Viability (morbidity and mortality) checks were performed twice daily. Cage side observation were conducted at least once daily during the dosing period (at approximately 2 to 3 hours post dose).
- Detailed clinical observations: Once weekly (at approximately 2 to 3 hours post dose) all animals were subjected to detailed clinical observations
- Body weight: Body weights of all male and female animals were recorded weekly. Additionally body weight was recorded on the day when they attain puberty (completion of preputial separation or vaginal patency).
- Food consumption: Food consumption was measured for F1 animals at least once weekly from weaning till termination.
- Sexual maturation: All selected F1 animals were observed for development landmarks including pinna unfolding, hair growth, eye opening, tooth eruption, nipple retention, testes descent, preputial separation, and vaginal opening
- Estrous cycle (Cohort 1A): After the onset of vaginal patency, vaginal smears were examined at least once daily for Cohort 1A until the first estrus stage is observed. From PND 75 to 88, vaginal smears were examined to determine estrus cycle for Cohort 1A.
- Clinical pathology: Prior to necropsy, blood was collected from F1 Cohort 1A study animals (10 animals/sex/group) for evaluation of hematology and clinical chemistry according to Table 1 and 2 (‘Any other information on materials and methods incl. tables’). Urine was collected from F1 Cohort 1A study animals (10 animals/sex/group) once just prior to necropsy and checked for volume, color and clarity, specific gravity, pH, glucose, microscopic examination of sediment, bilirubin, ketones, occult blood, protein, urobilinogen. Animals were fasted overnight prior to blood/urine collection.
- Terminal body weight (Cohort 1A and 1B): Terminal body weight was determined for all Cohort 1A and 1B animals prior to scheduled necropsy.
- T4 and TSH hormone determinations (Cohort 1A): At least 1.5 mL of blood (for TSH and T4 analysis) was collected from F1 Cohort 1A animals (10/sex/group surviving) from the abdominal aorta at scheduled necropsy as a terminal procedure after isoflurane anesthesia. Analysis was performed with commercially available ELISA kits. The ELISA was performed according to a validated method based on the manufacturer’s protocol.
- Sperm Analysis: At scheduled necropsy, epididymal sperm was derived from the left cauda epididymis of all male Cohort 1A animals of each group for evaluation of sperm motility, morophology and count.
- Immunox analysis: Splenic lymphocyte subpopulation analysis was performed in F1 Cohort 1A (10 animals/sex/group). At scheduled necropsy, after tissue collection for histopathology, the remaining tissue (one half of the spleen) for the spleen was collected and transferred on wet ice for processing and analysis. Lymphocyte subpopulation analysis was conducted using a validated flow cytometry method (method number: 385-0008-IM). For the flow cytometric analysis of the splenocytes, the cells were incubated with a specific antibody cocktail preceded by Fc block to minimize non-specific antibody binding to surface Fc receptors. The antibody cocktail consisted of anti-CD45RA, CD161, CD3, CD4 and CD8a antibodies in order to determine the frequency of T lymphocytes (i.e. T-helper and T-cytotoxic cells), B lymphocytes and natural killer cells by flow cytometry (FACS CANTO II). Addition of FVS dye enabled discriminating living and dead cells. Flowcytometric analysis was performed using a FACSCanto™ II flowcytometer and analyzed using BD FACSDiva™ 8.01 software. In addition to the absolute numbers of above-mentioned cells, frequencies were calculated as follows:
- % total T cells = (number of T cells / number of live cells) x 100%
- % T-helper cells = (number of T-helper cells / number of T cells) x 100%
- % T-cytotoxic cells = (number of T-cytotoxic cells / number of T cells) x 100%
- % B cells = (number of B cells / number of live cells) x 100%
- % NK cells = (number of NK cells / number of live cells) x 100%

GROSS EXAMINATION OF DEAD PUPS:
Necropsy was performed on stillborn pups and pups dying or sacrificed in a moribund state during the study and macroscopic abnormalities were recorded.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals were sacrificed within 4 days after all dams necropsied.
- Maternal animals: All surviving animals were euthanized and subjected to necropsy on LD22. Unmated females (if any) were necropsied 26 days after completion of mating period.
Treated study animals found dead or euthanized, the dams or their pups found dead or in moribund will be necropsied completely. The pregnancy should be confirmed for paired females, if applicable.

