Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test was performed with 1,1-isopropylidenebis(p-phenyleneoxy)dipropan-2-ol. Treatment by oral gavage in male and female Wistar Han rats revealed parental toxicity at 250 and 500 mg/kg/day, and reproduction toxicity at 500 mg/kg/day. No effects on reproductive parameters (estrous cycle length, spermatogenic staging profiles or histopathological study ) were observed in an oral 90-day repeated toxicity study up to 180 mg/kg/day.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 April 2011-21 June 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA, Health Effects Test Guidelines OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000.
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: Crl:WI(Han)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
Nulliparous and nonpregnant females and untreated animals were used at initiation of the study.
- Age at study initiation: Approximately 13 weeks.
At the start of treatment, animals were approximately 13 weeks old instead of approximately 14 weeks. Mating started shortly after the animals had attained full sexual maturity according to the OECD 422 guideline and thus a slight deviation in age does not affect the study integrity.
- Weight at study initiation: mean weight range at start of treatment was Males: 339-355 grams and Females: 231-234 grams.
- Fasting period before study: No
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).
Mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
Lactation: Pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm). During locomotor activity monitoring of the dams the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes.
General: Sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment/nesting material (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. Certificates of analysis were examined and then retained in the NOTOX archives. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment or bedding material.
- Diet (ad libitum): Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). Results of analyses for nutrients and contaminants were examined and archived.
- Water (ad libitum): Tap-water. Certificates of analysis (performed quarterly) were examined and archived.
- Acclimation period: At least 5 days prior to the start of treatment.
A health inspection was performed prior to commencement of treatment to ensure that the animals were in a good state of health.
Analysis of bedding, paper, diet and water did not reveal any findings that were considered to have affected the study integrity.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.1 – 21.6
- Humidity (%): 26 - 95
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
Temporary deviations from the maximum and minimum level of realtive humidity and light regime occurred in the animal room. Laboratory historical data do not indicate an effect of the deviations.

IN-LIFE DATES: From: 28 April To: 21 June 2011
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
specific gravity 1.125
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Method of formulation: Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level.
Adjustment was made for the specific gravity of the vehicle.
Storage condition of formulations: At ambient temperature.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at NOTOX.
- Concentration in vehicle: 25-100 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg (Dose volume). Actual dose volumes were calculated according to the latest body weight.
- Method of gavage: using a plastic feeding tube. Formulations were placed on a magnetic stirrer during dosing.
Details on mating procedure:
- M/F ratio per cage: 1/1. One female was cohabitated with one male of the same treatment group, avoiding sibling mating.
- Length of cohabitation: A maximum of 14 days was allowed for mating.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage, by the appearance of an intravaginal copulatory plug, and/or by determination of the estrous stage. This day was designated Day 0 post-coitum. Once mating occurred, the males and females were separated.
- After 14 days of unsuccessful pairing one female at 500 mg/kg who had not shown evidence of mating was separated from her male.
Mating was not detected for one animal Group 2 which did deliver live offspring. The mating date of this animal was estimated at 21 days prior to the actual delivery date. This day was designated Day 0 post-coitum.
Due to mortality, there were not enough Group 3 males available to mate all females on a 1 to 1 basis. As such, pairing for one female began one day later after than for all other females, when a pregnancy of a female in Group 3 was confirmed and that proven male could be used.

- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: Each pregnant female was caged individually in Macrolon cages (MIII type, height 18 cm).
- Any other deviations from standard protocol: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase (03 May 2011), according to a validated method (NOTOX Project 495511). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration).

The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was = 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

The concentrations analyzed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).

No test substance was detected in the Group 1 formulation. The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation = 10%) and formulations over the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 6 hours.

Duration of treatment / exposure:
Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 43-54 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy). One females of Group 1 and one of Group 3 were not dosed during littering.

Pups were not treated directly, but were potentially exposed to the test substance in utero and through lactational transfer.
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to the scheduled necropsy.
Details on study schedule:
- Age at mating of the mated animals in the study: Approximately 15 weeks.
Remarks:
Doses / Concentrations:
0, 125, 250, 500 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based on the results of a 10-day dose range finding study (NOTOX Project 496584; see attached results).
Based on the results of the range finding study, dose levels for the main study were: 125, 250 and 500 mg/kg body weight.
Since no clear peak effect of occurrence of clinical signs was observed in the range finding study, clinical observations in the main study were conducted immediately after dosing.

- Other:
Parturition: The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes and placentas cleaned up, nest build up and/or feeding of pups started). Females that were littering were left undisturbed.

Identification of pups: On Day 1 of lactation, all pups were randomized per litter and individually identified by means of subcutaneous injection of Indian ink.

Selection of animals for selected measurements: 5 animals/sex/group were randomly selected at allocation for functional observations, locomotor activity, clinical pathology, macroscopic examination (full list), organ weights (full list) and histopathology (with the exception of Group 4 where only 3 females could be selected for functional observations and motor activity and only 2 females could be selected for organ weights, macroscopic and histopathological examination). Only females with live pups were selected.
Positive control:
No
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Mortality at least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily, detailed clinical observations were made in all animals, at least immediately after dosing (based on the lack of a clear peak period for effects seen after dosing in the dose range finding study NOTOX Project 496584). Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena. Arena observations were not performed when the animals were mating, or housed individually. The time of onset, grade and duration of any observed sign was recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.
In order to monitor the health status Group 4 animals were weighed an extra time on 11 May 2011 (Treatment Day 13).

FOOD CONSUMPTION: Yes
Weekly, except for males and females which were housed together for mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.
food efficiency was calculated as: (average food consumption [per animal per day]/average body weight per cage)x100

WATER CONSUMPTION: No
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
-Time schedule for collection of blood: Immediately prior to scheduled post mortem examination, between 7.00 and 10.30 a.m.
-Anaesthetic used for blood collection: Yes (isoflurane)
-Animals fasted: Yes (with a maximum of 20 hours). Water was provided.
-How many animals: 5 animals/sex/group (except for Group 4, where only 2 females could be selected)
-Parameters checked were: White blood cells, Differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils) Red blood cells, Reticulocytes, Red blood cell distribution width, Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelets, Prothrombin time, Activated Partial thromboplastin time

CLINICAL CHEMISTRY: Yes
-Time schedule for collection of blood: Immediately prior to scheduled post mortem examination, between 7.00 and 10.30 a.m.
-Anaesthetic used for blood collection: Yes (isoflurane)
-Animals fasted: Yes (with a maximum of 20 hours). Water was provided.
-How many animals: 5 animals/sex/group (except for Group 4, where only 2 females could be selected)
-Parameters checked were: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Total Protein, Albumin, Total Bilirubin, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate, Bile acids.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
-Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females were tested during lactation (all before blood sampling).
-Dose groups that were examined: 5 animals/sex/group (except for Group 4, where only 3 females could be selected).
-Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, grip strength and locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway, USA).

During the motor actiivty test all animals were caged individually (pups were housed in their home cages and kept warm using bottles filled with warm water).
Oestrous cyclicity (parental animals):
Not determined.
Sperm parameters (parental animals):
Paramerters examined in all male parental animals:
Testis weight, epididymis weight.
In addition, for 5 males of the control and high dose group, slides of the testes were prepared to examine staging of spermatogenesis.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
The number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities.

-Mortality: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or the cause of death were evaluated.
-Clinical signs: At least once daily, detailed clinical observations were made in all animals.
-Body weights: live pups were weighed on Days 1 and 4 of lactation.
-Sex: Sex was determined for all pups on Days 1 and 4 of lactation (by assessment of the ano-genital distance).

