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

Developmental toxicity / teratogenicity

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

614-503-3

Cas Number:

68441-62-3

Molecular formula:

(C3H7O2)xC4H4O2Br2(C4H9O2)y with x + y = 2.5

IUPAC Name:

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

Test material form:

liquid: viscous

Details on test material:

- Name of test material (as cited in study report): Polyol IXOL B 350
- Substance type: Halogenated polyetherpolyol
- Physical state: viscous, dark brown liquid
- Analytical purity: ≥99%
- Lot/batch No.: 08-01-2013
- Expiration date of the lot/batch: 2015-01-08
- Storage condition of test material: ambient temperature, protected from light

Test animals

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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 1% (w/v) Gum tragacanth powder in purified water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared at least once weekly 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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

230 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

470 mg/kg bw/day

Doses / concentrations 3

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.

Examinations

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.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

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.

Effect levels (maternal animals)

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

Results (fetuses)

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.

Effect levels (fetuses)

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.

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.