GROSS NECROPSY
At unscheduled and/or scheduled necropsy, all male and female animals were anesthetized by isoflurane and euthanized by exsanguinations and/or necropsied. Animals were fasted overnight prior to scheduled necropsy.
An examination of the external features of the carcass, external body orifices, and cervical, abdominal, and thoracic viscera, organs and tissues was performed. Gross lesions and all tissues listed in Table 3 in 'Any other information on materials and methods incl. tables' were collected, fixed and retained for pathology evaluation.
The ovaries and uteri of dam on LD 22 were examined to determine:
♦ Number of corpora lutea on each ovary;
♦ Location and No. of implantation sites.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 3 in ‘Any other information on materials and methods incl. tables’ were prepared for microscopic examination and weighed, respectively. In addition, all tissue masses and gross lesions were collected. Tissues were trimmed and fixed in 10% neutral buffered formalin (NBF) with the following exceptions: eyes with optic nerve(s) and testes as well as epididymides will be fixed in Modified Davidson’s solution for 24 to 72 hours. After fixation, these tissues were transferred to 10% NBF.
Tissues for microscopic examination were embedded in paraffin wax, sectioned and stained with haematoxylin and eosin. Microscopic examination was performed on the collected organs of all animals of the control (group 1) and high dose group (group 4). For the lower dose groups from F0 animals, slides were prepared and examined microscopically only for 1) gross lesions and masses, 2) tissues with potential compound-related effects [target organs: Kidney (males only); Heart (males only); Liver (both sexes); Thyroid (both sexes); Pituitary (both sexes)] based on experimental findings.
Postmortem examinations (offspring):
SACRIFICE
- Grossly malformed pups were sacrificed and examined.
- The F1 pups that were not selected for the cohorts (non-selected pups) at PND 4 or at weaning (PND 22) were sacrificed.
- The Cohort 1A and 1B pups were sacrificed on scheduled necropsy.

GROSS NECROPSY
The unscheduled and/or scheduled terminated pups were euthanized by intraperitoneal injection of pentobarbital sodium (5 mg/fetus, supplement anesthetics when deemed necessary) before and including PND14. After PND14 (before and including weaning on PND 21), the unscheduled terminated study animals were euthanized by CO2 inhalation. The unscheduled and/or scheduled F1 Cohort 1A terminated animals were anesthetized by isoflurane and euthanized by exsanguinations and/or necropsied. Cohort 1A animals were fasted overnight prior to scheduled necropsy. The unscheduled and/or scheduled terminated F1 Cohort 1B animals were euthanized by exsanguination after CO2 inhalation and were not fasted prior to necropsy.

HISTOPATHOLOGY / ORGAN WEIGTHS
- F1 pups: The organs/tissues listed in Table 4 in ‘Any other information on materials and methods incl. tables’ field of 10 non-selected pups/sex/group at weaning were weighed and/or were subjected to microscopic examination. The remaining surplus pups were necropsied with gross observation only.
- Cohort 1A animals: A complete necropsy was conducted on Cohort 1A animals. Females were necropsied on Day 71.
- Cohort 1B animals: A complete necropsy was conducted on Cohort 1B animals on Day 78.

