2-Butyne-1,4-diol, polymer with 2-(chloromethyl)oxirane, brominated, dehydrochlorinated, methoxylated

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Based on these results of a GLP compliant OECD 443 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.

Link to relevant study records

Reference

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

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.

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

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

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

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%.

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: 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).

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

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

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.

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:

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.

- 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.

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

Reproductive effects observed:

no

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

450 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

GLP complaint OECD guideline study, klimisch 1

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

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 ratsat dose levels of 0, 75, 150 and 450 mg/kg bw/day. No treatment-related changes were observed in reproductive and developmental parameters at any of the dose levels investigated. Therefore, 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 for 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 at the low dose level.

Effects on developmental toxicity

Description of key information

The No-Observed-Adverse-Effect-Level (NOAEL) of Polyol IXOL B350 for maternal and embryo-fetal development toxicity in rats was considered to be 940 mg/kg/day.

The NOAEL of Polyol IXOL B350 for maternal toxicity in rabbits was considered to be 100 mg/kg/day, and the NOAEL for embryo-fetal developmental toxicity in rabbits was considered to be 200 mg/kg/day.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BioLASCO Taiwan Co., Ltd.
- Age at study initiation: Females, 9-10 weeks and males, 19-20 weeks,
- Weight at study initiation: 211 g (females) and 506-686 g (males),
- Housing: the animals were individually housed in shoebox cages with corncob bedding. During the mating period, the rats were housed on the basis of one male to one or two female(s). The mated females were housed individually.
- Diet: rodent feed, ad libitum
- Water: reverse-osmosis purified and chlorinated water, ad libitum
- Acclimation period: 15 days for females and 8 days for males

ENVIRONMENTAL CONDITIONS
- Temperature (°C): between 21.3° and 23.7°C,
- Humidity (%): between 41.7% and 68.3%,
- Air changes (per hr): 10 to 20
- Photoperiod (hrs dark / hrs light): 12/12

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 and were stirred until a homogeneous suspension was achieved. Homogeneity was determined in this study for the low- and high-dose formulations following the first preparation. 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.

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 dosing formulations according to a validated method (WuXi AppTec Study No.: 239-0002-AC).

HOMOGENEITY:
Homogeneity was determined for the low- and high-dose formulations following the first preparation. For homogeneity analysis, two sets of samples (1 mL each, one set for analysis, the other one for backup) were collected from the top, middle, and bottom layers of formulations.

STABILITY:
Stability in vehicle over 8 days at room temperature under normal laboratory light conditions was determined for the highest and lowest concentration.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: one or two female/one male
- Length of cohabitation: 14 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 post-coitum
- After successful mating each pregnant female was caged (how): Females were individually housed in shoebox cages.
- Any other deviations from standard protocol: no

Duration of treatment / exposure:

Females were exposed from GD5 to GD19 by oral gavage.

Frequency of treatment:

Once daily.

Duration of test:

15 treatment days.

Dose / conc.:

230 mg/kg bw/day

Dose / conc.:

470 mg/kg bw/day

Dose / conc.:

940 mg/kg bw/day

No. of animals per sex per dose:

25 mated females/dose.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In order to set the dose levels for the main study, a dose range finding study was performed. Groups of 4 females (7 weeks old) were dosed at 417, 625 or 938 mg/kg/day for 14 consecutive days (study No. 239-0003-TX) by oral gavage. No treatment related changes in clinical observations, body weight, and food consumption were observed at 938 mg/kg/day. Gross findings of enlarged liver and thyroid glands were noted in males at 417-938 mg/kg/day and females at 625-938 mg/kg/day. No treatment related changes in reproductive organ were observed macroscopically. Therefore, 940 mg/kg/day was selected as the highest dose level for this study. The low dose of 230 mg/kg/day was not expected to result in any significant test article-related effects. The intermediate dose of 470 mg/kg/day was selected to help evaluate the dose-response relationship of any potential adverse effects.

Maternal examinations:

VIABILITY:
Viability (morbidity and mortality) checks were made twice daily, except on animal arrival and the day of necropsy, where animals were examined at least once.

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily from GD 1 to GD 19. During dosage period, the cage side observations were conducted once at approximately 6 to 10 hours post dose.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily on GD 0 and from GD 5 until termination (including necropsy day). During dosage period, the detailed observations were conducted once at approximately 3 to 6 hours post dose.

