Reaction mass of 1,3-dioxane-5,5-dimethanol and 1,3-Propanediol, 2-(hydroxymethyl)-2-[(methoxymethoxy)methyl]-

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

An OECD 443 study in the rat is ongoing.

Effect on fertility: via oral route

Endpoint conclusion:

no study available

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

No evidence of an effect on the reproductive organs was seen in a 90-day study at dose levels of up to and including 1000 mg/kg bw/d. Similarly, no evidence of any adverse effects on the developing fetus was seen in prenatal developmental toxicity studies at the limit dose level of 1000 mg/kg bw/d. The absence of histopathological changes in reproductive organs in the repeated dose toxicity study indicate that the substance will not have any adverse effects on fertility. An extended one generation reproductive toxicity (EOGRT OECD 443) study is ongoing (waiver proposed). 

Short description of key information:
No evidence of any potential for reproductive toxicity was seen in a 90-day study at dose levels of up to and including 1000 mg/kg bw/d. Similarly, no evidence of any adverse effects on the developing fetus was seen in prenatal developmental toxicity studies at the limit dose level of 1000 mg/kg bw/d.

Extended one generation reproductive toxicity (EOGRT OECD 443) study is ongoing (waiver is proposed).

Justification for selection of Effect on fertility via oral route:
No study available - waiver proposed, as EOGRT (OECD 443) study is ongoing.

Effects on developmental toxicity

Description of key information

Two oral prenatal developmental toxicity studies are available for Polyol PX- in rat and rabbit; no adverse effects were observed up to and including the highest dose tested therefore the NOAELs were considered to be 1000 mg/kg bw/d for both maternal toxicity and embryofetal toxicity

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 09 November 2021 to November 2022

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Agricultural Production Bureau, November 24, 2000.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): Polyol PX; alternative name: Reaction mass of 1, 3-Propanediol, 2-(Hydroxymethyl)-2-[(Methoxymethoxy)methyl]-and 1, 3-Dioxcine 5, 5-Dimethanol
- Source: Sponsor
- Physical state: Solid
- Purity: 71.2% (dose calculations were corrected for purity; correction factor 1.40)
- Lot/batch No.: 90000029663
- Expiration date of the lot/batch: 26 August 2022
- Stability and homogeneity: Homogeneity and stability of the test material in the vehicle was determined as part of this programme of work in Labcorp Study No. 8459847. Storage was confirmed at ambient temperature (15 to 25ºC) for up to 1 day and following refrigeration (2 to 8°C) for up to 15 days following fresh preparation in purified water for the range from 2 mg/mL and 200 mg/mL.

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

The rabbit was chosen as the test species because of the requirement for a non-rodent species by regulatory agencies.

- Strain: New Zealand White rabbit - time mated females
- Source: Envigo RMS, UK
- Age at study initiation: 19 to 22 weeks old at the start of the study
- Weight at study initiation: 2.87 to 4.54 kg
- Housing: individually housed in suspended cages with perforated floor panels and mounted in batteries. Undertrays lined with absorbent paper were changed at least three times a week. Cages were also fitted with a plastic resting platform.
- Diet (e.g. ad libitum): Teklad 2930 diet, restricted (200 g/animal/day). A small supplement of autoclaved hay was given on a daily basis and a small amount of chopped fresh vegetables were given twice weekly.
- Water (e.g. ad libitum): water from the public supply, ad libitum
- Acclimation period: Five days from arrival on Day 1 after mating to commencement of treatment on Day 6 after mating.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15-21°C
- Humidity (%): 45-70%
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod (hrs dark / hrs light): 14 hour light : 10 hour dark

IN-LIFE DATES: 09 November 2021 to 10 December 2021

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was heated to 40°C in a water bath and the required amounts weighed. Approximately 50% of the required vehicle was added to the test item and magnetically stirred until all the test item was uniformly mixed. Further amounts of vehicle were gradually added and mixed to produce the required concentration. The formulation was finally mixed using a high-shear homogenizer and transferred, via syringe, into final containers. Start and finish times recorded in the raw data. The dosing formulations were prepared weekly, stored in a refrigerator set to maintain 2 to 8 °C.