OTHER:
- Since treatment-related effects were observed in the high-concentration group, evaluation of the Kidney (males only); Heart (males only); Liver (both sexes); Thyroid (both sexes); Pituitary (both sexes)] were examined in the lower dose groups.
Statistics:
The statistical procedures for analysis of the various parameters of this study are described in Table 4 in "Any other information on materials and methods incl. tables'.
Group Pair-Wise Comparisons
Whenever there were more than two groups, the homogeneity of the group variances was evaluated using the Levene’s test at the 0.05 significance level. If differences between group variances are not found to be significant (p>0.05), then a parametric one way analysis of variance (ANOVA) was performed. When significant differences among the means are indicated by ANOVA test (p≤0.05), the Dunnett’s test was used to perform the group mean comparisons between the control group and each treated group.
If Levene’s test indicates heterogeneous group variances (p≤0.05) and the data set contains just positive values, log transformation was performed. If transformed data still failed test for homogeneity of variance (p≤0.05) or where the data contains zero and/or negative values, then the non parametric Kruskal-Wallis test was used to compare all considered groups. When the Kruskal-Wallis test was significant (p≤0.05), the Dunnett’s test on ranks was used to perform the pairwise group comparisons of interest.
Whenever there are only two groups to compare, the Levene’s test was performed as desribed above but a two sample Students's t-test replaced the ANOVA, a Wilcoxon rank-sum test was performed instead of the Kruskal-Wallis and, no Dunnett’s tests or Dunnett’s tests on ranks was performed.
Each pairwise group comparisons of interest was conducted via a two-sided test at the 5% significance level. Significant results are reported as either p≤0.001, p≤0.01, or p≤0.05, where p represents the observed probability.
Reproductive indices:
Mating, fertility and fecundity indices will be calculated using the following formula:
♦ Male mating index: (No. of males with confirmed mating/No. of males cohabitated) ×100
♦ Male fertility index: (No. of males impregnating a female/No. of males cohabitated) ×100
♦ Female mating index: (No. of females with confirmed mating/No. of females cohabitated) ×100
♦ Female fertility index: (No. of pregnant females/No. of females cohabitated) ×100
♦ Fecundity index: (No. of pregnant females/ No. of females with confirmed mating) ×100
Offspring viability indices:
♦ %Viability Index (till PND 4) = Number live pups on PND 4 prior to pup culling / Number live pups on PND 0 x 100%.
♦ %Viability Index (till PND 21) = Number live pups on PND 21 / Number live pups on PND 4 after pup culling x 100%.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
For F0 animals, dose related abnormal stool (soft) were noted in 2 males (Days 15 to 18) and 1 female (Day 8) at 150 mg/kg/day and 11 males (Days 7 to 26) and 1 female (Days 18 to 26) at 450 mg/kg/day during premating period. These signs were considered non-adverse due to transient in nature. In addition, 1 female with hard mass in head (Days 59-63) at 150 mg/kg/day, 2 males with mass in head (hard mass) or anogenital region (soft mass) (Days 103-131) and 1 female with hard mass in axillary (GD 6 to LD 22) at 450 mg/kg/day were observed. This finding (mass) was of uncertain relationship of the test item due to low incidence and lack of clear dose response.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No test item-related mortality of F0 animals was observed during the study period. One F0 female administered 150 mg/kg/day was found dead on Day 28 (premating) and one female administered 75 mg/kg/day was found dead on LD 20 (during lactation period). The cause of death for the female at 150 mg/kg/day was attributed to moderate subacute inflammation in the lung which was consistent with gavage error. The cause of death for the female at 75 mg/kg/day was attributed to spontaneous lymphoma.

Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no test item related changes on body weights and body weight changes for F0 animals during premating (both males and females), gestation and lactation period. All the data in treated groups were comparable to concurrent control.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no test item related changes on food consumption for F0 animals during premating (both males and females), gestation and lactation period. All the data in treated groups were comparable to concurrent control.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
There were no test item-related changes in hematological parameters for F0 animals in this study. The variations in hematological parameters, including those that attained statistical significance were considered unrelated to test item treatment because they were small in magnitude, comparable with the control group, lack of dose-dependency effects, and/or sporadic incidence.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Increases in TP, ALB, GLB, TCHO and Ca were observed in F0 males at 450 mg/kg/day when compared to the control by the end of dosing phase. These changes were considered test item-related but non-adverse due to small magnitude. The results are provided in Table 3 (see attached document).

