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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date: 30 March 2022
Final report date: 4 January 2023 (see attached justification)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2022

Materials and methods

Test guidelineopen allclose all
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
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2'-isopropylidenebis(p-phenyleneoxy)diethanol
EC Number:
212-985-6
EC Name:
2,2'-isopropylidenebis(p-phenyleneoxy)diethanol
Cas Number:
901-44-0
Molecular formula:
C19H24O4
IUPAC Name:
2,2'-isopropylidenebis(p-phenyleneoxy)diethanol
Test material form:
solid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD(SD)
Details on test animals or test system and environmental conditions:
Supplier Females: Charles River (Italy).

Males: Stock animals obtained from Charles River (Italy). Males were unrelated to females.

Number of animals 98 females (88 on study and 10 spares).

Spare animals were removed from the study room after treatment commenced.

Duration of acclimatization 33 days before commencement of pairing.

Age of the animals at the start of the study (Day 0 of gestation) 83 to 89 days of age.

Weight range of the animals at the start of the study (Day 0 of gestation) 260 to 333 g.

Environmental Control
Animal facility Limited access - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.

Air supply Filtered fresh air which was passed to atmosphere and not recirculated.

Temperature and relative humidity Monitored and maintained within the range of 20-24°C and 40-70%.

There were no deviations from these ranges.

Lighting Artificial lighting, 12 hours light: 12 hours dark.

Electricity supply Public supply with automatic stand-by generators.

Animal Accommodation
Cages Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.

Solid (polycarbonate) bottom cages were used were used during the acclimatization and gestation periods.

Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing.

Cage distribution The cages constituting each group were blocked by group and mounted in batteries.

Bedding Cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.

Number of animals per cage
Acclimatization Up to four females.
During pairing One (stock) male and one female.
Gestation One female.

Environmental Enrichment
Aspen chew block A soft white untreated wood block: provided to each cage throughout the study and replaced when necessary.

Diamond twists (paper product) Provided to each cage throughout the study (except during pairing) and replaced at the same time as the cages.

Plastic shelter Provided to each cage throughout the study (except during pairing) and replaced at the same time as the cages.

Diet Supply
Diet SDS VRF1 Certified, pelleted diet.

The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.

Availability Non-restricted.

Water Supply
Supply Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.

Availability Non-restricted.

Supplier Certificates of Analysis
Certificates of analysis for the diet are scrutinized and approved before any batch of diet was released for use. Certificates of analysis were routinely provided by the water supplier.

Certificates of analysis were also received from the suppliers of the softwood based bark-free fiber bedding, Aspen chew blocks and Diamond twists.

No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% (w/v) methylcellulose 4000 cps aqueous solution
Details on exposure:
Method of preparation
Formulations were prepared under yellow light.

Suspension of the test item was performed following the steps below:

• Weighed the required quantity of test item.
• Ground the test item using a mortar and pestle.
• Added vehicle dropwise and mixed with the test item to obtain a homogeneous mixture.
• Progressively added the vehicle and transferred the mixture into a gauged flask, with rinsing of the mortar and pestle using vehicle.
• Homogenized the suspension by manual stirring before completing to final volume with vehicle.
• Homogenized the suspension by magnetic stirring for at least 10 minutes before further homogenisation using ultraturrax at 11000 rpm for 10 minutes.
• Further stirred the formulations by magnetic stirring at room temperature and protected from light for at least 15 minutes.

A series of formulations at the required concentrations were prepared by dilution of individual weighings of the test item.

Frequency of preparation Groups 1 to 3: On two occasions and dose formulations were prepared six days in advance of dosing.

Group 4: Daily on each morning of administration.

Storage of dose formulation Groups 1 to 3: Refrigerated (2 to 8°C) until required for use.

Group 4: Ambient (15 to 25°C).

Test item accounting
Detailed records of compound usage were maintained. The amount of test item necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability and homogeneity The homogeneity and stability of formulations during storage were confirmed as part of another study, Labcorp Study No. 8468033 In that study, formulations in the range 2 to 200 mg/mL were determined to be stable for:

• One day at ambient temperature (15 to 25°C)
• 15 days when stored refrigerated (2 to 8°C)

Achieved concentration Samples of each formulation prepared for administration in the first and last weeks of treatment were analyzed for achieved concentration of the test item.
Details on mating procedure:
Mating
Male/female ratio 1:1 with identified stock males.

Daily checks for evidence of mating Ejected copulation plugs in cage tray and vaginal smear.

Day 0 of gestation When positive evidence of mating was detected.

A colony of stock males from the same strain and source (CD IGS) was maintained specifically for the purpose of mating. These animals were not part of the study and were maintained as stock animals.