The dosing volume was 5 mL/kg.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical method involved extraction and dilution in methanol followed by gas
chromatographic analysis with flame ionization detection. Sample concentrations were
determined with reference to external standards prepared in the concentration range
10 μg/mL to 250 μg/mL.

The formulations for First Week, Second Week, Third Week, and Last Week were sampled for all groups, 1 × 10 mL (accurately weighed) was sampled from the middle of the formulation by Dose Formulation personnel. For the formulations for the Second Week, dose residues were sampled for all groups from the middle of the formulation by Dose Formulation personnel.
Duplicate aliquots (1 mL) from all groups were analyzed in accordance with the analytical procedure.

The mean concentrations for the second week, third week and fourth weeks’ preparations were within ±10% of the nominal concentration, confirming the accuracy of formulation. The difference from mean and coefficient of variation for these samples remained within 4%, confirming precise analysis.

For the first week’s formulations, Groups 1 and 2 sample extracts were analyzed beyond stability due to system failures, therefore results are reported for information only. Contingency analysis was not performed as formulations were out of stability.

For the analyzed concentrations for the first week, Groups 3 and 4 were out of specification originally. The original extracts were re-diluted in duplicate, and the contingency samples were analyzed. All re-dilutions and contingency samples were in agreement with each other however they did not confirm the original out of specification results, showing that a possible dilution error took place during original analysis. Re-dilutions and contingency samples were analyzed beyond established stability therefore re-dilution (first sample) and contingency samples were reported for information only. The dose preparation records were reviewed and there were found to be no discrepancies in the preparation of the dose formulations, further suggesting the initial out of specification results were due to a dilution error during analysis.

For the last week’s samples, no results were available from the original analysis due to a system issue. Samples extracted were beyond stability therefore contingency samples were analyzed and reported.

As the out of specification results for the first week’s samples were considered likely to be the result of a dilution error, when taken with the results for the second, third and last weeks’ samples that were within the acceptance criteria, it is considered that the formulations were prepared accurately throughout the study.

Homogeneity and stability of the test material in the vehicle was determined as part of this programme of work in Labcorp Study No. 8459847. Storage was confirmed at ambient temperature (15 to 25ºC) for up to 1 day and following refrigeration (2 to 8°C) for up to 15 days following fresh preparation in purified water for the range from 2 mg/mL and 200 mg/mL

Details on mating procedure:

Natural mating with New Zealand White bucks of established fertility at the supplier’s facility. Males and females not closely related. After mating, each female will be injected intravenously with 25 i.u. lutenising hormone.

Duration of treatment / exposure:

Days 6 to 28 after mating.

Frequency of treatment:

Females were treated from Day 6 to Day 28 (inclusive) after mating, once daily at approximately the same time each day.

Duration of test:

Day 6 to Day 28 of gestation

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

300 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

24 animals/dose

Control animals:

yes, concurrent vehicle

Details on study design:

Dose levels for this study (0, 100, 300 and 1000 mg/kg/day) were selected in conjunction with the Sponsor based on the results from the preliminary embryo-fetal study in the rabbit, (Renaut 2022, Labcorp Study No. 8459841). In that study, body weight gain and body weight loss, when adjusted for the gravid uterine weight, was low for females at 1000 mg/kg/day. There was no clear effect at 300 or 600 mg/kg/day, with no dose response apparent for body weight gain or adjusted body weight loss. Similarly, fetal and placental weight were slightly lower than Control at 1000 mg/kg/day, and while 300 or 600 mg/kg/day fetal and placental weights were also lower than Control, there was no clear dose response to treatment. Macroscopic examination of fetuses on GD29 revealed three with major abnormalities, one of which was in the Control group: ascending aorta/aortic arch dilated, ascending aorta/pulmonary trunk fused, pulmonary trunk dilated, cranium brow ridge and head frontal region misshapen, in a single fetus of a Control female; head acephalostomia, lower jaw misshapen associated with major abnormality, pinna bilateral present associated with major abnormality, tongue present associated with major abnormality and forepaw(s) bilateral flexure, in a single fetus of a female that received 600 mg/kg/day; and head meningocele, pinna left small and forepaw(s) bilateral flexure, in a single fetus of a female that received 1000 mg/kg/day. Based on the effects of treatment being limited to body weight loss at 1000 mg/kg/day and the presence of major abnormalities being restricted to single fetuses in the previous study, 1000 mg/kg/day was selected as the high dose for this study. 100 and 300 mg/kg/day were selected to assess the dose responsiveness of Polyol PX administration.