Thyroid hormone analyses:
TSH was increased in a number of males dosed at 450 mg/kg/day. Compared with mean control data, the increment was 1.5 fold. TSH was also increased in one male receiving 150 mg/kg/day (Animal No. 3001, 2 fold compared with controls), but the group mean values were comparable to the controls. Test item-related and significant decreases in T4 were noted in males at ≥75 mg/kg/day when compared with the control. Since thyroxine (T4) was decreased in the same group animals, these data could reflect a hypothyroidism induced by the test item. Detailed results are provided in Table 5 (see attached document).
Endocrine findings:
effects observed, treatment-related
Description (incidence and severity):
Test item-related changes on thyroid hormone included increased TSH in F0 males at ≥ 150 mg/kg/day, and decreased T4 in F0 males at ≥ 75 mg/kg/day. These changes correlated with thyroid hypertrophy/hyperplasia (non adverse changes) in F0 males and females at ≥ 75 mg/kg/day and thyroid adenomas (adverse changes) in F0 males at ≥ 75 mg/kg/day, and one F0 female at 450 mg/kg/day. These data could reflect a hypothyroidism induced by the test item.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no test item-related changes in urinalysis and urine sediment parameters for F0 animals in this study.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The detailed results on the histopathological findings of the F0 animals are provided in Table 10 (see attached document).

Liver: In F0 animals, minimal hepatocellular hypertrophy was observed in seven males and two females administered 450 mg/kg/day. Increases in group mean hepatic organ weight parameters correlated with these observations at a dose level of 450 mg/kg/day. Hepatocellular hypertrophy was considered an adaptive response and was not considered adverse.

Thyroid: Increased incidence and/or severity of thyroid hypertrophy/hyperplasia was observed in males and females at doses ≥ 75 mg/kg/day. Diffuse thyroid gland hypertrophy/hyperplasia was characterized by increased follicular cell size and cellular density with nearly no colloid within the follicles diffusely across the glands in mildly affected animals. Minimally affected animals were similarly affected; however, more colloid was present in the follicles. In some animals, thyroid gland hypertrophy/hyperplasia had a nodular morphology that was characterized by multifocal expansion of hyperplastic follicular structures resulting in a nodular appearance to the gland. Notably, minimal thyroid hypertrophy (characterized by increased cellular size and occasional small projections in to the luminal of the follicles) was observed at a high incidence in both male and female control animals as a background finding; therefore, all tabulated findings in test item-treated animals were above this threshold. Thyroid hypertrophy/hyperplasia was considered an adaptive response and was not considered adverse.

Kidneys: In the kidneys, the test item was associated with a slight increase in severity of background/spontaneous alterations. The background/spontaneous alterations changes consisted of an increased incidence and severity of focal and multifocal tubular basophilia and multiple proteinaceous casts in males at doses ≥ 75 mg/kg/day. Tubular basophilia was characterized by segmental basophilia of tubular epithelium in scattered tubules of the cortex and outer stripe and proteinaceous casts were characterized by hyaline homogeneous material within tubules with attenuated cytoplasm. Given the minimal magnitude of the tubular basophilia and proteinaceous casts, and similarity to common background findings in rats of this age, these findings were not considered adverse in the context of this study. Additionally, there were three 450 mg/kg/day males with minimal focal hemorrhage within single tubules in the cortex that was consistent with damage to the glomerular basement membrane. These changes correlated with increases in renal weight parameters in males administered 450 mg/kg/day and were considered an adverse finding.

Pituitary: In the pituitary, increased amphophilic cells were present in males at doses ≥150 mg/kg/day; and in one 75 mg/kg/day female and two 450 mg/kg/day females. This change was characterized by scattered individual or small groups of pituicytes with slightly increased amounts of amphophilic cytoplasm. The dose relationship in males made it clear that increased amphophilic cells in the pituitary was related to the test item; however, in females, the low incidence and severity coupled with the lack of a clear dose response made the relationship of the observations in females to the test item uncertain. Given the lack of degenerative changes in this cellular population, this change was considered an adaptive response and not an adverse change.