GROSS EXAMINATION OF DEAD PUPS: Yes
If possible, defects or the cause of death were evaluated.
Postmortem examinations (parental animals):
SACRIFICE
Necropsy was conducted on the following days:
Females which delivered: Lactation Days 5-7.
Females which failed to deliver: Post-coitum Day 26 (one animal with evidence of mating) or approximately 21 days after the last day of the mating period (one animal without evidence of mating).
Females with total litter loss: Within 24 hours of litter loss.
Males: Following completion of the mating period (a minimum of 28 days of dose administration).
Spontaneous deaths: As soon as possible after death and always within 24 hours.
Euthanized in extremis: When pain, distress or discomfort is considered not transient in nature or is likely to become more severe.

GROSS NECROPSY
All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.

The numbers of former implantation sites and corpora lutea were recorded for all paired females.

Samples of the following tissues and organs were collected of selected 5 animals/sex/group (except for Group 4, where only 2 females could be selected; see 6.3 Allocation) and all animals that died spontaneously or were killed in extremis:
Adrenal glands, (Aorta), Brain - cerebellum, mid-brain, cortex, Caecum, Cervix, Clitoral gland, Colon, Coagulation gland, Duodenum, Epididymides, Eyes (with optic nerve (if detectable) and Harderian gland), Female mammary gland area, Femur including joint, Heart, Ileum, Jejunum, Kidneys, (Lacrimal gland, exorbital), (Larynx), Liver, Lung, infused with formalin, Lymph nodes - mandibular, mesenteric, (Nasopharynx), (Esophagus), Ovaries, (Pancreas), Peyer's patches [jejunum, ileum] if detectable, Pituitary gland, Preputial gland, Prostate gland, Rectum, (Salivary glands - mandibular, sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, (Skin), Spinal cord -cervical, midthoracic, lumbar, Spleen, Sternum with bone marrow, Stomach, Testes, Thymus, Thyroid including parathyroid if detectable, (Tongue), Trachea, Urinary bladder, Uterus, Vagina, All gross lesions

All remaining animals, females which failed to deliver and the female with a total litter loss: Cervix, Clitoral gland, Coagulation gland, Epididymides, Ovaries, Preputial gland, Prostate gland, Seminal vesicles, Testes, Uterus, Vagina, All gross lesions.

Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.


ORGAN WEIGHTS
The following organ weights and terminal body weight were recorded from the following animals on the scheduled day of necropsy:
Selected 5 animals/sex/group (except for Group 4, where only 2 females could be selected):
Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Spleen, Testes, Thymus, Uterus (including cervix), Prostate, Seminial vesicles including coagulating glands, Thyroid including parathyroid

All remaining males:
Epididymides, Testes

HISTOPATHOLOGY: yes
From the selected 5 males of the control and high dose group, and all males suspected to be infertile, additional slides of the testes were prepared to examine staging of spermatogenesis.
The following slides were examined by a pathologist:
- The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4 (except for Group 4, where only 2 females could be selected).
- The additional slides of the testes of the selected 5 males of Groups 1 and 4 (see 6.3 Allocation) and all males suspected to be infertile to examine staging of spermatogenesis.
- The preserved organs and tissues of the animals of all dose groups which died spontaneously or were killed in extremis.
- All gross lesions of all animals (all dose groups).
- The following tissues of all selected 5 animals of Groups 2 and 3 (males and/or females), based on (possible) treatment-related changes in these organs in Group 4:
Selected animals of Groups 2 and 3:
Males and Females: Heart, Lung, Thymus, Stomach, Duodenum, Liver, Spleen, Kidney, Urinary bladder, Adrenals, Brain, Pituitary gland, Skeletal muscle, Sternum.
Males: Testes, Epididymides, Prostate, Seminal vesicles, Coagulation gland, Preputial glands
Females: Ovaries

- The full list of organs was collected for two females that were never mated and non-pregnant, respectively. The tissues will be retained for possible future histopathological examination.
- The reproductive organs* of males of Group 4; males that failed to sire and females of Group 4; females that failed to deliver healthy pups.
* Reproductive organs included the cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, and vagina.

Inadvertantly, a few organs were not available for histopathology. Reasons for missing tissues included that these tissues were not discernable at necropsy or trimming, or were erroneously not collected at necropsy. Missing tissues are listed in the raw data and pathology report. Additionally, a limited necropsy was perfomed on one animal where a full necropsy was required and no organ weights were measured for this animal. Sufficient data was available for a thorough evaluation.
Postmortem examinations (offspring):
SACRIFICE
Pups surviving to planned termination were killed by decapitation on lactation Days 5-7 of lactation.

GROSS NECROPSY
All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for te presence of milk. If possible, defects or cause of death were evaluated.

HISTOPATHOLOGY / ORGAN WEIGHTS
Not performed
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.

The following additional methods of statistical analysis were used:
Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences followed by the Wilcoxon test to compare the treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.

Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
Reproductive indices:
For each group, the following calculations were performed:

Mating index (%): Number of females mated/Number of females paired x 100

Fertility index (%): Number of pregnant females/Number of females paired x 100

Conception index (%): Number of pregnant females/Number of females mated x 100

Gestation index (%): Number of females bearing live pups/Number of pregnant females x 100

Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:
Percentage live males at First Litter Check: Number of live male pups at First Litter Check/Number of live pups at First Litter Check x 100

Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100

Percentage of postnatal loss Days 0-4 of lactation: Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check x 100

Viability index: Number of live pups on Day 4 of Lactation/Number of pups born alive x 100
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 500 mg/kg, clinical signs for the 9 animals that were killed in extremis included lethargy, hunched or flat posture, piloerection, yellow staining of the genital region, salivation, lean appearance, uncoordinated movements, laboured respiration, feces containing mucus and rales. There were no clinical signs noted for one female that died spontaneously.
For surviving animals, salivation was noted for all animals, and piloerection and hunched posture were each noted for 6/10 animals that survived to the end of the treatment period.
At 250 mg/kg, three animals were euthanized in extremis. Their clinical signs included lethargy, hunched posture, uncoordinated movements, diarrhoea, piloerection, salivation and lean appearance.
For surviving animals, salivation was noted for all animals and piloerection was noted for 4 females. Rales was noted for a single animal and hunched posture, piloerection, lean appearance and chromodacryorrhoea of both eyes were noted in one animal.
There were no toxicologically relevant clinical signs noted at 125 mg/kg.
At 125 mg/kg, a single male was noted with salivation. At the singular incidence observed, it was not considered to be toxicologically relevant.
Alopecia was an incidental finding noted for control and treated animals, which occurred within the range of background findings to be expected for rats of this age and strain. It was not considered to be toxicologically relevant.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
Two males and one female at 250 mg/kg were killed in extremis. At 500 mg/kg, five males and four females were killed in extremis and an additional female (no. 71) died spontaneously.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 500 mg/kg, male absolute body weights and body weight gains (males at this dose level had weight loss) were significantly lower than controls from Day 8 of the pre-mating period through the entire mating period. Female body weights were significantly lower than controls on Day 8 of the pre-mating period (absolute body weights and body weight gains) and on lactation Day 4 (absolute weights only).
At 250 mg/kg, absolute body weights and body weight gains were significantly lower on Day 8 of the pre-mating period, and body weight gains were significantly lower on Days 1 and 15 of the mating period for males. Body weight gains for females at this dose level were significantly lower on Day 4 of the post coitum period only.
Body weights and body weight gains were unaffected at 125 mg/kg.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption (absolute and relative) was lower for males and females at 500 mg/kg during the pre-mating period only. At 250 mg/kg, the same relationship was seen to a lesser degree for males and females during the pre-mating period only. During the mating period, absolute and relative food consumption was higher than controls for males at 250 and 500 mg/kg. At 500 mg/kg, relative food consumption was slightly higher for females from Days 7-20 of the post coitum period (significantly higher from Days 14-17).
Absolute and relative food consumption was unaffected by treatment at 125 mg/kg.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The following (statistically significant) changes in haematology parameters distinguished treated animals from control animals:
-Lower reticulocytes (500 mg/kg, males only)
-Lower red blood cells (500 mg/kg, females only)
-Lower haemoglobin (250 and 500 mg/kg, females only)
-Lower haematocrit (250 and 500 mg/kg, females only)
-Lower prothrombin time (PT; 250 and 500 mg/kg, females only)
-Lower activated partial thromboplastin time (APTT; 250 mg/kg females only)