Allocation and Identification
Allocation On day of positive evidence of mating. Only females showing at least two copulation plugs were allocated.

Method To group and cage position in the sequence of mating. Females mating 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 treatment group.

Identification of animals Each animal was assigned a number and identified uniquely within the study by a microchip.

Identification of cages Each cage label was color-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupant(s).
Duration of treatment / exposure:
Females Day 6 to 20 after mating
Frequency of treatment:
Females were treated from Day 6 to Day 20 (inclusive) after mating, once daily at approximately the same time each day
Duration of test:
Study initiation (Study Plan signed by Study Director) 24 March 2022
Experimental start date (Animal arrival) 30 March 2022
Pairing commenced 02 May 2022
Treatment commenced 09 May 2022
Necropsy 24 to 27 May 2022
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Group 1 - Control. Vehicle only
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Group 4
No. of animals per sex per dose:
22 females per dose group, including control
Control animals:
yes, concurrent vehicle
Details on study design:
Rationale for Dose Level Selection
Selection of the doses was based on the results of the preliminary embryo-fetal study (Labcorp study no. 8468035). During this study, the test item was administrated at doses of 500, 750 or 1000 mg/kg bw/day.

No mortality was recorded at all dose-levels. There were no clear treatment-related changes in clinical condition at any dose levels investigated, with only non-adverse and transient signs observed at 750 or 1000 mg/kg bw/day.

Following the commencement of treatment on Day 6 of gestation, when compared to Controls, a group mean body weight loss was observed following the first dose administration in pregnant females at all dose levels investigated, generally followed by slightly low body weight gains throughout the remainder of treatment at 750 or 1000 mg/kg bw/day. An overall low mean body weight gain (GD 6-21) for all groups of treated females (7%, 13% or 24% lower than Controls at 500, 750 or 1000 mg/kg bw/day, respectively) was recorded.

In accordance with the OECD Testing Guideline 414, the highest dose level was chosen with the aim to induce some developmental and/or maternal toxicity (clinical signs or a decrease in body weight) but not death or severe suffering.

Based on the outcome of the preliminary study, the highest dose level was 1000 mg/kg bw/day, as it was the limit dose for the OECD TG 414, and it did not induce death nor severe suffering. 250 and 500 mg/kg bw/day were selected as the low and intermediate dose-levels, respectively.

Animal Model
The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Sprague-Dawley [Crl:CD(SD)] strain was used because of the historical control data available at this laboratory.

Route of Administration
The oral gavage route of administration was chosen to simulate the conditions of potential human exposure.

Thyroid Hormone Analysis
Blood samples were collected at the following occasion:

Occasion Animals
At termination All adults surviving to scheduled termination on GD 21

Parameters Triiodothyronine (T3)
Thyroxine (T4)
Thyroid stimulating hormone (TSH)

Sequence of blood sampling on each occasion
To minimize any potential confounding effect of the time of day of blood sampling, the time of blood sampling was controlled to allow satisfactory inter-group comparisons.

Conditions No overnight deprivation of food.

Blood sample site Sublingual vein.

Anesthetic Isoflurane.

Anticoagulant None.

Blood tubes Greiner Minicollect tubes with clotting activator.

Blood volume 1.0 mL.

Treatment of samples Samples were kept at ambient temperature (15 to 25°C) for a minimum of 30 minutes prior to centrifugation.

Centrifugation conditions At 2000g for ten minutes at 4°C.

Serum was transferred to appropriately labelled polypropylene “cryo” tubes using plastic disposable pipettes and then mixed by gentle ten-fold inversion. Following mixing, each serum sample was divided into two aliquots.

Number of aliquots
Two per animal. Aliquot 1: 0.2 mL serum for T3/T4
Aliquot 2: residual serum for TSH

Final storage conditions Deep frozen (approximately -60°C to -90°C) pending analysis.

Fate of samples Aliquot 1 (T3 and T4): dispatched to the Department of LC MS/MS Bioanalysis, Labcorp.

Aliquot 2 (TSH): dispatched to the Department of Immunology and Immunotoxicology, Labcorp.

T3 and T4 Performed by the Department of LC-MS/MS Bioanalysis, Labcorp.

TSH Performed by the Department of Immunology and Immunotoxicology, Labcorp.

:

Examinations

Maternal examinations:
Serial Observations

Mortality
A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary.

A complete necropsy was performed in all cases.

Clinical Observations
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.

Signs Associated with Dosing
Detailed observations were recorded daily during the treatment period 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

Clinical Signs
A detailed physical examination was performed on each animal, weekly during acclimatization and on Days 0, 3, 5, 12, 18 and 21 after mating to monitor general health.