Heart: In the heart, a test item-related decreased incidence of myocardial degeneration and mononuclear cell infiltration was observed in males administered 450 mg/kg/day compared with male controls. Given the low incidence of these findings in control females, a similar effect in females could not be established. In control males, minimal or mild degeneration of the myocardium was characterized by single or multiple foci of cardiomyocytes with granular, fragmented sarcoplasm infiltrated by mononuclear cells. In animals without active degeneration, minimal mononuclear cell infiltration was characterized by small foci of mononuclear cells within the interstitium.
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Thyroid adenomas occurred in individual males administered doses ≥75 mg/kg/day and in one female administered 450 mg/kg/day (see Table 10 of the attached document). These nodules compressed and/or displaced normal thyroid tissue peripherally. The adenomas varied in appearance from single small focal encapsulated or unencapsulated nodules with abnormally shaped follicles or collapsed follicles and piling of cuboidal (occasionally basophilic) follicular cells to multiple large foci nearly completely replacing normal glandular tissue. The presence of adenomas correlated with increased thyroid organ weight parameters on an individual animal level. Thyroid adenomas of the follicular epithelium were considered adverse due to permanent alteration to the organ structure.
In the rat, thyroid hypertrophy/hyperplasia and/or increased incidences of thyroid follicular adenomas occur in response to chronic receptor-mediated TSH stimulation of the thyroid gland, particularly in males (Capen, 1997). This occurs secondary to interference with thyroid hormone synthesis or secretion, increased thyroid hormone excretion into the bile, or disruption of conversion of T4 to T3. It should be noted that rodent thyroid tumors resulting from continuous stimulation of the thyroid gland by increased TSH levels are not relevant to humans (US FDA, EFSA). Consequently, compounds that induce such tumors do not warrant classification as carcinogenic.
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
F0-Females: Prolonged dioestrum (>5 consecutive days) was noted in 9/28, 10/28, 15/27, 17/28 F0 females at control, 75, 150 and 450 mg/kg/day respectively. The mean estrus cycle at 75 mg/kg/day, 150 mg/kg/day and 450 mg/kg/day (attained statistical significance) was 7.4, 8.2, 9.8 days respectively, which was higher than the concurrent control (6.2 days). In addition, there were 2/27, 4/28 acyclic F0 females at 150 and 450 mg/kg/day respectively, which were also higher than control (1/28). The changes correlated with increased number of cyclic animals with length of the longest cycle (>6 days) at 150 and 450 mg/kg/day. The mean length of the longest cycle at 75 mg/kg/day, 150 mg/kg/day and 450 mg/kg/day was 7.8, 9.1, 10.4 days respectively, which was higher than the concurrent control (6.7 days).
These changes were considered test item-related. However, these effects were not considered adverse in absence of effects on cohabitation duration or fertility.
Irregular estrous cycles maybe a consequence of the observed hypothyroidism induced by the test item since thyroid hormones play a role in the regulation of reproductive hormones secretion in the cyclic rat ovary.
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
Slight decreases in rapid sperm was noted in F0 males at 450 mg/kg/day (14%, attained statistical significance) when compared to the control. However, the motile sperm in this group was comparable to the control. These changes were considered test item-related but non-adverse due to small magnitude and lack of reproductive organs histopathology correlates.
Reproductive performance:
no effects observed
Description (incidence and severity):
- Fertility data: There were no test item-related changes in fertility data, including male mating index, male fertility index, female mating index, female fertility index, and fecundity index in this study. Detailed results are provided in Table 1 (see attached document).
- Cohabitation Duration: There were no test item-related cohabitation duration changes in the study. All the data in test item treated groups were comparable to the control. With exception of one paired group at 450 mg/kg/day, all other paired males and females mated successfully within 2 weeks mating period. One female at control, one female at 75 mg/kg/day, and two females at 450 mg/kg/day mated but did not result in pregnancy.
- Gestation duration: There was no effect on gestation duration in the present study. All the data in the treated groups were comparable to the control.
- Parturition observation: No abnormal signs were observed at parturition in this study.
- Litter data for F0 animals: There were no test item related changes on litter data, for F0 animals, including number of corpora lutea, implantation sites, live pups (litter size) and pre- and post-implantation loss. Detailed results on litter data are provided in Table 2 (see attached document).