BODY WEIGHT: Yes
- Time schedule for examinations: at least once during the period of pre-mating. The study animals were weighed on GD 0, 3, 5, 7, 9, 12, 15, 18 and 20.

FOOD CONSUMPTION AND COMPOUND INTAKE
- Time schedule for examinations: Daily food consumptions on GD 0-1, 3-4, 5-6, 7-8, 9-10, 12-13, 15-16, and 18-19 were measured.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day: All surviving main study animals were euthanized and subjected to necropsy and caesarean section on GD 20. The thoracic, abdominal, and pelvic viscera were examined macroscopically. Gross lesions were collected, fixed and retained (one control female was randomly selected for corresponding control tissue collection) for possible pathology evaluation.

Ovaries and uterine content:

The ovaries and uteri were examined to determine:
- Number of corpora lutea;
- Weight of intact gravid uterus;
- Location and number of implantation sites, resorptions, fetal death, dead fetuses;
- Number, sex, body weight, and crown-rump length of all live fetuses;
- Gross evaluation of placentae.
Uteri without visible implantations was immersed in a solution of ammonium sulfide to reveal evidence of early resorptions.
Dead foetuses were retained with 10% buffered formalin (10% NBF) for possible further evaluation.

Fetal examinations:

All live fetuses were examined for external malformations. Approximately 1/2 of the live fetuses in each litter were fixed in modified Davidson’s fixative for soft tissue examination. The remaining live fetuses were stained with Alizarin red S for subsequent skeletal examination.

Statistics:

Quantitative data from each treatment group will be compared with the controls (Group 1). Where necessary, the homogeneity of the group variances will be evaluated using the Levene’s test at the 0.05 significance level.
Incidences of fetal (litters) abnormalities (including malformations and variations) will be analyzed using Chi-squared test, and using Fisher’s Exact test in place of Chi-squared test when expected numbers of animals will be less than or equal to 1.
Statistical analyses of in-life data collected using Provantis system, including bodyweight, food consumption, etc., will be conducted in Provantis system. The other study data will be analyzed using the SAS System for Windows.

Indices:

Pre-implantation loss were calculated as a percentage from the formula:
(No. of corpora lutea-No. of implantations)/No. of corpora lutea ×100 %
Post-implantation loss were calculated as a percentage from the formula:
(No. of implantations-No. of live fetuses)/No. of implantations ×100 %
Sex ratios of the live fetuses were calculated as the percentage of males per litter.

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
Mortality of Females
One animal was found dead at the first dosing time. This death was considered to be due to a dosing error and was not considered to be treatment related. No other mortalities occurred.

Clinical observations
There were no test article-related observations during the study.

Body weight and Absolute weight gain
Body weight changes increased significantly during GD3-5 at 470 mg/kg/day; this was considered unrelated with test article since the increase happened at term prior to dosing. Body weight changes increased significantly by 56% during GD7-9 at 940 mg/kg/day; this was correlated with increase of food consumptions at term, and was considered test article-related. Dosage related increase of absolute weight gain during gestation from GD0 to GD20 was observed in treated groups, and statistical significances were observed at 470 and 940 mg/kg/day (increased by 13% and 15%, respectively). These were considered test article-related but not adverse due to their small magnitude. There were no test article-related changes on body weights and absolute weight gain at 230 mg/kg/day.

Food consumption
At 940 mg/kg/day, food consumptions increased significantly by 9.6%-12.2% on GD7-8, GD9-10, GD 12-13, and GD 18-19 when compared to control. At 470 mg/kg/day, food consumptions increased significantly by 9.2 and 9.4% on GD12-13 and GD 18-19, respectively, when compared to control. These increases were dosage related, correlated with the absolute weight gain during gestation at 470 and 940 mg/kg/day, and were considered to be test article-related but not adverse due to their small magnitude. There were no test article-related effects on food consumption at 230 mg/kg/day.

Macroscopic Observation of females
No gross abnormalities were observed in this study.

Key result

Dose descriptor:

NOAEL

Effect level:

940 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: No treatment related effects on maternal toxicity at the highest dose level

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There were no test article-related changes on the number of corpora lutea, implantation sites, live fetuses, resorptions, fetal death, and death fetuses, pre- and post-implantation loss, sex ratio, fetal crown-rump length, and fetal weight.