Animals were allocated randomly to group and cage position on the day of arrival. Females mated on any one day were evenly distributed amongst the groups. Allocation was controlled to prevent any stock male from providing more than one mated female in each treated group and to prevent more than one sibling female in each group, where possible.

Maternal examinations:

CLINICAL OBSERVATIONS: Yes
- Time schedule: at least twice daily
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health. Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate. During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

PHYSICAL EXAMINATION: Yes
-Time schedule: A detailed physical examination was performed on each animal on Days 1, 6, 12, 18, 23 and 29 after mating to monitor general health.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed observations were recorded at the following times in relation to dose administration:
Pre-dose observation.
One to two hours after completion of dosing.
As late as possible in the working day.

BODY WEIGHT: Yes
- The weight of each adult was recorded on the day of arrival (Day 1), on Day 3 and on Days 6 to 29 after mating.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: yes
- The weight of food supplied to each animal, that remaining and an estimate of any spilled was recorded daily from Day 2 after mating to termination.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No (ad libitum)

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on day 29 after mating.
-All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed.

Ovaries and uterine content:

Gravid uterine weight (including cervix and ovaries) was recorded for all females surviving to term. For each ovary/uterine horn the number of Corpora lutea, Implantation sites, resorption sites (assessed as early or late), fetuses (live and dead) were recorded. For non-pregnant females and for apparently empty uterine horns the absence or number of uterine implantation sites was confirmed.

Fetal examinations:

Examination of all viable fetuses and placentae
Fetuses and placentae were dissected from the uterus and weighed individually. Fetuses were identified within the litter using a coding system based on their position in the uterus and examined externally with abnormalities recorded, sampled as appropriate and retained in appropriate fixative. All fetuses were subject to a gross internal examination of the viscera of the neck, thorax and abdominal cavities and the sex of each fetus was also recorded.

Fetal, Litter and Placental Weights
Mean fetal weights were calculated for each litter. Values were presented for male, female and overall fetal weight. Litter weight was calculated as the sum of all fetal weights. Mean placental weight was also calculated for each litter. Group mean values and SD were calculated using individual litter mean values.

Detailed Fetal Examination
Findings from external, visceral and skeletal examination of fetuses are tabulated on an individual basis for affected litters and fetuses, linking the results of initial external examinations with subsequent visceral and/or skeletal examinations to fetal weight.

Group incidences of observations on fetuses and litters are summarized in terms of major or minor abnormalities or as skeletal variants. The incidence of structural changes are presented as numeric fetal and litter incidences. Findings observed were classified, according to severity and incidence, as:
- Major abnormalities: normally rare, definitely detrimental to normal subsequent development, possibly lethal, e.g. partially open eyelids, absent kidney/ureter.
- Minor abnormalities: minor differences from normal that are detected relatively frequently considered to have little detrimental effect and may be a transient stage in development e.g. bipartite centrum, dilated ureter.
-Variants: alternative structures or stages of development occurring regularly in the control population, e.g. number of ribs and thoracolumbar vertebra, incomplete ossification of 5th and 6th sternebrae.

Statistics:

For adult parameters, the analyses were carried out using the individual animal as the basic experimental unit. For litter/fetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.