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
<= 75 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: neoplastic
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: neoplastic
Dose descriptor:
NOAEL
Effect level:
450 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects observed in reproductive and developmental parameters and general toxicity at any of the dose levels

Target system / organ toxicity (P0)

Critical effects observed:
yes
Lowest effective dose / conc.:
75 mg/kg bw/day (nominal)
System:
endocrine system
Organ:
thyroid gland
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no test item related clinical signs for F1 animals in this study.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
There was no effect on pup viability and count at lactation (PND0 to PND21). All F1 animals after weaning survived to scheduled necropsy dates.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no test item related changes on body weights and body weight changes for F1 animals during lactation period and after weaning. There were no test item related changes on F1 body weights when puberty was attained. All the data in treated groups were comparable to concurrent control.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no test item related changes on food consumption for F1 animals after weaning to termination. All the data in treated groups were comparable to concurrent control.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
There were no test item-related changes in hematological parameters for F1 animals in this study.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Increases in TP, ALB, TCHO and Ca were observed in F1 males at 450 mg/kg/day when compared to the control by the end of dosing phase. These changes were considered test item-related but non-adverse due to small magnitude. The results are provided in Table 4 (see attached document).

Thyroid hormone analyses:
F1 Animals-PND 4: No differences between control and treated animals were observed for T4. TSH was not measured at PND4.
F1 Animals-PND 22: No differences between control and treated animals were observed for TSH and T4.
F1 Animals-Cohort 1A - Day 71: F1 males showed an increase of TSH at 450 mg/kg/day. TSH was also increased in a number of females receiving 450 mg/kg/day. The increments were 3.7 in males and 3.3 in females. Two males dosed at 150 mg/kg/day showed similar increase of TSH (5.0 and 5.3 fold, respectively, compared with controls). Thyroxine (T4) decrease was recorded in almost all animals of both sexes dosed at 450 mg/kg/day, therefore the above findings confirmed the hypothyroidism observed for F0 animals. Detailed results are provided in Table 6 (see attached document).
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no test item-related changes in urinalysis and urine sediment parameters for F1 animals in this study.
Sexual maturation:
no effects observed
Description (incidence and severity):
- Vaginal opening: There were no effects on the mean age of vaginal opening in F1 females
- Time to first estrous: There were no effects on the mean time from vaginal opening to first oestrous in F1 females.
- Estrous cycles: There were no effects on means estrous cycle parameters in F1 females.
- Preputial separation: Slightly prolonged group mean day of preputial separation was noted at 450 mg/kg/day (+2 days) when compared to control. These changes were considered test item-related but non-adverse due to small magnitude and the individual data in each group including the control group was in the same range (38 to 49 postnatal days).
- Sperm measures: there were no test item-related changes in sperm motility, sperm count, or sperm morphology (incidence of sperms with morphological abnormalities) for F1 animals in this study. All data in test item treated groups were comparable to the control.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
No changes were observed on the anogenital distance of pups measured on PND 4.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
No nipples were retained in male pups on PND 11/12/13.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
In F1 animals on PND 22, test item-related increases in absolute and relative thyroid gland weight parameters were observed in animals administered ≥150 mg/kg/day. The results are provided in Table 8 (see attached document).
At terminal sacrifice of cohort 1A, F1 animals on Day 71, test item-related increases in liver and thyroid organ weight parameters were observed at all doses. These increases were most pronounced at a dose level of 450 mg/kg/day. The results are provided in Table 9 (see attached document). No test item-related differences in organ weight parameters were observed in the weighed organs on Day 78 for Cohort 1B (brain, epididymis, prostate, testes, seminal vesicles and coagulating glands, ovaries, uterus and pituitary gland).
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
For F1 surplus pups on PND 4 and F1 surplus pups on PND 22, no abnormal macroscopic observations were noted.
For F1 necropsy of Cohort 1A on Day 71, test item-related macroscopic observations of enlarged liver were made for one male administered 75 mg/kg/day and two males administered 450 mg/kg/day. Test item-related bilateral enlargement of the thyroid glands was observed in four males and one female administered 75 mg/kg/day, four males administered 150 mg/kg/day and five males and one female administered 450 mg/kg/day. One male administered 450 mg/kg/day had a focal dark red discoloration of the glandular mucosa that correlated microscopically with a peracute focal erosion. This finding was not considered related to the test item due to the low incidence and severity of this observation.
For F1 necropsy of Cohort 1B on Day 78, an increased incidence of renal pelvic dilation was observed in males across all dose levels, including controls and were therefore not considered test item-related. Test item-related bilateral enlargement of the thyroid glands was observed in three males administered 75 mg/kg/day, three males administered 150 mg/kg/day and six males administered 450 mg/kg/day with two control males also affected. These tissues were not examined microscopically.
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
The detailed results on the histopathological findings of the F1 animals are provided in Table 11 (see attached document).