External examination of fetuses
There were no test article-related changes on fetal external examination in the study. Cranial meningocele was observed in one fetus at 230 mg/kg/day. No other fetuses with external abnormality were found in the treated groups and control. This fetal external malformation was considered incidental due to low incidence.

Visceral examinations of fetuses
There were no test article-related changes on fetal visceral examination in the study. Dilated lateral ventricle was observed in one fetus at 230 mg/kg/day. No other fetuses with visceral abnormality were found in the treated groups and control. This fetal visceral malformation was considered incidental due to low incidence

Skeletal examination of fetuses
The incidence of fetuses and litters with skeletal abnormalities (with any variations or malformations) at treated groups was comparable to control.
The incidence of fetuses with wave rib at 940 mg/kg/day (4.9%) was significantly higher than at control. The incidence of fetuses with bipartite ossification of thoracic centrum at 230 mg/kg/day (5.0%) and at 940 mg/kg/day (3.3%), respectively, was significantly higher than at control. The incidence of litters with bipartite ossification of thoracic centrum at 940 mg/kg/day (25%) was significantly higher than at control. In the historical control data compiled by the
Middle Atlantic Reproduction and Teratology Association from 154 developmental studies (a total of 27121 fetuses examined on GD20) conducted using Sprague–Dawley rats (MARTA, 1993; Ref. 3), the max incidence of fetus with wave rib was 28.40%, the max incidence of fetus with bipartite ossification of thoracic centrum was 11.19%, and the max incidence of litters with bipartite ossification of thoracic centrum was 34.78%. Therefore, the above significantly increases were considered incidental due to low incidence (within the range of historical control data).
The malformation of fused cervical arch was observed in one fetus at 230 mg/kg/day and one fetus at 940 mg/kg/day; these were considered incidental due to low incidence.
There were some skeletal variations, including hyoid unossified, incomplete ossification of ischium, incomplete ossification of pubis, dumbbell-shaped lumbar centrum, nodulated rib, short rib, short supernumerary rib (cervical or thoracolumbar), sternebrae unossified, incomplete ossification of sternebrae, unilateral ossification of sternebrae, dumbbell-shaped thoracic centrum, bipartite ossification of thoracic centrum were observed at treated groups. These were
considered incidental due to low incidence (within the range of historical control data), and/or similar findings were observed in the control animals.

Key result

Dose descriptor:

NOAEC

Effect level:

940 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment related effect on embryo-fetal development in rats.

Abnormalities:

no effects observed

Developmental effects observed:

no

Analysis of Dose Preparations

The concentrations of Polyol IXOL B350 analysed in the dosing formulations of were in agreement with target concentrations (i.e. mean accuracies between 95% and 103%). No test substance was detected in the control formulation.

Homogeneities of 23 and 94 mg/mL formulations at the first preparation of dosing formulations met the acceptance criteria. Concentration of the top, middle, and bottom portions were within 94% to 102% of nominal values for the 23 and 94 mg/mL formulations. The relative standard deviation (RSD) of top, middle, and bottom samples were 3% and 4% at 23 and 94 mg/mL, respectively.

Test article formulations at 23 mg/mL and 94 mg/mL were stable at room temperature for at least 8 days.

Conclusions:

The No-Observed-Adverse-Effect-Level (NOAEL) of Polyol IXOL B350 for maternal and embryo-fetal development toxicity in rats was considered to be 940 mg/kg/day.

Executive summary:

A GLP compliant prenatal developmental toxicity study with Polyol IXOL B350 was conducted in Sprague Dawley rats according to OECD Guideline 414. Mated females were treated with Polyol IXOL B350 by oral gavage at dose levels of 230, 470, and 940 mg/kg/day during gestation (from Gestation Day 5 to 19). A control group of 25 females was included and the animals were given the vehicle (1% (w/v) Gum tragacanth powder in purified water).

The study animals were observed daily for mortality and clinical signs. On GD 20, all the surviving study animals were necropsied and examined macroscopically. The ovaries and uteri were examined for determination of litter data. All the live fetuses were weighed and examined for external abnormalities; their crown-rump length was measured and their sex was determined. Approximately 1/2 of the live fetuses in each litter were fixed with modified Davidson’s fixative for soft tissue examination and the remaining fetuses were stained with Alizarin red solution for subsequent skeletal examination.