For females at 250 mg/kg, the significant increase in neutrophils and the decrease in lymphocytes were attributable to relatively low and high control values for these parameters, respectively. As such, they were not considered toxicologically relevant.
Effects on haematology parameters showed a clear dose response effect. Thus, despite the limited n for females at 500 mg/kg, the changes in haematology parameters were considered toxicologically relevant because they were also seen at 250 mg/kg.
Haematology parameters were unaffected at 125 mg/kg.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The following (statistically significant) changes in clinical biochemistry parameters distinguished treated animals from control animals:
-Higher alanine aminotransferase (ALAT, males at 250 and both sexes at 500 mg/kg)
-Higher aspartate aminotransferase (ASAT, females at 250 mg/kg and males at 500 mg/kg)
-Lower albumin (all treatment levels, females only)
-Higher creatinine (500 mg/kg males only)
-Higher cholesterol (all treatment levels for males, and at 250 and 500 mg/kg for females)
-Lower potassium (500 mg/kg, males only)
-Higher calcium (500 mg/kg, males only)
-Higher inorganic phosphate (500 mg/kg, males only)
The significantly higher chloride seen for males at 125 and 250 mg/kg and the lower total protein seen for females at 250 mg/kg were not dose-dependent and remained within the range of available historical control data considered normal for this age and strain. These were not considered to be toxicologically relevant.
Similarly, the significantly lower bile acids seen for males of all treatment groups was primarily attributable to a high mean obtained for control animals. The high mean was due to a very high value obtained for animal no. 5, and the differences in treated values compared to controls was not considered treatment-related.
Based on the absence of any treatment related microscopic findings noted at 125 mg/kg, the changes in cholesterol, albumin and bile acids were not considered toxicologically relevant.
NOTE: Because the sample size for selected females at 500 mg/kg is only 2 animals, any statistical significance must be interpreted with the limited number of animals in mind.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
The hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals.
The number of ambulatory counts measured for females at 125 and 500 mg/kg was significantly lower than controls. The toxicological relevance of this difference is doubted, however, because no clear dose response effect was evident and there were no signs like lethargy noted for these females at the time of motor activity testing. Additionally, there were no clinical signs noted for females at 125 mg/kg during the entire treatment period. Furthermore, the motor activity profile of female control animals was unusual, given they did not show a normal habituation profile with very high activity counts in the first interval that decreased over the testing interval. Their activity remained steady or increased slightly throughout the entire testing period. No explanation for this can be given. Taken together, the lower ambulatory counts for females at 125 and 500 mg/kg were not considered to be biologically relevant.
All treated groups showed a similar habituation profile with very high activity in the first interval that decreased over the duration of the test period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
There were three unscheduled deaths at 250 mg/kg (Group 3; all killed in extremis) and ten at 500 mg/kg (Group 4; one spontaneous death and nine killed in extremis). One Group 4 male died after 26 treatment days. All the other ones died between 4 to 14 treatment days, before mating had occurred.
No cause of moribundity could be established for a female from one Group 3 female, one Group 4 female and one Group 4 male. For the remaining ten animals which were sacrificed in extremis an increase in the number and/or severity of one of more of the following microscopic lesions were thought to contribute to their moribundity:
- Heart: 2 males from Group 3, 3 males from Group 4
(Mineralization of aorta and/or myocard and/or arteries, myocardial degeneration and/or necrosis and/or inflammation)
- Stomach: 2 males from Group 3 and from Group 4
(Mineralization of the glandular mucosa and/or muscular wall)
- Kidney: 2 males from Group 3, 5 males and 4 females from Group 4
(Mineralization of tubules and/or pelvis, tubular dilation and /or degenerative vacuolation and/or basophilia, granular casts, hyperplasia of the urothelium)
- Brain: one male from Group 3 and Group 4 and 2 females from Group 4
(Necrosis of the choroid plexus)
- Pituitary gland: two males from Group 3 and Group 4
(Vacuolation pars nervosa)
- Skeletal muscle: one male and two females from Group 4
(Myofiber necrosis and/or vacuolation and/or inflammation)
Furthermore, there were adaptive changes like hepatocellular hypertrophy of the liver and diffuse cortical hypertrophy of the adrenals, presumably secondary changes like (lymphoid) atrophy in thymus and/or spleen and/or bone marrow, hyperkeratosis of the forestomach and treatment related findings, mainly regarding the reproductive organs.

There were treatment-related microscopic findings in the rats that survived to the scheduled necropsy from Groups 3 and 4.
Heart:
- Aortic mineralization was noted at minimal to slight degree in 2/2 Group 4 females.
Thymus:
- Lymphoid atrophy was recorded in 2/5 Group 3 males (minimal), 2/5 Group 4 males (minimal-slight) and 2/2 Group 4 females (minimal).
Stomach:
- Mucosal mineralization was recorded at increased incidence and severity in the females of Group 4 (3/3 up to moderate degrees).
- Muscular mineralization was noted in 1/5 males and 1/3 females of Group 4 (minimal).
- Giant cells in the glandular mucosa were noted in 1/5 Group 3 females (minimal) and 1/3 Group 4 females (slight).
- Myofiber degeneration was recorded at slight degree in 1/3 Group 4 females.
- Hyperkeratosis of the forestomach was recorded at minimal degree in one Group 4 male.
Liver:
- Centrilobular (and sometimes midzonal) heptocellular hypertrophy was noted in 1/5 males and 1/5 females of Group 3 and 2/5 males of Group 4 (all at minimal degree).

Kidneys:
- Tubular dilation was recorded at higher severity in 2/5 Group 3 females (slight), 3/5 males and 3/3 females of Group 4 (slight-moderate).
- Tubular mineralization was recorded at higher severity in 2/5 Group 3 and 1/2 Group 4 females (slight).
- Pelvic mineralization was recorded at higher incidence and severity in Groups 3 and 4: 1/7 Group 1 males and 1/5 Group 2 females at minimal degree, compared with 1/8 males (slight) and 3/5 females (minimal–slight) in Group 3 and 5/5 males and 3/3 females in Group 4 (minimal–slight).
- Hyperplasia of the urothelium was noted in 1/8 Group 3 (slight), 1/10 males (minimal) and 2/3 females (slight) of Group 4.
- Interstitial inflammation was noted at a somewhat higher degree (slight) in 1/5 males and 1/3 females of Group 4.
- Pelvic inflammation was noted in 1/5 Group 4 males at slight degree.
- Tubular basophilia was noted at higher incidence and/or severity in all five males (2 slight, 3 moderate) and all three females (slight, moderate, marked) of Group 4.
Skeletal muscle:
- Myofiber necrosis was noted in 1/5 males (minimal) and 1/5 females (moderate) of Group 3 and in 1/2 females (minimal) of Group 4.
- Myofiber vacuolation was seen in 1/5 Group 3 females (minimal).
- Myofiber inflammation (lymphocytic) was increased up to moderate degree in 1/5 Group 3 females.
Epididymides:
- Cellular debris was seen at increased severity (minimal to slight) in 3/5 Group 4 males.
Testes:
- Change in sperm release was noted in 1/5 Group 4 males (minimal).
Prostate gland:
- Reduced contents was noted in 1/5 Group 4 males (minimal).
Seminal vesicles:
- Reduced contents was noted in 4/5 Group 4 males (minimal-slight).
Coagulation glands:
- Reduced contents was noted in 1/5 Group 3 males (minimal) and 3/5 Group 4 males.
Ovaries:
- Hypertrophy/hyperplasia of the interstitial gland was noted at higher severity (moderate) in 2/5 Group 3 and 3/5 Group 4 females.
One finding of note was recorded in one Group 4 male. At necropsy a (unilateral) thickening of the cranial nerves (area of cranial nerves III-VI) was noted. The microscopic correlate of this findng was a benign tumor of the nerve sheath: Schwannoma. Although it is possible to induce a schwannoma, this finding is not considered to be test article related based on the short study duration (this animal was on test for 29 days) and was therefore considered to be an unusual incidental finding.
All remaining microscopic findings recorded were considered to be within the normal range of background pathology encountered in Wistar-Han rats of this age and strain.
No abnormalities were seen in the reproductive organs which could account for the infertility of the Group 4 rats:
Incipient kidney changes in one female are considered to be responsible for the total litter loss of this dam, one female did not mate and one female had no offspring.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
The assessment of the integrity of the spermatogenetic cycle in the testes did provide evidence of impaired spermatogenesis in Groups 3 and 4. This evidence consisted of:
- Changes in sperm release in 2/7 Group 3 (slight) and 5/10 (minimal or slight) Group 4 males.
- Spermatid retention in tubule stage X/XI in 2/5 Group 3 males (minimal or slight).