The following data types were analyzed at each timepoint separately:
- Body weight, using absolute weights and gains over appropriate study periods
- Food consumption, over appropriate study periods
- Gravid uterine weight and adjusted body weight
- C-section litter data (corpora lutea, implantations, pre/post implantation loss, live young and sex ratio - percentage male)
- Placental, litter and fetal weights

The following comparisons were performed: Group 1 vs 2, 3 and 4

Parametric/Non-Parametric tests include:
- Bartlett's test, Willams' test, t-tests, Dunnett's tests, Shirley's test Kruskal-Wallis' tests, Wilcoxon rank sum tests and Steel's test.

Indices:

Pre-implantation loss (%) = (Number of corpora lutea - Number of implantations)/(Number of corpora lutea) x 100

Post-implantation loss (%) = (Number of implantations - Number of live fetuses)/(Number of implantations) x 100

Historical control data:

HCD for New Zealand White rabbit are available at the test laboratory in which the study was conducted.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

One control female (No. 6) was euthanized for welfare reasons on GD 16. The animal was found without the use of its hind limbs and was uncoordinated. The macroscopic examination showed the animals left hind femur was broken and there was a hemorrhage. Prior to GD 16 the animal had been in good clinical condition. The animal was pregnant with eight live fetuses.

A second control female (No. 19) was euthanized for welfare reasons on GD 17. The animal was observed to have decreased food intake from GD 12, which resulted in the animal being thin, decreased number and size of fecal pellets and loss in bodyweight. No macroscopic findings were observed, and the animal was not pregnant.

Signs observed in association with dosing, including flattened posture, underactive behavior, and rapid breathing, were apparent at a low incidence in females receiving 100, 300 and 1000 mg/kg/day; these signs were generally only observed on one day of dosing and for up to two days in a small number of animals.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Two control females were euthanized for welfare reasons.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weight gain for females receiving 1000 mg/kg/day from GD 6-29 was statistically significantly higher than controls (167%). Similarly, females receiving 100 or 300 mg/kg/day body weight gain was also higher than controls (160 and 153% respectively) but did not attain statistical significance.

Gravid uterine weight and maternal body weight loss following adjustment for the gravid uterine weight was unaffected by treatment.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption was statistically significantly higher on GD 15-20, being 26%, 33% or 20% higher than Control for females that received 100, 300 or 1000 mg/kg/day, respectively. Overall food consumption (GD 6-29) was subsequently slightly higher than Control for females at all dose levels, although without statistical significance or a dose dependent increase.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

not examined

Details on maternal toxic effects:

The once daily oral administration of Polyol PX to pregnant New Zealand White rabbits throughout organogenesis and the fetal growth phase at dose levels of 100, 300 or 1000 mg/kg/day was maternally well tolerated. Overall, body weight for all treated groups was slightly higher than Control (160%, 153% or 167% at 100, 300 or 1000 mg/kg/day, respectively), but without statistical significance. This may be attributed to a statistically significant increase in food consumption in comparison to Control on GD 15-20.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: Other:

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

At 1000 mg/kg/day, there was an increase in incidence of the minor abnormalities of full supernumerary 13th ribs (fetal incidence only), 20 thoracolumbar vertebrae and unilateral shift of pelvic girdle compared to Control (all within HCD range except 20 thoracolumbar vertebrae). This indicates a slight shift in rib/vertebral configuration, which, as anatomical variants are not considered adverse, but may be related to treatment. A dose response was not observed however for these minor skeletal fetal abnormalities.

At 300 mg/kg/day, the incidence of short and full supernumerary cervical ribs was slightly higher compared to concurrent control (outside HCD range), but as this was not seen at 1000 mg/kg/day, no effect of treatment is inferred.

At 100 mg/kg/day, there was an increase in fetal and litter incidence of delayed ossification/unossified 5th sternebrae, compared to concurrent control (outside fetal HCD range), but as there was no increase in the number of litters with this finding at 300 or 1000 mg/kg/day, no effect of treatment is inferred. As incomplete ossification is a transient stage of fetal development, this is not considered adverse

Visceral malformations:

no effects observed

Key result

Dose descriptor:

NOEL

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Sex:

not specified

Basis for effect level:

skeletal malformations

Key result

Abnormalities:

not specified

Localisation:

skeletal: supernumerary rib

skeletal: vertebra

skeletal: pelvic girdle

Description (incidence and severity):

An increase in incidence of the minor abnormalities of full supernumerary 13th ribs (fetal incidence only), 20 thoracolumbar vertebrae and unilateral shift of pelvic girdle at 1000 mg/kg/day compared to concurrent control (all within HCD range except 20 thoracolumbar vertebrae).