Test article-related changes (increases) on body weight gains, absolute weight gain during gestation, and food consumptions were observed at 470 and 940 mg/kg/day when compared to control; these were not considered adverse due to their small magnitude. There were no treatment related changes in clinical observations, macroscopical observations, litter data, sex ratio, fetal crown-rump length, fetal weight, fetal external, visceral and skeletal examinations.

Based on these results, the No-Observed-Adverse-Effect-Level (NOAEL) of Polyol IXOL B350 for maternal and embryo-fetal development toxicity in rats was considered to be 940 mg/kg/day.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Pizhou Dongfang Breeding Co., Ltd., Guanhu Town, Pizhou, Jiangsu Province
- Age at study initiation: 4-7 months of age (females)
- Weight at study initiation: 3.0 to 4.1 kg (females)
- Housing: the animals were single housed in stainless steel cages elevated off the floor. The animals were provided with manipulatives for environmental enrichment and with hay as dietary enrichment.
- Diet: rabbit growth breeding feed (Lot No. 16034113 and 16064113) from contract vendor, ad libitum.
- Water: reverse-osmosis purified and chlorinated water, ad libitum
- Acclimation: The study animals arrived at the test facility on GD3 and were acclimated until GD6. All animals were examined after receipt by the veterinary staff.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): targeted range 16°C to 22°C (the actual temperature ranged between 18.6°C to 20.5°C at room 2102A; 19.3°C to 22.7°C at room 2103 and 17°C to 21.2°C at room 2104). The temperature in Room 2103 exceeded the limit of 22°C and reached 22.7°C for 1 hour but this deviation is not considered to adversely affect the outcome of the study.
- Humidity (%): targeted range 40% to 70% (the actual humidity ranged between 35.4% to 68.9% at room 2102A; 51.7% to 67% at room 2103; 36% to 72.3% at room 2104). The humidity was occasionally out of the target range, but each of these excursions lasted less than 1 hour and had no adverse impact on the study outcome.
- Air changes (per hr): 10 to 20 times per hour
- Photoperiod (hrs dark / hrs light): 12/12

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 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 preparation. 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): 5.0 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

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 the mid-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.

HOMOGENEITY:
Homogeneity was determined for the low- and high-dose formulations following the first preparation. 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.

STABILITY:
At the first preparation, 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 (20 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.

Details on mating procedure:

The female rabbits were inseminated at the vendor facility. The day of insemination was considered Gestation Day 0 (GD0). The study animals arrived at the test facility on GD3 and were acclimated until GD6.

Duration of treatment / exposure:

Females were exposed from GD6 to GD28 by oral gavage.

Frequency of treatment:

Once daily.

Duration of test:

23 treatment days.

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

200 mg/kg bw/day

Dose / conc.:

400 mg/kg bw/day

No. of animals per sex per dose:

25 mated females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dosages were selected based on the data from the previous dose range finding study in rabbits (study No. 385 0001 TX). In this study, decreased food consumption (up to 70%) was observed at 1000 mg/kg/day throughout the dosing phase, and decreased food consumption (up to 54%) was also observed at 500 mg/kg/day from GD 15 to 28, when compared to control. Correlated with decreased food consumption, decreased body weights were observed at 500 and 1000 mg/kg/day. No notable changes on clinical signs, food consumption, and body weights were noted at 250 mg/kg/day during the course of study.
Based on the above data, the rabbit dosage of 400 mg/kg/day was selected as the high dose level for this study.

Maternal examinations:

VIABILITY:
Viability (morbidity and mortality) checks were made twice daily, except on animal arrival and the day of necropsy, where animals were examined at least once.

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily from GD 5 until necropsy.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed observations were conducted once on GD5 for all animals before randomization, and once daily after dosing until necropsy. During dosage period, the detailed observations were conducted once daily at approximately 1 to 4 hours post dose.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed once on GD5 for randomization. The study animals were weighed once on GD6 prior to dosing, and once on GD9, 12, 15, 18, 21, 24, 27, and 29. No unscheduled body weight was conducted in the study.