Instead of the normal physiologic resorption of little residual bodies by sertoli cells, there were rats with big eosinophilic, finely vacuolated, cytoplasmic blebs, which were shed into the tubular lumen. This is defined for this study as: Changes in sperm release. Normally sperm will be released at stage VII and VIII of the spermatogenic cycle. In this study there were rats with sperm visible at the basis of the tubules in stage X and XI. This is called: Spermatid retention in tubule stage X/XI.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
At 500 mg/kg the mating, fertility and conception indices were lower for females with values of 80, 60 and 75%, respectively. The aforementioned indices were all 100% for control and other treated females. Additionally, the number of corpora lutea was lower for females at 500 mg/kg. Contributing to the reduced number of corpora lutea, there were only four females available for assessment (one female had not mated and was thus not included in the corpora lutea count), and of these, one was not pregnant.
The mating, fertility and conception indices were unaffected up to 250 mg/kg.
The gestation index, precoital time, and the number of implantation sites were unaffected by treatment up to 500 mg/kg.
Dose descriptor:
NOAEL
Effect level:
125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
food efficiency
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Remarks:
Reproduction
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive function (sperm measures)
reproductive performance
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
VIABILITY (OFFSPRING)
The number of dead pups at first litter check was higher at 500 mg/kg than for controls. There were four dead pups at first litter check, all from one female, compared to no dead pups at first litter check in the control group. The number of living pups was also lower at 500 mg/kg, with a mean of 8.7 pups compared to a mean of 11.8 pups in the control group. This difference is due, in part, to the limited number of litters available (3 litters at 500 mg/kg and 10 litters for the control group). In this group, one female only had 2 living pups at the first litter check, which also contributed to the low mean. The other two females at 500 mg/kg had 12 pups each. As such, the higher incidence of dead pups at first litter check and the low number of living pups was entirely attributable to this female; thus the difference from controls was not considered toxicologically relevant.

Additionally, the postnatal loss was also higher at 500 mg/kg. While the absolute number of pups lost was not affected (2 pups were lost in both the control and 500 mg/kg groups), the corresponding percentage of living pups was higher, with 2 pups representing only 1.7% of living pups for the control group, but 7.7% of living pups at 500 mg/kg. The limited number of litters also contributed to the high percentage seen at 500 mg/kg. Lastly, the viability index was 92.3 at 500 mg/kg, which was lower than 98.3 for controls. Since the two pups lost were also from this female, no toxicological significance was attributed to the higher postnatal loss.

The sex ratio was unaffected by treatment.

The above mentioned parameters were completely unaffected up to 250 mg/kg.

Two pups from the control group, two pups at 125 mg/kg, three pups at 250 mg/kg and six pups at 500 mg/kg were found dead or were missing during the first days of lactation. Missing pups were most likely cannibalized. All of the dead pups at 500 mg/kg were from one animal, who had a total litter loss by Day 4 of lactation. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence at 500 mg/kg was entirely due to this one female, and in the other treatment groups the mortality did not show a dose-related trend and remained within the range considered normal for pups of this age. At microscopic examination, this one female was found to have incipient kidney changes, which are likely responsible for her total litter loss.

CLINICAL SIGNS (OFFSPRING)
Incidental clinical symptoms of pups consisted of a wound or scab on the right flank, purple discoloration of the tail apex, purple discoloration and thickened left hind paw, no milk in the stomach, pale and/or a lean appearance. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore not considered to be toxicologically relevant.

BODY WEIGHT (OFFSPRING)
Body weights of pups were unaffected by treatment up to 500 mg/kg.

GROSS PATHOLOGY (OFFSPRING)
No milk was noted for pups that were found dead. For surviving pups, a wound on the right flank was noted for a single pup from the control group. This was the only macroscopic finding noted for surviving pups, and was not attributed to treatment with the test substance since it was noted only for a control animal.
Dose descriptor:
NOAEL
Remarks:
Developmental
Generation:
F1
Effect level:
>= 500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Highest dose tested
Reproductive effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
In an OECD 421/422 study with rats, the reproduction NOAEL was set at 250 mg/kg bw/day due to effects on reproductive performance and impaired spermatogenesis, The parental NOAEL was 125 mg/kg bw/day.
Executive summary:

4,4’-Isopropylidenediphenol, propoxylated (BPA+2PO) was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 125, 250 and 500 mg/kg/day. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 29 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 43-54 days). Formulation analysis showed that the formulations were prepared accurately, were homogenous, and were stable for at least 6 hours at room temperature.

Parental results:

Treatment related toxicity was evident at 250 and 500 mg/kg including mortality (3 animals at 250 mg/kg and 10 animals at 500 mg/kg), clinical signs (hunched posture, salivation, piloerection and lethargy, among others), changes in body weights, food consumption, haematology and clinical biochemistry parameters, and organ weight and organ to body weight ratios. Macroscopic and microscopic findings in the heart, stomach, brain, pituitary gland, kidneys, liver thymus, skeletal muscle, prostate gland, seminal vesicles, coagulation gland and ovaries were also noted. Additionally, impaired spermatogenesis were noted for males at 250 and 500 mg/kg. No toxicologically significant changes were noted in functional observations, and no toxicologically relevant effects were seen in any parameter at 125 mg/kg.

Reproductive results:

The mating, fertility and conception indices were lower for females at 500 mg/kg, along with a lower number of corpora lutea. The high mortality and limited number of litters available for evaluation likely contributed to this, though when also taking into account the evidence of impaired spermatogenesis, a treatment related effect cannot be excluded. There were no toxicologically significant changes noted in any of the remaining reproductive parameters investigated in this study (i.e. gestation index, precoital time, and number of implantation sites).

Developmental results:

No developmental toxicity was observed up to 500 mg/kg. Due to mortality there was a lower number of litters available for assessment at 500 mg/kg, and a higher incidence of pup mortality, a lower number of living pups, and a higher number of dead pups was seen at this dose level. However, all pup mortality was attributable to a single dam and was secondary to maternal toxicity.

No treatment-related changes were noted in any of the remaining developmental parameters investigated in this study (i.e. duration of gestation, parturition, maternal care and clinical signs, body weights and macroscopy of pups).

In conclusion, treatment with 4,4’-Isopropylidenediphenol, propoxylated (BPA+2PO) by oral gavage in male and female Wistar Han rats at dose levels of 125, 250 and 500 mg/kg body weight/day revealed parental toxicity at 250 and 500 mg/kg body weight/day, and reproduction toxicity at 500 mg/kg body weight/day.

Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:

Parental NOAEL: 125 mg/kg/day

Reproduction NOAEL: 250 mg/kg/day

Developmental NOAEL: >=500 mg/kg/day

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP study which meets the Annex IX chapter 8.7.1 requirements.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

1,1'-isopropylidenebis(p-phenyleneoxy)dipropan-2-ol was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 125, 250 and 500 mg/kg/day. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 29 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 43 -54 days). Formulation analysis showed that the formulations were prepared accurately, were homogenous, and were stable for at least 6 hours at room temperature.

Parental results:

Treatment related toxicity was evident at 250 and 500 mg/kg including mortality (3 animals at 250 mg/kg and 10 animals at 500 mg/kg), clinical signs (hunched posture, salivation, piloerection and lethargy, among others), changes in body weights, food consumption, haematology and clinical biochemistry parameters, and organ weight and organ to body weight ratios. Macroscopic and microscopic findings in the heart, stomach, brain, pituitary gland, kidneys, liver thymus, skeletal muscle, prostate gland, seminal vesicles, coagulation gland and ovaries were also noted. Additionally, impaired spermatogenesis were noted for males at 250 and 500 mg/kg. No toxicologically significant changes were noted in functional observations, and no toxicologically relevant effects were seen in any parameter at 125 mg/kg.

Reproductive results:

The mating, fertility and conception indices were lower for females at 500 mg/kg, along with a lower number of corpora lutea. The high mortality and limited number of litters available for evaluation likely contributed to this, though when also taking into account the evidence of impaired spermatogenesis, a treatment related effect cannot be excluded. There were no toxicologically significant changes noted in any of the remaining reproductive parameters investigated in this study (i.e. gestation index, precoital time, and number of implantation sites).

In conclusion, treatment with 1,1'-isopropylidenebis(p-phenyleneoxy)dipropan-2-ol by oral gavage in male and female Wistar Han rats at dose levels of 125, 250 and 500 mg/kg body weight/day revealed parental toxicity at 250 and 500 mg/kg body weight/day, and reproduction toxicity at 500 mg/kg body weight/day.

However, in the OECD 408 study (Sub-chronic toxicity study (90-day), oral route (gavage) in rats) performed with 1,1'-isopropylidenebis(p-phenyleneoxy)dipropan-2-ol at the dose-levels of 0, 20, 60 and 180 mg/kg, some reproductive parameters were examined. In this study, oestrous cycle length was normal for all examined females in any group except for one control female showing an extended oestrous cycle length. Minimal interstitial cell hypertrophy in the interstitial glands of the ovaries of females starting in the 60 mg/kg group was observed and considered as non-adverse. In addition, the spermatogenic staging profiles were normal for all males assessed at any dose-level, confirming that the impaired spermatogenesis and histopathological findings in the prostate gland, seminal vesicles, coagulation gland and ovaries observed concomitantly with non reproductive organs in the parental generation of the OECD 422 study were linked to the observed high toxicity.


Effects on developmental toxicity

Description of key information

A prenatal developmental toxicity study (OECD 414) was performed in rats by oral gavage with 1,1'-isopropylidenebis(p-phenyleneoxy)dipropan-2-ol. The maternal No Observed Adverse Effect Level (NOAEL) was established as being 100 mg/kg bw and the developmental NOAEL was established as 250 mg/kg bw.

A prenatal developmental toxicity study (OECD 414) was performed in rabbits by oral gavage with 1,1'-isopropylidenebis(p-phenyleneoxy)dipropan-2-ol. A maternal and developmental No Observed Adverse Effect Level (NOAEL) of at least 75 mg/kg was established.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 April 2017 - 27 July 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Justification for Test System and Number of Animals:

The New Zealand White rabbit was chosen as the animal model for this study as it is an accepted non-rodent species for developmental toxicity testing by regulatory agencies. Charles River Den Bosch has historical data on the background incidence of fetal malformations and developmental variations in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of developmental toxicants.
The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test item. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives.
At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.
This type of study plan was reviewed and agreed by the Laboratory Animal Welfare Officer and the Ethical Committee of Charles River Den Bosch as required by the Dutch Act on Animal Experimentation (February 1997).
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2001
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
- Storage condition of test material: At room temperature protected from light
- Stability of the test substance in the solvent/vehicle 0.5% CMC + 1.25% Tween 80: Stability for at least 6 hours at room temperature, 8 days in the refrigerator and 3 weeks in freezer is confirmed over the concentration range 1 mg/g (1 mg/mL) to 469 mg/g (500 mg/mL), Test Facility Study No.517011.
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (Chatillon sur Chalaronne, France)
- Age at study initiation: 17-19 weeks
- Weight at study initiation: between 2965 and 4307 g
- Fasting period before study: no
- Housing: individually in cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) equipped with water bottles
- Diet: Pelleted diet for rabbits (Global Diet 2030 from Harlan Teklad®, Mucedola, Milanese, Italy) was provided ad libitum throughout the study, except during designated procedures. In addition, pressed hay (Tecnilab-BMI bv, Someren, The Netherlands) and wooden sticks (Swedish aspen wood, Bioservices, Uden, The Netherlands) were provided during the study period.
- Water: tap water, ad libitum
- Acclimation period: at least 5 days prior to treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 55 to 97%
- Air changes (per hr): Ten or greater
- Photoperiod (hrs dark / hrs light):12 hour light/12 hour dark cycle

IN-LIFE DATES: From: 5 June 2017(first delivery of mated females) To: 6 July 2017
Route of administration:
oral: gavage
Vehicle:
other: 0.5% aqueous carboxymethyl cellulose with 1.25% Tween 80
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
When solidified, the test item was heated to a maximum of 75±5°C for around 2 hours to reach the liquid state and obtain visual homogeneity. Test item dosing formulations (w/w) were homogenized to visually acceptable levels. The dosing formulations were prepared daily and dosed within 6 hours after completion of the preparation of the test item. Formulations were heated to a maximum of 75±5°C for maximally 25±5 minutes to obtain visual homogeneity. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment were made for specific gravity of the test item. No correction was made for the purity/composition of the test item.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were performed on two days during the treatment phase (12 and 26 June 2017) by using a validated analytical procedure. Samples of formulation were analyzed for homogeneity (lowest and highest concentration) and accuracy of preparation (all concentrations).
The accuracy of preparation was considered acceptable if the mean measured concentrations within or equal to ± 15% for suspensions of target concentrations. Homogeneity was demonstrated if the coefficient of variation of concentrations was smaller or equal to 10%.
Details on mating procedure:
- Impregnation procedure: rabbits were purchased timed pregnant, arriving at test facility on Day 0 or Day 1 post-coitum (Day 0 post-coitum is defined as the day of successful mating).
Duration of treatment / exposure:
From Days 6 to 28 post-coitum, inclusive
Frequency of treatment:
Daily
Duration of test:
Necropsy on Day 29 post-coitum
Dose / conc.:
0 mg/kg bw/day
Remarks:
concurrent vehicle control
Dose / conc.:
8 mg/kg bw/day
Dose / conc.:
25 mg/kg bw/day
Dose / conc.:
75 mg/kg bw/day
No. of animals per sex per dose:
22 females/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on the results of the dose range finder. In the dose-range finder pregnant females (n=6/group) were dosed at 0, 40, 75 or 100 mg/kg bw/d from day 6 to 28 post-coitum, inclusive. At 100 mg/kg bw/day, 1 female was sacrificed in extremis on Day 24 post-coitum due to a reduced food consumption, no body weight gain and the persistence of piloerection and lean appearance, in addition to severely reduced feces production, pale appearance, alopecia and red fluid on manure tray. Except for alopecia, no macroscopic alterations were noted at necropsy.
A dose-dependent increased incidence and severity of reduced feces production was observed (up to a severe degree at 75 and 100 mg/kg bw/day). Piloerection was observed for two females at 75 mg/kg bw/day and for all females at 100 mg/kg bw/day. Pale appearance was noted for one female at 75 mg/kg bw/day and two females at 100 mg/kg bw/day, which had a lean appearance in addition.
A slight body weight loss and reduced body weight and body weight gain was observed at 75 and 100 mg/kg bw/day. Reduced food consumption was observed in all treatment groups. Macroscopic observations at necropsy did not reveal any alterations that were considered to be toxicologically relevant.
All females were pregnant and had litters with viable fetuses. No treatment related changes for the number of post-implantation loss were observed. Litter sizes, sex ratio and fetal body weights were within normal limits for all groups.External examination of the fetuses did not show any abnormalities.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily for mortality and viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily, beginning on Day 3 post-coitum and lasting up to the day prior to necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: Days 3, 6, 9, 13, 16, 20, 23, 26 and 29 post-coitum

FOOD CONSUMPTION: Yes
- Food consumption was quantitatively measured for Days 3-6, 6-9, 9-13, 13-16, 16-20, 20-23, 23-26 and 26-29 post-coitum.