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day

Treatment related:

not specified

Relation to maternal toxicity:

developmental effects in the absence of maternal toxicity effects

Dose response relationship:

no

Relevant for humans:

not specified

Conclusions:

It is concluded, based on these data, that the maternal No-Observed-Adverse-Effect-Level (NOAEL) was 1000 mg/kg/day. Due to an increase in incidence of the minor abnormalities of full supernumerary 13th ribs (fetal incidence only), 20 thoracolumbar vertebrae and unilateral shift of pelvic girdle at 1000 mg/kg/day compared to concurrent control (all within HCD range except 20 thoracolumbar vertebrae), the NOEL for embryo fetal development was 300 mg/kg/day, as the relation to treatment for these findings could not be excluded, and the NOAEL for embryo fetal survival and growth is 1000 mg/kg/day.

Executive summary:

The purpose of this study was to assess the influence of Polyol PX (an industrial chemical) on embryo-fetal survival and development when administered during the organogenesis and fetal
growth phases of pregnancy in the New Zealand White Rabbit. Three groups of 24 females received Polyol PX at doses of 100, 300 or 1000 mg/kg/day by oral gavage administration, from Day 6 to 28 after mating. A similarly constituted Control group received the vehicle, purified water at the same dose volume as treated groups. Animals were killed on Day 29 after mating for reproductive assessment and fetal examination. Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 29 after mating and the gravid uterus weight was recorded. All fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination of the head or skeletal examination.  There were no treatment related deaths during the study.  Signs observed in association with dosing, including flattened posture, underactive behavior, and rapid breathing, were apparent at a low incidence in females receiving 100, 300 and 1000  mg/kg/day, generally on only one day of dosing. Mean overall body weight gain (GD 6-29) for females at 100, 300 or 1000 mg/kg/day was relatively higher, at 160%, 153% or 167% of Control, respectively, and mean food consumption was relatively higher on GD 15-20 for these females, at 26%, 33% or 20% of Control, respectively. Mean overall food consumption was subsequently slightly higher than Control for all treated groups, although without significance or a dose dependent increase. Gravid uterine weight was unaffected by treatment and there were no maternal abnormalities related to treatment at the macroscopic examination. The incidence of major fetal abnormalities and minor visceral abnormalities showed no relationship to treatment. At 1000 mg/kg/day, there was an increase in incidence of the minor abnormalities of full supernumerary 13th ribs (fetal incidence only), 20 thoracolumbar vertebrae and unilateral shift of pelvic girdle compared to concurrent control (all within HCD range except 20 thoracolumbar vertebrae). This indicates a slight shift in rib/vertebral configuration, which, as variants are not considered adverse but may be related to treatment with Polyol PX.  Embryo fetal survival and growth were unaffected by treatment. Based on these data, the maternal NOAEL is 1000 mg/kg/day, the NOEL for embryo fetal development is 300 mg/kg/day and the NOAEL for embryo fetal survival and growth is 1000 mg/kg/day.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 September 2015 to 24 March 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Modern GLP and guideline compliant study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): Polyol PX; alternative name: Reaction mass of 1, 3-Propanediol, 2-(Hydroxymethyl)-2-[(Methoxymethoxy)methyl]-and 1, 3-Dioxcine 5, 5-Dimethanol
- Source: Sponsor
- Physical state: Solid
- Purity: 83.9% (dose calculations were corrected for purity; correction factor 1.19)
- Lot/batch No.: 441258479C
- Expiration date of the lot/batch: 02 June 2016

Stability analyses performed previously in conjunction with Charles River Study No 425834
demonstrated that the test item is stable in the vehicle when prepared and stored under the
same conditions at concentrations bracketing those used in the present study for at least 8
days at 2-8°C, in the dark.