FOOD CONSUMPTION AND COMPOUND INTAKE
- Time schedule for examinations: Daily food consumption was measured (weighed) on GD 6-7, 9-10, 12-13, 15-16, 18-19, 21-22, 24-25, and 27-28 for study animals.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day: All surviving study animals were euthanized and subjected to necropsy and caesarean section on GD 29. The thoracic, abdominal, and pelvic viscera were examined macroscopically. No gross lesions were observed at scheduled necropsy. Unscheduled necropsies were conducted for 3 rabbits of the high dose level with observed abortions. The does were euthanized and the thoracic, abdominal, and pelvic viscera were examined macroscopically. Gross lesions were retained. An ovarian and uterine examination was conducted (the corpora lutea and implantation types were counted and recorded) on all does at an unscheduled interval.

Ovaries and uterine content:

For the pregnant animals, the ovaries and uteri were examined to determine:
• Number of corpora lutea;
• Weight of intact gravid uterus;
• Location and No. of implantation sites, resorptions, fetal death, dead fetuses, and live fetuses;
• Sex, body weight, and crown-rump length of all live fetuses;
• Gross evaluation of placenta.
Uteri without visible implantations were immersed in a 10% solution of ammonium sulfide overnight to reveal evidence of early resorptions.
The dead fetuses were retained with 10% neutral buffered formalin for possible further evaluation.

Fetal examinations:

All live fetuses were examined for external abnormalities. Fetus with external malformation was saved in 10% neutral buffered formalin for possible further evaluation. The thoracic, abdominal, and pelvic viscera were examined macroscopically for each fetus. The head collected from approximately 1/2 of the fetuses in each litter was fixed with Modified Davidson’s Fixative for soft tissue examination. The remaining fetuses or bodies were stained with Alcian Blue Solution and Alizarin Red Solution for subsequent skeletal examination. For the fetus with head collected for visceral examination, the skull and cervical vertebrae were not examined.

Statistics:

The statistical evaluation of data was performed with the program package SAS System for Windows (v9.1.3). 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 were 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 were 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 fail test for homogeneity of variance (p≤0.05) or where the data contains negative values and/or 0, 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.

Indices:

Pre-implantation loss was calculated as a percentage from the formula:
(No. of corpora lutea-No. of implantations)/No. of corpora lutea ×100%
Post-implantation loss was calculated as a percentage from the formula:
(No. of implantations-No. of live fetuses)/No. of implantations ×100%
Sex ratios of the live fetuses were calculated as the percentage of males per litter.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Inappetance was observed in 3/19 pregnant rabbits at 200 mg/kg/day, 5/18 pregnant rabbit at 400 mg/kg/day during late dosing period (mainly observed on GD 20 to GD 29), the incidence being dosage related. Decreased defecation was also observed in the pregnant rabbits observed with inappetance at 400 mg/kg/day. All these signs correlated with decreased food consumption.
Materials in pan/bedding (small to medium) were observed in 4 animals at 400 mg/kg/day, out of which 3 animals were found abortion. Moderate inappetance and decreased defecation were noted in the other one animal. These changes were considered test item related and adverse.

Mortality:

mortality observed, treatment-related

Description (incidence):

No spontaneous death occurred. Unscheduled necropsies were conducted for 3 rabbits with observed abortions.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Slightly decreased body weights (attained statistical significance) were observed by up to 6.6% at 400 mg/kg/day when compared to control animals during late gestation (GD 27 to 29). As this correlated with decreased food consumption (up to 54%) and observed inappetance in some animals at 400 mg/kg/day, the slight decreases in body weights were considered test item-related but not adverse due to small magnitude.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

There were no test item related changes on food consumption noted at ≤100 mg/kg/day.
Progressively decreased food consumption (up to 54%) was observed at 400 mg/kg/day, when compared to the concurrent controls throughout the study. As this correlated with decreased body weights and observed inappetance in some animals at 400 mg/kg/day, it was considered test item-related and adverse due to large magnitude.
Decreased food consumption (up to 26%) was observed at 200 mg/kg/day, when compared to the concurrent controls during the late-gestation (GD 21 to GD 28). These changes were considered test item-related but non-adverse due to small magnitude.

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

No gross abnormalities were observed at scheduled necropsy.
Discoloration (dark red) in the left/right caudal lobes of lung, and/or in all lobes of lung was observed in two unscheduled terminated high dose animals due to abortion noted.