WATER CONSUMPTION: Yes
- Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles/containers.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29 or within 24 hours of early delivery

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
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
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and reported at the 1% and 5% levels.
Group means were calculated for continuous data and medians were calculated for discrete data (scores). Test statistics were calculated on the basis of exact values for means and pooled variances.
Parametric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Non-Parametric: Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test). Mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss, and sex distribution were compared using the Mann Whitney test. Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.
Incidence: An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using a two-sided Fisher’s exact test at the 5% significance level if the overall test was significant. No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.
Indices:

Pre-implantation loss (%) = ((number of corpora lutea - number of implantation sites)/number of corpora lutea) X 100
Post-implantation loss (%) = ((number of implantation sites - number of live fetuses)/number of implantation sites) x 100
Viable fetuses affected/litter (%) = ((number of viable fetuses affected/litter)/(number of viable fetuses/litter)) x 100
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Piloerection and a lean appearance were observed incidentally in individual females in all dose groups, including controls. In addition, a hunched posture was noted on a few occasions in one high dose female. At the incidence observed, these signs were considered to be unrelated to treatment. Reduced feces and/or absence of feces was noted for almost all females across the groups, including controls. In the absence of a dose-relationship, this finding was not considered to represent a sign of toxicity. A slightly shallow respiration was observed for one control female on post-coitum Days 14 and 15. This animal was therefore preventively not dosed on Day 14. In addition, a necrotic wound in the throat area was observed for this animal from post-coitum Day 24 onwards, which was considered to be background finding. No other relevant signs were noted for this control animal.
All other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rabbits of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female at 8 mg/kg bw/day and one female at 75 mg/kg bw/day were sacrificed in extremis on post-coitum Day 20 and 16, respectively, due to their detoriating physical condition. Prior to their early sacrifice, these females had completely stopped eating for a period of 10 consecutive days, with consequent body weight losses of 10-13%, when compared to start of treatment. In addition, up to severely reduced feces production along with piloerection (low dose female only) and a lean appearance were noted for these animals prior to sacrifice. No abnormalities were noted at necropsy. In the absence of a dose-relationship these decedents were considered to have occurred by chance and not to be related to treatment.
In addition, two females at 8 mg/kg bw/day died on post-coitum Day 26 directly after dosing, due to complications of the oral gavage procedure. No abnormal signs, food intake values or body weights were noted for these animals prior to their moribund state. Except for red fluid around the mouth and nose and in the trachea noted for both females, no macroscopic abnormalities were observed at necropsy. Complications during the oral gavage procedures are incidentally observed in pregnant rabbits and in the absence of any other relevant signs, these decedents were considered to be unrelated to treatment with the test item.
One control female and one female at 8 mg/kg bw/day were sacrificed on post-coitum Day 21 and 25, respectively, as they delivered their offspring early.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Slightly (but statistically significantly) lower body weight gain was noted for females at 75 mg/kg bw/day on post-coitum Days 9 and 13, when compared to controls. On post-coitum Day 9, almost all females at 75 mg/kg bw/day showed a minimal body weight loss, with an average weight loss of 1%, whereas in the other groups including controls, this was noted for incidental females only.
In addition, body weight gain corrected for gravid uterus was also slightly lower for these high dose females: Mean corrected body weight gain was -202 gram (-5%), compared to -156 gram (-4%) in controls. These changes were only minimal and remained within the normal range .
Body weights, body weight gain and weight gain corrected for gravid uterus of animals treated at 8 and 25 mg/kg bw/day remained in the same range as controls over the study period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption before and after correction for body weight was statistically significantly lower over post-coitum Days 6-9 and 9-13 for females at 75 mg/kg bw/day, when compared to controls. Average relative food consumptions were 41% and 35% lower than controls.
No toxicologically relevant changes in food consumption before or after correction for body weight were recorded for females at 8 and 25 mg/kg bw/day.
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
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.
Incidental findings were noted among control and treated animals, including wounds, foci located on the lungs or stomach, and cysts on the oviducts. These are occasionally seen among rabbits used in these types of study and at the incidences observed in absence of a dose-relationship, they were considered to be of no toxicological significance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
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:
effects observed, non-treatment-related
Description (incidence and severity):
One control female and one female at 8 mg/kg bw/day delivered their offspring early on Day 21 and 25 post-coitum, respectively. Prior to early delivery, piloerection and reduced food intake with slight body weight loss (6%) were noted for the control female. For the low dose female, only slightly reduced food intake was noted prior to early delivery. No abnormalities were noted at necropsy for either female, or their fetuses. In the absence of a dose-relationship and at the incidence observed, these early deliveries were not considered to be related to treatment with the test item.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
At scheduled necropsy, there were 17, 14, 19 and 16 pregnant females which had litters with viable fetuses in respectively the control, 8, 25 and 75 mg/kg bw/day group. In total, including the premature decedents which were all pregnant, there were 18, 18, 19 and 17 pregnant females in respectively the control, 8, 25 and 75 mg/kg bw/day group.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
In total, 16 females across the dose groups were not pregnant, based on no visually observed implantation sites at necropsy: 4, 4, 3 and 5 at 0, 8, 25 and 75 mg/kg bw/day, respectively. Despite the high number of non-pregnancy, this was considered to be unrelated to treatment, as treatment started on post-coitum Day 6.
The numbers of corpora lutea and implantation sites, and pre-implantation loss in the treatment groups remained in the same range as controls.
Key result
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean combined (male and female) fetal body weights were 38.3, 42.0, 37.0 and 38.9 grams for the control, 8, 25 and 75 mg/kg bw/day groups, respectively. The relatively high value at 8 mg/kg bw/day, reaching statistical significance for male fetal body weights, was mainly caused by one litter, having an average fetal body weight of 54.2 gram. As this was observed for one single litter in the lowest dose group, this was considered to have occurred by chance and not to be related to treatment.
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
One fetus at 8 mg/kg bw/day was found dead at scheduled necropsy.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The male:female ratio was unaffected by treatment up to 75 mg/kg bw/day. Mean sex ratios (males:females) were 49:51, 46:54, 51:49 and 54:46 for the control, 8, 25 and 75 mg/kg bw/day groups, respectively.
Changes in litter size and weights:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related effects on litter size of any group. Mean litter sizes were 9.6, 8.9, 9.3 and 8.7 viable fetuses/litter for the control, 8, 25 and 75 mg/kg bw/day groups, respectively. The mean litter incidences of viable fetuses, early and late resorptions and post-implantation loss were slightly lower in the 75 mg/kg bw/day group, which was caused by two litters. As the changes were only minimal and not statistically significant and as the incidences of the other 20 litters of the 75 mg/kg bw/day group remained in the same range as controls, this was considered not to be related to treatment with the test item.
Description (incidence and severity):
n/a
External malformations:
no effects observed
Description (incidence and severity):
The numbers of fetuses (litters) available for fetal morphological examination were 164 (17), 124 (14), 176 (19) and 139 (16) in the control, 8, 25 and 75 mg/kg bw/day groups, respectively.
There were no external malformations and variations seen for any fetus in any group.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related effects on skeletal morphology following treatment up to 75 mg/kg bw/d. Skeletal malformations in fetuses were a rib anomaly in one fetus each at 25 and 75 mg/kg bw/day and a vertebral anomaly with associated rib anomaly in one fetus at 25 mg/kg bw/day. Because these malformations occurred singly and were noted previously in historical controls, they were not considered to be treatment related. One other skeletal malformation (caudal vertebral anomaly) was observed in a control fetus, and as such considered to be spontaneous in origin.
Skeletal variations occurred at an incidence of 60.5%, 79.3%, 75.5% and 77.6% per litter at 0, 8, 25 and 75 mg/kg bw/day, respectively. All the variations noted, were not considered treatment related as they occurred infrequently, in the absence of a dose-related incidence trend and/or at frequencies that were within the range of available historical control data.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related effects on visceral morphology following treatment up to 75 mg/kg bw/day. Four different visceral malformations were observed that occurred singly 0, 8 and 25 mg/kg bw/day. No malformations were observed at 75 mg/kg bw/day. At 25 mg/kg bw/day, three fetuses were affected and these either had a malpositioned kidney, absence of the accessory lung lobe or tetralogy of Fallot. A malpositioned kidney was also found at 8 mg/kg bw/day, whereas tetralogy of Fallot was seen in one control fetus as well. The only other malformation observed was a large intestine in one control fetus. As the above visceral malformations occurred singly in one or two groups, including the control group, and not in the high dose group, all were considered to be chance findings.
All the variations noted were considered not treatment related as they occurred infrequently, occurred at frequencies that were within the range of available historical control data or were observed in control fetuses only.
Other effects:
not examined
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Developmental effects observed:
no