Body Weight
The weight of each adult was recorded weekly during acclimatization and on Days 0, 3 and 6 to 21 after mating.

Food Consumption
The weight of food supplied to each adult, that remaining and an estimate of any spilled was recorded for the periods Days 0-2, 3-5, 6-8, 9-11, 12-14, 15-17 and 18-20 after mating inclusive.

Necropsy
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. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

Schedule Animals surviving until the end of the scheduled study period were killed on Day 21 after mating.

Sequence To allow satisfactory inter-group comparison.

Tissue and regions examined
Abnormalities
Gravid uterine weight (including cervix and ovaries)
Thyroid

Organ Weights
For bilateral organs, left and right organs were weighed together.

Light Microscopy
Tissues preserved for examination were examined as follows:

Category Animals Tissues
Premature deaths All animals from all groups. Thyroid
Scheduled kill All animals from all groups. Thyroid

Findings were either reported as "present" or assigned a severity grade. In the latter case one of the following five grades was used - minimal, slight, moderate, marked or severe. A reviewing pathologist undertook a peer review of the microscopic findings.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes

For each ovary/uterine horn
Number of:
Corpora lutea.
Implantation sites.
Resorption sites (classified as early or late).
Fetuses (live and dead).

Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Apparently non pregnant animals and for apparently empty uterine horns The number of uterine implantation sites were checked after staining with ammonium sulphide [modification of the Salewski staining technique (Salewski, 1964)].

Blood sampling:
Blood samples were collected at the following occasion:
Occasion Animals
At termination All adults surviving to scheduled termination on GD 21

Blood volume 1.0 mL.
Fetal examinations:
Fetal Examination and Processing
Examination of all viable fetuses and placentae Dissected from the uterus, individually weighed and identified within the litter using a coding system based on their position in the uterus. Fetuses examined externally with abnormalities recorded. Particular attention was paid to the external genital organs of male fetuses. The sex and ano-genital distance of each fetus was recorded.

50% of fetuses in each litter Sexed internally, examined for visceral abnormalities by fresh microdissection (Modified Staples technique) and subsequently fixed in Bouin’s solution.

50% of fetuses in each litter Sexed internally, eviscerated and fixed in Industrial Methylated Spirit (IMS).

Processing Heads were removed from Bouin’s fixed fetuses and were subject to Wilsons free-hand serial sectioning. Torsos were retained in Bouin’s solution.

IMS fixed fetuses were processed and stained with Alizarin Red and Alcian Blue.

Fetal Pathology Examination
Bouin’s fixed fetuses Serial sections were examined for visceral abnormalities.
Alizarin Red and Alcian Blue stained fetuses Assessed for skeletal development, cartilage and abnormalities.

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.

Ano-Genital Distance
Ano-genital distance were presented both as absolute/unadjusted and adjusted for fetal body weight, using the weight recorded at necropsy.

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. ventricular septal defect.

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, incomplete ossification of 5th and 6th sternebrae.
In the fetal examination appendix, observations on repeated structures like ribs, vertebrae and sternebrae are reported as the first and last affected element, in the form ‘5th 13th bilateral ribs’, which should be interpreted as ‘5th to 13th bilateral ribs’.
Statistics:
Please refer to "any other information on materials and methods"
Indices:
Reproductive Assessment
Prenatal losses are separated into pre- and post-implantation phases. Pre-implantation loss was considered to reflect losses due to non-fertilization of ova and failure to implant. It was calculated from the formula:

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

Where the number of implantations exceeded the number of corpora lutea observed, pre implantation loss was assumed to be zero (i.e. no pre-implantation loss was considered to have occurred).

Post-implantation loss was calculated from the formula:

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

All group values and SD were calculated from the individual litter values.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Administration of 2,2'-isopropylidenebis(p-phenyleneoxy)diethanol to the surviving females given 1000 mg/kg/day or in females given 250 or 500 mg/kg/day was generally well-tolerated with no clear treatment-related changes in clinical condition observed. A slight increased incidence of piloerection immediately following dosing was generally observed from Gestation Day 10 in females given 1000 mg/kg/day and from Gestation Day 18 in females given 500 mg/kg/day although this was not associated with further clinical signs, transient and not considered adverse.
Mortality:
mortality observed, treatment-related
Description (incidence):
Two premature deaths occurred during the study:
Female 4F 75 given 1000 mg/kg/day was euthanized for welfare reasons on Gestation Day 8 following post-dose observations on Gestation Day 7 that consisted of piloerection, hunched posture and decreased activity and a 20 grams body weight loss. Macroscopic examination revealed firm abnormal contents in the cecum and rectum with dark areas in the stomach glandular mucosa and this female was pregnant with 11 live fetuses.
Female 4F 82 given 1000 mg/kg/day was euthanized for welfare reasons on Gestation Day 12 due to poor clinical condition with post-dose observations previously seen were piloerection and unsteady gait with an overall body weight loss of 51 grams and was further associated with low food consumption. Macroscopic examination did not reveal any abnormalities although this female was found with 15 early resorptions (total litter resorption).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Following the commencement of treatment on Day 6 of gestation, when compared to Controls, a group mean body weight loss was observed following the first dose administration in pregnant females given 1000 mg/kg/day and was followed by periods of slightly low body weight gains during the remainder of treatment. These differences resulted in an overall low group mean body weight gain (GD 6-21) for females given 1000 mg/kg/day attaining statistical significance (9% lower than Controls). Females receiving 500 mg/kg/day showed a statistically significantly lower overall body weight gain when compared to Controls on GD 6-7 (p<0.05). There was no effect of treatment on body weight or body weight gain in females given 250 mg/kg/day.

Mean gravid uterine weight on Day 21 of gestation was not affected by treatment at any dose level investigated. When overall group mean body weight gain was adjusted for the contribution of the gravid uterus, net mean body weight gain was low at 1000 mg/kg/day attaining statistical significance when compared to Controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Following the commencement of treatment, when compared to Controls, females given 1000 mg/kg/day showed a statistically significantly low group mean food consumption during Day 6-9 of gestation with food intake during the remainder of treatment was similar to Control values although these differences resulted in an overall low group mean food consumption at this dose level. Food consumption in females given 250 or 500 mg/kg/day was not affected by treatment.
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
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The assessment of thyroid gland organ weight parameters at scheduled termination did not reveal any differences that were considered related to treatment at any dose level investigated.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Macroscopic examination at scheduled termination did not reveal any treatment-related findings in maternal females at all dose levels investigated.

All macroscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls), and/or were as expected for Sprague-Dawley rats of this age.

Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related microscopic findings in the thyroid gland.
All microscopic findings were considered spontaneous and/or incidental because they
occurred at a low incidence, were randomly distributed across groups (including concurrent controls), and/or their severity was as expected for Sprague-Dawley rats of this age.
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
Not applicable as rats do not abort
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There was no evidence of an effect of maternal treatment on litter data, as assessed by the mean number of implantations, resorptions (early or late), live young, sex ratio or the levels of pre- and post-implantation loss at all dose levels investigated.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
There was no evidence of an effect of maternal treatment on litter data, as assessed by the mean number of implantations, resorptions (early or late), live young, sex ratio or the levels of pre- and post-implantation loss at all dose levels investigated.
Early or late resorptions:
no effects observed
Description (incidence and severity):
There was no evidence of an effect of maternal treatment on litter data, as assessed by the mean number of implantations, resorptions (early or late), live young, sex ratio or the levels of pre- and post-implantation loss at all dose levels investigated.
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed

Effect levels (maternal animals)

Remarks on result:
other: Not yet confirmed as report in draft stage

Maternal abnormalities

Abnormalities:
not specified

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
There was no effect of maternal treatment of mean placental weights, total litter weights, or male, female and overall fetal weights at any dose level investigated.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There was no evidence of an effect of maternal treatment on live young
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There was no evidence of an effect of maternal treatment on sex ratio
Changes in litter size and weights:
no effects observed
Anogenital distance of all rodent fetuses:
no effects observed
Description (incidence and severity):
There was no effect of treatment of fetal ano-genital distance.
External malformations:
no effects observed
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The incidence of major abnormalities, minor skeletal abnormalities and skeletal variants showed no relationship to treatment.
Across the treated groups, there was an increased fetal incidence of variant of short supernumerary 14th ribs, when compared to concurrent Control, and was outside the fetal Historical Control Data (HCD) range at 1000 m/kg/day (30 fetuses in 10 litters) but was within litter HCD range; this variant was not considered an adverse finding.
At 1000 mg/kg/day, there was an increase in fetal incidence of variant of incompletely ossified 5th and/or 6th sternebrae, when compared to concurrent Control, but was within fetal and litter HCD range. Incomplete ossification is a transient stage in fetal development, indicative of fetal immaturity and not considered adverse.
Visceral malformations:
no effects observed

Effect levels (fetuses)

Remarks on result:
other: Not yet confirmed as report in draft stage

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The conclusion will be added upon completion of the examinations currently conducted on the test item.