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Han Wistar (Crl:WI(Han)) - time mated females
- Source: Charles River UK Limited, Margate, UK
- Age at study initiation: 9 weeks old at time of mating
- Weight at study initiation: 177-267 g (170 -250 g on arrival)
- Housing: 2 per cage in suspended polycarbonate/polypropylene cages with stainless steel grid tops and solid bottoms. Bedding was sterilised white wood shavings.
- Diet (e.g. ad libitum): SDS VRF-1 breeder diet, ad libitum
- Water (e.g. ad libitum): water from the public supply, ad libitum
- Acclimation period: 3-5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-23°C
- Humidity (%): 38-62%
- Air changes (per hr): Ten or more with 100% fresh air
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

IN-LIFE DATES: From: 25 September 2015 To 14 October 2015

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of test item was accurately weighed and transferred into a pre-labelled container according to instructions from the formulation computerised system. The appropriate amount of control item was then added to the container and the formulation was magnetically stirred until visibly homogenous. The dosing formulations were prepared weekly, stored in a refrigerator set to maintain 4°C, and dispensed daily. The dosing formulations were removed from the refrigerator and were stirred for at least 30 minutes before dosing and continuously during dosing.

The dosing volume was 10 mL/kg.

VEHICLE
- Concentration in vehicle: 0, 10, 30, 100 mg/mL

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration analyses were performed from all dosing formulations during Week 1 and Week 2 by HPLC (reverse phase) with ELSD detection using a validated analytical procedure. Homogeneity was determined in all test substance groups during Week 1 and Week 2.

Duplicate top, middle and bottom samples (middle only for control) of 1 mL for each sampling time point were collected and sent to the analytical laboratory for analysis. Triplicate sets of top, middle and bottom samples (middle only for control) of the same volume were also collected and retained as backup samples. Concentration results were considered acceptable if mean sample concentration results were within 10% of theoretical concentration. Homogeneity results were considered acceptable if the relative standard deviation of the mean value at each sampling location was ≤10% for each group.

Stability analyses were performed during a previous study at the test facility and confirmed that the test substance is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study for at least 8 days at 2-8°C, in the dark.

Details on mating procedure:

Time-mated female rats were delivered to the test facility

Duration of treatment / exposure:

Days 6-19 of gestation

Frequency of treatment:

Daily

Duration of test:

Days 6-19 of gestation (scheduled euthanasia on Day 20)

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

300 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

24 females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels were chosen by the Sponsor following review of data from a 14 day study using Polyol PX (Charles River Study Number 528596) and in attempt to produce graded responses to the test item. On Study 528596, there were no treatment related clinical observations or any body weight or food consumption effects at dose levels of up to 1000 mg/kg bw/d.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily; once in the morning and once towards the end of the working day for general health/mortality and moribundity. Animals were also observed prior to dosing and regularly throughout the day for reaction to treatment on each day of dosing.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once pretreatment, and daily from Day 6 of gestation until study termination

BODY WEIGHT: Yes
- Time schedule for examinations: on Day 4 gestation and daily from Days 6 to 20 of gestation

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION): Yes
- Time schedule for examinations: daily visual inspection of water bottles

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: External examination, contents of cranial, thoracic and abdominal cavities examined macroscopically (samples of abnormal tissues fixed in neutral buffered 10% formalin). The reproductive tract was examined separately (see below).

OTHER:

Ovaries and uterine content:

Gravis uterus weight was recorded and the fetuses were removed. The ovaries and uterus were examined for number and distribution of corpora lutea, implantation sites, placentae (size, colour or shape - only abnormalities were recorded), live and dead fetuses, early and late resorptions.