Dose formulation analyses:

Accuracy

The concentrations analyzed in the formulations of low and high dose group from both week 1 and 3 of treatment were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). 

For the formulation of the mid dose group prepared for use in week 1 of treatment, the mean accuracy was above the target concentration (i.e. 120% of target). Out of scope investigation was performed and no analytical issue was found. The concentrations analyzed in the formulations of the mid dose group from week 3 of treatment were in agreement with target concentrations (i.e. mean accuracy between 85% and 115%).

No test item was detected in the control group formulations.

Homogeneity

The formulations of low dose group from both week 1 and 3 of treatment were homogeneous (i.e. coefficient of variation10%).

For the formulations of high dose group prepared for use in week 1 of treatment the coefficient of variation was above the target (i.e. 20%). Out of scope investigation was performed and no analytical issue was found. For the formulations of high dose group prepared for use in week 3 of treatment, the coefficient of variation was marginally above the target homogeneity of =10% (i.e. 10.6%), which was considered to be acceptable as the accuracies were within target concentrations.

The out of target results in week 1 of treatment were related to the duration of the heating period of the formations. On the first three days of the treatment period, including the day of sampling in week 1, and on Day 9 of treatment formulations were heated for about 15 minutes. The remainder treatment period, including the day of sample analysis in week 3, formulations were heated for about 30 minutes. As the formulations were sufficiently heated for 22 out of 26 days of the treatment period resulting in acceptable accuracy and homogeneity levels, it was considered that the formulations were appropriate for the purpose of the study.

Conclusions:
Based on the results of a prenatal developmental toxicity study performed according to OECD/EC guidelines and GLP principles, the maternal and developmental No Observed Adverse Effect Level (NOAEL) for 1,1’-isopropylidenebis(p-phenyleneoxy)dipropan-2-ol was established as being at least 75 mg/kg bw/day.
The high dose of 75 mg/kg bw/day was considered to be the highest tolerated dose, based on the results of a dose range finding study.
Executive summary:

A prenatal developmental toxicity study (OECD 414) was performed in rabbits by oral gavage with 1,1'-isopropylidenebis(p-phenyleneoxy)dipropan-2-ol. The objectives of this study were todetermine the potential of the substance to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given orally by gavage to time-mated female New Zealand White rabbits from Day 6 to 28 post-coitum, inclusive. In addition, theNo Observed Adverse Effect Levels(NOAELs) for maternal toxicity and developmental toxicity were evaluated.

The dose levels in this study were selected to be 0 (0.5% aqueous carboxymethyl cellulose with 1.25% Tween 80), 8, 25 and 75 mg/kg/day, based on the results of a dose range finder.

Chemical analyses of formulations were conducted twice during the study to assess accuracy and homogeneity.

The following parameters and end points were evaluated in this study for the F0-generation: mortality/moribundity, clinical signs, body weights, food consumption, gross necropsy findings, number of corpora lutea, (gravid) uterine weight and uterine contents.

In addition, the following parameters were determined for the F1-generation: the number of fetuses, early and late resorptions, total implantations, fetal body weights, sex ratio, and external, visceral and skeletal malformations and developmental variations.

No mortality occurred during the study period that was considered to be related to treatment with the test item. Treatment at 75 mg/kg resulted in slight body weight loss (average weight loss of 1%) on post-coitum Day 9 and significantly reduced food consumption over post-coitum Days 6-13 (relative food consumption about 38% lower than controls). As during the remaining of the treatment period, body weight and food consumption values returned back to normal, these effects were transient and therefore considered not to be adverse.

No maternal toxicity was observed in the 8 and 25 mg/kg groups.

No developmental toxicity was observedin the 8, 25 and 75 mg/kg groups.

In conclusion, based on the results in this prenatal developmental toxicity study in rabbit a maternal and developmental No Observed Adverse Effect Level (NOAEL) for 1,1’-isopropylidenebis(p-phenyleneoxy)dipropan-2-ol of at least 75 mg/kg was established.

The high dose of 75 mg/kg was considered to be the highest tolerated dose, as in the dose range finder treatment at 100 mg/kg resulted in mortality of one female, piloerection noted for all females, body weight loss with an average of -4% on post-coitum Days 9 and 13, and significantly reduced food consumption of 70% over post-coitum Days 6 to 20.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 January - 17 February 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
January 2001
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
August 1998
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Wistar (Han)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Sex: females, nulliparous, nonpregnant and untreated fenimals were used at initiation of the study.
- Age at study initiation: Approximately 12 weeks.
- Weight at study initiation: 222-225g.
- Fasting period before study: no
- Housing:
Acclimatization: Animals were housed in groups of 5 animals/cage in Macrolon cages.
Mating: Females were caged together with stock males on a one-to-one-basis or two-to-one-basis in Macrolon cages.
Post-coitum: Females were individually housed in Macrolon cages.
General: Sterilised sawdust as bedding material and paper as cage enrichment/nesting material were supplied.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

IN-LIFE DATES: From: 09 January - 17 February 2014
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on exposure:
- Method of formulation: Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the vehicle. No correction was made for the purity/composition of the test substance.
- Storage conditions of formulations: At ambient temperature.
- Dose volume: 5 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:
Analyses were conducted on a single occasion during the treatment phase (16 January 2014), according to a validated method (Project 495511). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was = 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
Details on mating procedure:
- M/F ratio per cage: 1/1 or 2/1 (one or two females were cohabitated with one stockmale).
- Age at start of mating of the animals in the study: Approximately 12 weeks
- Length of cohabitation: until sufficient mated females had been obtained for each dose group.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage and/or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
Duration of treatment / exposure:
Duration of treatment: From Days 6 to 19 post-coitum, inclusive.
Frequency of treatment:
Once daily for 7 d/w.
Duration of test:
Duration of test: From Day 0 to 20 post-coitum, inclusive.
Remarks:
Doses / Concentrations:
0, 40, 100, 250 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
22
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: Dose levels were selected based on results of the dose range finding study (Project 503423).
Maternal examinations:
CAGE SIDE OBSERVATIONS
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS
- Time schedule: At least once daily from Day 0 post-coitum onwards up to the day prior to necropsy. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity.