Number of abortions:

effects observed, treatment-related

Description (incidence and severity):

Abortions were observed in 3/18 pregnant rabbits (animal No. 4512, 4514, and 4517) at 400 mg/kg/day. For these rabbits, slightly decreased body weight and almost zero or significantly decreased food consumption were observed from GD 12 to termination, accordingly, clinical observation such as inappetance and decreased defecation was observed in the animal 4512 during in-life period. Gross lesions were observed in two animals (4512 and 4517) at necropsy.

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

There were no test item-related changes on the number of corpora lutea, implantation sites and pre-implantation loss.
Significantly increased post implantation loss (31.2%) was observed at 400 mg/kg/day, when compared to the control (post implantation loss is 6.9%).

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

clinical signs

food consumption and compound intake

number of abortions

pre and post implantation loss

other: Decreased gravid uterine weight (17.7% and 15.3% respectively) and absolute gestation weight gain (49.4% and 154.9% respectively) were noted at 200 and 400 mg/kg/day.

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Significantly decreased fetal weight (16.9%) and fetal crown-rump length (6.0%) were observed at 400 mg/kg/day, when compared to the control. This is an indication of fetal developmental retardation toxicity, and was considered adverse due to large magnitude.
Slightly decreased fetal weight (8.4%, attained statistical significance) was observed at 200 mg/kg/day, when compared to the control but no significant changes on fetal crown-rump length were observed in this group. Therefore, this change was considered test item-related but non-adverse due to small magnitude.

Reduction in number of live offspring:

effects observed, treatment-related

Description (incidence and severity):

Decreased No. of live fetuses (6.8/litter) and increased No. of nonviable embryo-fetuses (2.94/litter, attained statistical significance) were observed at 400 mg/kg/day, when compared to the concurrent controls (8.3 live fetuses/litter and 0.65 dead embryo-fetuses/litter respectively). Accordingly, significantly increased post implantation loss (31.2%) was observed at 400 mg/kg/day, when compared to the control (post implantation loss is 6.9%).

Changes in sex ratio:

no effects observed

External malformations:

no effects observed

Description (incidence and severity):

One of 192 fetuses with external malformation of omphalocele was observed in a control litter. This single observation was not considered test item related.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

One of 192 fetuses with fused sternebra (located between the 4th and 5th sternebra) were observed at control. One of 138 fetuses with fused rib (located between the 6th and 7th rib, and between the 8th and 9th rib), branched rib (located in the 9th rib, approximately divide into two ribs), and malpositioned thoracic centrum were observed at 200 mg/kg/day. These single observations (skeletal malformations) were not considered test item-related due to isolated occurrence.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

One of 192 fetuses with large ventricle (left) in heart (visceral malformation) was observed at control. One of 182 fetuses with caudal lobe absent in liver (visceral malformation) was observed at 100 mg/kg/day. One of 122 fetuses with additional fissure in the median/right of liver (visceral malformation) was observed at 400 mg/kg/day. These single observations were not considered test item-related due to low incidence.

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

fetal/pup body weight changes

Developmental effects observed:

no

Results of Dose Formulations Analysis

Polyol IXOL B350 in dosing formulation at 20 mg/mL was stable for at least 8 days at room temperature.

Concentrations of Polyol IXOL B350in dosing formulations fell within 90% to 101% of nominal. The control samples were tested and there was no detectable peak observed at the retention time of Polyol IXOL B350. The data were reported as <LOQ × DF (Dilution Factor).

Homogeneities of Polyol IXOL B350 in dosing formulations at 20 and 80 mg/mL met acceptance criteria. For each concentration, concentrations of the top, middle, and bottom portions fell within 90% to 103% of nominal values. The relative standard deviations (RSD) of top, middle, and bottom samples were within 1% to 3%.

Conclusions:

The No-Observed-Adverse-Effect-Level (NOAEL) of Polyol IXOL B350 for maternal toxicity in rabbits was considered to be 100 mg/kg/day, and the NOAEL of Polyol IXOL B350 for embryo-fetal developmental toxicity in rabbits was considered to be 200 mg/kg/day.

Executive summary:

A GLP compliant prenatal developmental toxicity study with Polyol IXOL B350 was conducted in New Zealand White rabbits according to OECD Guideline 414.