Liver: Minimal hepatocellular hypertrophy was observed in thirteen males and six females administered 450 mg/kg/day. Hepatocellular hypertrophy was as previously described for F0 animals and correlated with increases in group mean hepatic organ weight parameters at a dose level of 450 mg/kg/day.

Thyroid: Increased incidence and/or severity of diffuse and nodular thyroid hypertrophy/hyperplasia was observed in males and females at doses ≥75 mg/kg/day. These changes microscopically were as previously described for the F0 animals. Notably, minimal thyroid hypertrophy was observed at a high incidence in both male and female control animals as a background finding; therefore, all tabulated findings in test item-treated animals were above this threshold. Increased thyroid weight parameters occurred on a group level in males at all dose levels. A single thyroid adenoma occurred in one male administered 75 mg/kg/day and correlated with increased thyroid organ weight parameters in that individual.

Pituitary: Increased amphophilic cells were present in males at a dose level 450 mg/kg/day compared with controls. This finding was a previously described for the F0 animals.Notably, one control male had mildly increased amphophilic cells compared to the remainder of the controls.



Developmental neurotoxicity (F1)

Behaviour (functional findings):
no effects observed
Description (incidence and severity):
For F1 animals, there were no test item related changes on reflexological and sensory functions, including surface righting reflex, air righting reflex, cliff avoidance, negative geotaxis, and auricular startle response in this study. All the data in treated groups were comparable to concurrent control.

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
no effects observed
Description (incidence and severity):
There were no test item-related changes in spleen lymphocyte subpopulations percentage (including Total T cells, Helper T cells, Cytotoxic T cells, B cells and NK cells) in this study.

Details on results (F1)

- Sex-ratio: Sex ratio was comparable between treated and control groups.
- Developmental landmarks: For F1 animals, there were no test item related changes on development landmarks including pinna unfolding, hair growth, eye opening, tooth eruption and testes descent. All the data in treated groups were comparable to concurrent control.
- Sperm measures: there were no test item-related changes in sperm motility, sperm count, or sperm morphology (incidence of sperms with morphological abnormalities) for F1 animals in this study. All data in test item treated groups were comparable to the control.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
450 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects observed in reproductive and developmental parameters and general toxicity at any of the dose levels

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based on these results of the present study, the no observed adverse effect level (NOAEL) of POLYOL IXOL B350 on fertility and development toxicity was considered to be 450 mg/kg/day. The NOAEL of POLYOL IXOL B350 on systemic toxicity was considered to be 150 mg/kg/day for F0 females, and 450 mg/kg/day for F1 females, and less than 75 mg/kg/day for F0 and F1 males.
Executive summary:

In a GLP compliant Extended One Generation Study performed according to OECD 443, POLYOL IXOL B350 was adminstered daily by oral gavage to Sprague-Dawley rats.Two hundred and twenty-four rats (112/sex) were randomly assigned to 4 groups of 28/sex/group. F0 males were treated 10 weeks prior to mating and throughout mating to termination (total of 128 to 131 days). F0 females were treated 10 weeks prior to mating and continued throughout mating, pregnancy and lactation until termination after the weaning of their litters (total of up to 113 to 127 days). During the lactation period, the F1 animals were not administered with test item directly. The F1 animals consisted of 2 cohorts (Cohort 1A and Cohort 1B) from weaning and were treated with the test item directly from weaning (postnatal day, PND 21) to termination for 10 weeks (F1 Cohort 1A) or 11 weeks (F1 Cohort 1B). Cohort 1A was allocated for primary assessment of effects upon reproductive systems and of general toxicity, and Cohort 1B for possible histopathology follow-up assessment of reproductive system. F0 and F1 animals were administered POLYOL IXOL B350 at dosages of 0 (vehicle, 1% (w/v) Gum tragacanth powder in purified water), 75, 150, and 450 mg/kg/day by oral gavage once daily. The dose volume was 10 mL/kg.

Criteria for evaluation included viability (morbidity/mortality), clinical observations, body weight, food consumption, estrus cycle, cohabitation duration, sperm analysis, fertility data, gestation duration, parturition observation, litter evaluation, litter data, development landmarks, reflexological and sensory development, clinical pathology (hematology, serum chemistry, urinalyses), thyroid hormone analysis (TSH and T4), spleen lymphocyte

subpopulations, gross (necropsy) evaluation, organ weight, and histopathological evaluation (F1 Cohort 1B animals was not examined microscopically).

Results

No test item-related mortality was observed in both F0 and F1 animals during the study period. There were no test item-related changes on body weight or food consumption. There were no test item-related changes in cohabitation duration, fertility data (male mating index, male fertility index, female mating index, female fertility index, and fecundity index), gestation duration,parturition observation, litter evaluation, litter data (number of corpora lutea, implantation sites, and litter size, pre- and post-implantation loss, sex ratio, pup viability). There were no test item-related changes on developmental landmarks (including pinna unfolding, hair growth, eye opening, tooth eruption, nipple retention, testes descent, and vaginal opening), anogenital distance, reflexological and sensory functions, hematology, urinalysis, spleen lymphocyte subpopulations percentage (including Total T cells, Helper T cells, Cytotoxic T cells, B cells and NK cells) in this study.

In F0 animals, test item-related microscopic changes consisted of hepatocellular hypertrophy at a dose level of 450 mg/kg/day in both sexes, an increased incidence of multifocal tubular basophilia and/or single or multiple proteinaceous casts in the kidneys of males at doses ≥ 75 mg/kg/day, an increased incidence and/or severity of thyroid hypertrophy/hyperplasia in males and females at doses ≥ 75 mg/kg/day with related thyroid gland adenomas in males administered ≥ 75 mg/kg/day and one female administered 450 mg/kg/day, increased amphophilic cells in the pituitary of males at doses ≥150 mg/kg/day, and a decreased incidence of background degeneration and mononuclear infiltration in the heart in males administered 450 mg/kg/day.

In F1 animals, test item-related microscopic changes consisted of hepatocellular hypertrophy at a dose level of 450 mg/kg/day in both sexes, an increased incidence and/or severity of thyroid hypertrophy/hyperplasia in males and at doses ≥ 75 mg/kg/day with related thyroid gland adenomas in one male administered 75 mg/kg/day, and increased amphophilic cells in the pituitary of males at a dose level of 450 mg/kg/day. Minimal hepatocellular hypertrophy was observed in thirteen males and six females administered 450 mg/kg/day. Hepatocellular hypertrophy correlated with increases in group mean hepatic organ weight parameters at a dose level of 450 mg/kg/day.

Conclusion

Based on these results of the present study, the no observed adverse effect level (NOAEL) of POLYOL IXOL B350 on fertility and development toxicity was considered to be 450 mg/kg/day. The NOAEL of POLYOL IXOL B350 on systemic toxicity was considered to be 150 mg/kg /day for F0 females, and 450 mg/kg/day for F1 females, and less than 75 mg/kg/day for F0 and F1 males based on thyroid gland adenomas observed at the low dose level.