Fetal examinations:

Each fetus was examined for external abnormalities. Late resorptions and dead fetuses were examined for external abnormalities to the extent possible. Each implant was classified as being live, or a dead fetus (dead full term fetus that showed no signs of maceration), or a late embryonic death (macerated tissue identifiable as an embryofetus, with recognizable external features such as tail, limbs, mouth and nares present; attached to distinct identifiable placentae), or an early embryonic death (discrete, formless, discoloured tissue mass attached to the internal uterine wall; may have been of varying size). The body weight of each fetus was recorded and fetuses were individually identified within each litter.

Visceral examination and sex: Half of the viable fetuses from each uterus were fixed in methylated ethyl alcohol, examined internally for sex and eviscerated following fixation; the viscera were not examined from fetuses prior to disposal. The remaining half of viable fetuses from each uterus were fixed in Bouins’ fluid. The fetuses fixed in Bouins’ fluid were examined for soft tissue abnormalities and sex by a freehand sectioning technique derived from Wilson.

Skeletal examination: The eviscerated carcasses were then macerated in potassium hydroxide, the skeletons stained with Alizarin Red S, and then the fetuses cleared with aqueous glycerol solutions. These preparations were examined for the presence of skeletal abnormalities and for the extent of ossification.

Statistics:

Means and standard deviations were calculated for body weight, food consumption and selected pregnancy data. Numerical data collected on scheduled occasions were analysed according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) are reported whenever possible. Inferential statistics were performed when possible, but excluded semi-quantitative data and any group with less than 3 observations. Levene’s test was used to assess the homogeneity of group variance parametric assumption at the 5% significance level. Datasets with at least three groups were compared using an overall one-way ANOVA F-test or Kruskal-Wallis test (if parametric assumptions were not met) at the 5% significance level. The pairwise comparisons were conducted using a twosided Dunnett’s or Dunn’s test, respectively, if the overall test is significant. Datasets with two groups were compared using a two-sided t-test or Wilcoxon Rank-Sum test, respectively. All significant pairwise comparisons were reported at the 0.1, 1 and 5% significance levels.

Indices:

Not applicable.

Historical control data:

Available at the test facility.

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Other effects:

not specified

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
There were no deaths and no clinical signs of toxicity. Group mean bodyweights and group mean food consumption were similar between all groups throughout the study. There were no abnormalities detected at necropsy.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

At 300 or 1000 mg/kg/day, a slightly lower number of fetuses with 13 complete ribs and
slightly increased number of fetuses with vestigial/reduced supernumerary ribs on the 1st
lumbar vertebra were noted when compared with Controls. These findings are normal
variation seen within litters.

Visceral malformations:

no effects observed

Other effects:

not specified

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Pregnancy performance and fetal weights were similar between all groups. Slight intergroup differences were considered to be incidental as they did not follow treatment related patterns and/or were too small to be attributed to treatment with Polyol PX.
The type and distribution of major and minor fetal abnormalities and skeletal ossification parameters did not indicate any association with treatment. At 300 or 1000 mg/kg/day, a slightly lower number of fetuses with 13 complete ribs and slightly increased number of fetuses with vestigial/reduced supernumerary ribs on the 1st lumbar vertebra were noted when compared with Controls. These findings are normal variation seen within litters; therefore, since the number of litters with these findings was generally similar across all groups, and there was no maternal toxicity or other correlating fetal findings to indicate that the differences were treatment related effects, the findings were considered to be incidental and unrelated to treatment with Polyol PX. Slight intergroup differences in other fetal parameters did not follow treatment related patterns and were also considered to be incidental and unrelated to treatment with Polyol PX.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Key result

Abnormalities:

effects observed, non-treatment-related

Key result

Developmental effects observed:

no

Dose formulation analyses: All analysed Group 2, 3 and 4 formulations from Study Weeks 1 and 2 were within the concentration acceptance criteria of ±10% of theoretical concentration, with the exception of Group 4 Week 2 samples, which were -11.6% of theoretical concentration. Back samples Group 4 Week 2 samples were therefore analysed, which were found to be within the acceptance criteria. A low relative standard deviation (≤3.2%) indicated that all formulations were homogenous. From review of data, no obvious reason could be found for the original Group 4 Week 2 results being out of acceptance; however, as the deviation was minor and backup samples were within acceptance, it is considered that animals likely received accurate formulations. The absence of Polyol PX from analysed control formulations was also confirmed.