Groups of 25 vendor-mated female rabbits were treated daily with the test substance by oral administration at three dose levels of 100, 200 and 400 mg/kg bw/day from Gestation Day (GD) 6 up to 28. A control group of 25 pregnant females was included and the animals were given the vehicle (1% (w/v) Gum tragacanth powder in purified water).

During the study, mortality was checked for and clinical observations were performed. Body weight and food consumption of the dams were also recorded. A Caesarean section and gross pathology were performed on gestational day 29. The ovaries and uteri were examined for determination of gravid uterine weight,the number of corpora lutea, the location and the number of implantation sites, embryonic resorptions, fetal death, dead fetuses, live fetuses, fetal sex, fetal body weight, fetal crown-rump length and fetal external abnormalities. The thoracic, abdominal, and pelvic viscera were freshly examined macroscopically for each live fetus. The head collected from approximately 1/2 of the live fetuses in each litter were fixed with modified Davidson’s fixative for soft tissue examination. The remaining fetuses or bodies were stained with Alcian Blue Solution and Alizarin Red Solution for subsequent skeletal examination.

In this study, 23/25, 22/25, 19/25, and 18/25 mated females were pregnant at 0, 100, 200 or 400 mg/kg/day, respectively. Test item-related abortion was noted in 3/18 pregnant rabbits at 400 mg/kg/day. Test item-related and adverse changes on clinical signs including materials in pan/bedding, decreased defecation, and inappetance were observed at 400 mg/kg/day, and inappetance were also noted at 200 mg/kg/day. Test item-related and adverse changes also including decreased food consumption (up to 54%) was observed at 400 mg/kg/day, decreased gravid uterine weight (17.7% and 15.3% respectively) and absolute gestation weight gain (49.4% and 154.9% respectively) were noted at 200 and 400 mg/kg/day. These changes were considered as an indication of maternal toxicity. Decreased number of live fetuses and increased number of nonviable embryo-fetuses were observed at 400 mg/kg/day. The post‑implantation loss (31.2%) at 400 mg/kg/day was higher than the control (post‑implantation loss is 6.9%). This is an indication of embryo lethal toxicity, and was considered adverse. Significantly decreased fetal weight (16.9%) and fetal crown-rump length (6.0%) were observed at 400 mg/kg/day, when compared to the control. This is an indication of fetal developmental retardation toxicity, and was considered adverse due to large magnitude.

Test item-related but non-adverse changes including slightly decreased body weights (up to 6.6%) at 400 mg/kg/day and decreased food consumption (up to 26%) and fetal weight (8.4%) at 200 mg/kg/day were observed.

There were no test item-related changes on the number of corpora lutea, implantation sites, pre‑implantation loss, sex ratio, fetal external, visceral, and skeletal examinations in the present study.

Despite a context of severe maternal toxicity at the mid and high-dose level, there was no indication of any teratogenic potential.

Based on these results, the No-Observed-Adverse-Effect-Level (NOAEL) of Polyol IXOL B350 for maternal toxicity in rabbits was considered to be 100 mg/kg/day, and the NOAEL of Polyol IXOL B350 for embryo-fetal developmental toxicity in rabbits was considered to be 200 mg/kg/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Quality of whole database:

GLP complaint OECD guideline study, klimisch 1

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Prenatal Developmental Toxicity Study with rats

A GLP compliant prenatal developmental toxicity study with Polyol IXOL B350 was conducted in Sprague Dawley rats according to OECD Guideline 414 ( Hongping Wan, 2014b). Mated females were treated with Polyol IXOL B350 by oral gavage at dose levels of 230, 470, and 940 mg/kg/day during gestation (from Gestation Day 5 to 19).A control group of 25 females was included and the animals were given the vehicle (1% (w/v) Gum tragacanth powder in purified water).

The study animals were observed daily for mortality and clinical signs. On GD 20, all the surviving study animals were necropsied and examined macroscopically. The ovaries and uteri were examined for determination of litter data. All the live fetuses were weighed and examined for external abnormalities; their crown-rump length was measured and their sex was determined. Approximately 1/2 of the live fetuses in each litter were fixed with modified Davidson’s fixative for soft tissue examination and the remaining fetuses were stained with Alizarin red solution for subsequent skeletal examination.

Test article-related changes (increases) on body weight gains, absolute weight gain during gestation, and food consumptions were observed at 470 and 940 mg/kg/day when compared to control; these were not considered adverse due to their small magnitude. There were no treatment related changes in clinical observations, macroscopical observations, litter data, sex ratio, fetal crown-rump length, fetal weight, fetal external, visceral and skeletal examinations.

Based on these results, the No-Observed-Adverse-Effect-Level (NOAEL) of Polyol IXOL B350 for maternal and embryo-fetal development toxicity in rats was considered to be 940 mg/kg/day.

Prenatal Developmental Toxicity Study with rabbits

A GLP compliant prenatal developmental toxicity study with Polyol IXOL B350 was conducted in New Zealand White rabbits according to OECD Guideline 414 ( Shuijin Yang, 2017a).

Groups of 25 vendor-mated female rabbits were treated daily with the test substance by oral administration at three dose levels of 100, 200 and 400 mg/kg bw/day from Gestation Day (GD) 6 up to 28. A control group of 25 pregnant females was included and the animals were given the vehicle (1% (w/v) Gum tragacanth powder in purified water).

During the study, mortality was checked for and clinical observations were performed. Body weight and food consumption of the dams were also recorded. A Caesarean section and gross pathology were performed on gestational day 29. The ovaries and uteri were examined for determination of gravid uterine weight,the number of corpora lutea, the location and the number of implantation sites, embryonic resorptions, fetal death, dead fetuses, live fetuses, fetal sex, fetal body weight, fetal crown-rump length and fetal external abnormalities. The thoracic, abdominal, and pelvic viscera were freshly examined macroscopically for each live fetus. The head collected from approximately 1/2 of the live fetuses in each litter were fixed with modified Davidson’s fixative for soft tissue examination. The remaining fetuses or bodies were stained with Alcian Blue Solution and Alizarin Red Solution for subsequent skeletal examination.

In this study, 23/25, 22/25, 19/25, and 18/25 mated females were pregnant at 0, 100, 200 or 400 mg/kg/day, respectively. Test item-related abortion was noted in 3/18 pregnant rabbits at 400 mg/kg/day. Test item-related and adverse changes on clinical signs including materials in pan/bedding, decreased defecation, and inappetance were observed at 400 mg/kg/day, and inappetance were also noted at 200 mg/kg/day. Test item-related and adverse changes also including decreased food consumption (up to 54%) was observed at 400 mg/kg/day, decreased gravid uterine weight (17.7% and 15.3% respectively) and absolute gestation weight gain (49.4% and 154.9% respectively) were noted at 200 and 400 mg/kg/day. These changes were considered as an indication of maternal toxicity. Decreased number of live fetuses and increased number of nonviable embryo-fetuses were observed at 400 mg/kg/day. The post‑implantation loss (31.2%) at 400 mg/kg/day was higher than the control (post‑implantation loss is 6.9%). This is an indication of embryo lethal toxicity, and was considered adverse. Significantly decreased fetal weight (16.9%) and fetal crown-rump length (6.0%) were observed at 400 mg/kg/day, when compared to the control. This is an indication of fetal developmental retardation toxicity, and was considered adverse due to large magnitude.

Test item-related but non-adverse changes including slightly decreased body weights (up to 6.6%) at 400 mg/kg/day and decreased food consumption (up to 26%) and fetal weight (8.4%) at 200 mg/kg/day were observed. There were no test item-related changes on the number of corpora lutea, implantation sites, pre‑implantation loss, sex ratio, fetal external, visceral, and skeletal examinations in the present study. Despite a context of severe maternal toxicity at the mid and high-dose level, there was no indication of any teratogenic potential.

Based on these results, the No-Observed-Adverse-Effect-Level (NOAEL) of Polyol IXOL B350 for maternal toxicity in rabbits was considered to be 100 mg/kg/day, and the NOAEL of Polyol IXOL B350 for embryo-fetal developmental toxicity in rabbits was considered to be 200 mg/kg/day.

Justification for classification or non-classification

Based on the results of the extended one-generation toxicity study and the prenatal developmental toxicity studies in rat and rabbit, no classification for fertility developmental effects is necessary according to Regulation (EC) No 1272/2008.