Conclusions:

The NOAEL for maternal and developmental toxicity was considered to be 1000 mg/kg bw/d.

Executive summary:

The effects of oral administration of Polyol PX in pregnant rats during the embryo-fetal period was investigated in a study according to OECD 414. Time-mated female Han Wistar rats were administered the test substance in water at 0, 100, 300 or 1000 mg/kg bw/d by once daily by gavage. Animals were dosed over Days 6-19, inclusive, of gestation (where the day of detection of mating was Day 0 of gestation). The animals were checked regularly for clinical signs of toxicity, body weight and food consumption performance and were killed on Day 20 of gestation for examination of pregnancies and embryo-fetal development.

Dosing with Polyol PX at dose levels of up to 1000 mg/kg/ bwd was not associated with any treatment related clinical signs, body weight or food consumption effects or any macroscopic necropsy findings. Pregnancy performance and fetal weights were similar between Controls and treated groups, and the type and distribution of major and minor fetal abnormalities and variants, including skeletal ossification parameters, did not indicate any association with treatment with Polyol PX. It was, therefore, concluded from the results of this study, that the maternal and fetal no observed adverse effect levels were 1000 mg/kg bw/d.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

other: Rat and Rabbit

Quality of whole database:

Two modern GLP and guideline-compliant studies are available for Polyol PX

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

A modern GLP and guideline compliant (OECD 414) prenatal developmental toxicity study was conducted with Polyol PX in the rat (McConnachie, 2016). Four groups of 24 females Sprague-Dawley rats were dosed orally by gavage on Days 6-19 of gestation (where Day 0 was the day of detection of mating), at dose levels of 0, 100, 300 or 1000 mg/kg bw/d in water for irrigation. At dose levels up to and including 1000 mg/kg/day were no test item-related effects on clinical observations, body weight gain, food consumption, gross pathology findings, pregnancy performance parameters, embryofetal survival or fetal weights, fetal abnormalities and variants. In conclusion, under the conditions of this study, the maternal and embryofetal no observed adverse effect levels (NOAEL) were considered to be 1000 mg/kg bw/d.

 

A modern GLP and guideline compliant (OECD 414) prenatal developmental toxicity study was conducted with Polyol PX in the rabbit. Four groups of 24 females received Polyol PX at doses of 0, 100, 300 or 1000 mg/kg/day by oral gavage administration, from Day 6 to 28 after mating. Mean overall body weight gain (GD 6-29) for females at 100, 300 or 1000 mg/kg/day was relatively higher  and mean overall food consumption was subsequently slightly higher than Control for all treated groups, although without significance or a dose dependent increase. Gravid uterine weight was unaffected by treatment and there were no maternal abnormalities related to treatment at the macroscopic examination. The incidence of major fetal abnormalities and minor visceral abnormalities showed no relationship to treatment. At 1000 mg/kg/day, there was an increase in incidence of the minor abnormalities of full supernumerary 13th ribs (fetal incidence only), 20 thoracolumbar vertebrae and unilateral shift of pelvic girdle compared to concurrent control (all within HCD range except 20 thoracolumbar vertebrae). This indicates a slight shift in rib/vertebral configuration, which, as variants are not considered adverse but may be related to treatment with Polyol PX.  Embryo fetal survival and growth were unaffected by treatment. Based on these data, the maternal NOAEL is 1000 mg/kg/day, the NOEL for embryo fetal development is 300 mg/kg/day and the NOAEL for embryo fetal survival and growth is 1000 mg/kg/day.

Justification for selection of Effect on developmental toxicity: via oral route:
Modern GLP and guideline compliant study in the rat and rabbit

Justification for classification or non-classification

No evidence of maternal toxicity or developmental toxicity potential was observed in prenatal developmental toxicity studies both in rats and in rabbits, at dose levels up to and including 1000 mg/kg bw/d.

 

No classification is therefore required according to EC Regulation 1272/2008.