BODY WEIGHT
- Time schedule for examinations: Days 0, 3, 6, 9, 12, 15, 17 and 20 post-coitum.

FOOD CONSUMPTION
- Time schedule for examinations: Days 0-3, 3-6, 6-9, 9-12, 12-15, 15-17 and 17-20 post-coitum.

WATER CONSUMPTION
No. Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

GENERAL REPRODUCTION DATA
- Mating date and confirmation of pregnancy were recorded.
- Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of these females was examined to detect signs of abortion or premature birth.

POST-MORTEM EXAMINATIONS
- Sacrifice on Day 20 post-coitumusing an oxygen/carbon dioxide procedure and subsequently subjected to a to an external, thoracic and abdominal examination.

ORGAN WEIGHTS
- Kidneys, liver and thymus were weighed at necropsy.

HISTOPATHOLOGY
- Slides of the thymus were examined from 10 pregnant females per group
Ovaries and uterine content:
Each ovary and uterine horn of animals surviving to planned necropsy were dissected and examined
as quickly as possible to determine:
- The number of corpora lutea.
- The weight of the (gravid) uterus.
- The number and distribution of live and dead fetuses.
- The number and distribution of embryo-fetal deaths.
- The weight of each fetus.
- The sex of each fetus from the ano-genital distance (during necropsy) and also from gonadal inspections (during further fetal examination).
- Externally visible macroscopic fetal abnormalities.
Fetal examinations:
External, visceral and skeletal fetal findings were recorded as developmental variations or malformations.
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: Groups 1 and 4
- Head examinations: Yes: half per litter
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Mann Whitney test was used to compare mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and sex distribution.
- Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test (Ref. 10) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.
Indices:
For each litter the following calculations were performed:
Pre-implantation loss (%) = (number of corpora lutea - number of implantation sites) / number of corpora lutea x 100
Post-implantation loss (%) = (number of implantation sites - number of live fetuses) / number of implantation sites x 100
The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison, calculating the number of affected fetuses as a mean litter proportion on a total group basis, where:
Viable fetuses affected / litter (%) = number of viable fetuses affected/litter / number of viable fetuses/litter x 100
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 250 mg/kg piloerection and salivation were noted for several animals. Laboured respiration, rales
and hunched posture were also seen, though these were noted for only one or two females for one or
two days.
No clinical signs were noted for the control, low and mid dose groups.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no premature deaths with treatment up to 250 mg/kg.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Females at 250 mg/kg had statistically significantly lower body weights and weight gains than controls
from post coitum Days 12-20 (the absolute weight on Day 20 was not statistically significantly different
from controls). In addition, for uterus corrected body weight gain was statistically significantly
decreased at 250 mg/kg.
At 40 and 100 mg/kg, body weights and body weight gain remained in the same range as controls
over the treatment period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
At 250 mg/kg, food consumption before or after allowance for body weight was decreased on Days 6-
17 post-coitum (this was statistically significant on Days 6-15 post-coitum).
The slightly reduced food consumption at 100 mg/kg on Days 9-12 post-coitum was not considered
toxicologically relevant as the change was very slight and showed a quick recovery during the
treatment period.
Food consumption before or after allowance for body weight was similar for animals treated at 40
mg/kg and control animals.
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:
effects observed, treatment-related
Description (incidence and severity):
Slightly lower thymus weights were noted for females at 250 mg/kg. This was statistically significantly
for absolute weights only. Slight lymphoid atrophy was the microscopic correlate noted for this finding
(see 7.2.7).
Higher absolute and relative thymus weights were recorded for females at 40 mg/kg. This was not
considered to be toxicologically relevant as it occurred in the absence of a dose response and there
were no correlating findings noted at the microscopic examination.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no treatment related effects on macroscopic findings up to 250 mg/kg.
Incidental findings included reddish focus on the thymus, dark red discoloration of the thymus,
accessory liver lobe and pelvic dilation of the kidneys.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Minor test article-related microscopic findings after treatment with 4, 4’-Isopropylidenediphenol
propoxylated were noted in the thymus of the 250 mg/kg/day group females and consisted of lymphoid
atrophy in 3/10 females at 250 mg/kg/day (2:minimal, 1:slight) and a slightly higher incidence of
increased lymphocytolysis. Increased lymphocytolysis was recorded at a minimal degree in 3/10
females at 250 mg/kg/day, compared to 1/10 females per group in the remaining dose groups (control,
40 mg/kg/day and 100 mg/kg/day).
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Number of abortions:
no effects observed
Description (incidence and severity):
There were no abortions or premature births.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There were no differences between control and treated animals in the numbers of pre- or post-implantation loss.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
There were no differences between control and treated animals in the numbers of corpora lutea,
implantation sites, viable or dead fetuses, early or late resorptions, or in pre- or post-implantation loss.
Early or late resorptions:
no effects observed
Description (incidence and severity):
There were no differences between control and treated animals in the numbers of early or late resorptions.
Dead fetuses:
no effects observed
Description (incidence and severity):
There were no differences between control and treated animals in the numbers of viable or dead fetuses.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
There were 18, 20, 19 and 20 pregnant females in the control, 40, 100 and 250 mg/kg groups, respectively. There were 4, 2, 3, and 2 non pregnant animals in the same respective groups.
Other effects:
not examined
Details on maternal toxic effects:
Maternal toxic effects:yes
MORTALITY: There were no premature deaths with treatment up to 250 mg/kg.

CLINICAL SIGNS: At 250 mg/kg piloerection and salivation were noted for several animals. Laboured respiration, rales and hunched posture were also seen, though these were noted for only one or two females for one or two days. No clinical signs were noted for the control, low and mid dose groups.

BODY WEIGHTS: Females at 250 mg/kg had statistically significantly lower body weights and weight gains than controls from post coitum Days 12-20 (the absolute weight on Day 20 was not statistically significantly different from controls). In addition, for uterus corrected body weight gain was statistically significantly decreased at 250 mg/kg. At 40 and 100 mg/kg, body weights and body weight gain remained in the same range as controls over the treatment period.

FOOD CONSUMPTION: At 250 mg/kg, food consumption before or after allowance for body weight was decreased on Days 6-17 post-coitum (this was statistically significant on Days 6-15 post-coitum). The slightly reduced food consumption at 100 mg/kg on Days 9-12 post-coitum was not considered toxicologically relevant as the change was very slight and showed a quick recovery during the treatment period. Food consumption before or after allowance for body weight was similar for animals treated at 40 mg/kg and control animals.

MACROSCOPIC EXAMINATION: There were no treatment related effects on macroscopic findings up to 250 mg/kg. Incidental findings included reddish focus on the thymus, dark red discoloration of the thymus, accessory liver lobe and pelvic dilation of the kidneys.

ORGAN WEIGHTS: Slightly lower thymus weights were noted for females at 250 mg/kg. This was statistically significantly for absolute weights only. Slight lymphoid atrophy was the microscopic correlate noted for this finding. Higher absolute and relative thymus weights were recorded for females at 40 mg/kg. This was not considered to be toxicologically relevant as it occurred in the absence of a dose response and there were no correlating findings noted at the microscopic examination.

MICROSCOPIC EXAMINATION: Minor test article-related microscopic findings after treatment with 4,4’-Isopropylidenediphenol, propoxylated (BPA+2PO) were noted in the thymus of the 250 mg/kg/day group females and consisted of lymphoid atrophy in 3/10 females at 250 mg/kg/day (2:minimal, 1:slight) and a slightly higher incidence of increased lymphocytolysis. Increased lymphocytolysis was recorded at a minimal degree in 3/10 females at 250 mg/kg/day, compared to 1/10 females per group in the remaining dose groups (control, 40 mg/kg/day and 100 mg/kg/day).

MATERNAL PREGNANCY DATA: There were 18, 20, 19 and 20 pregnant females in the control, 40, 100 and 250 mg/kg groups, respectively. There were 4, 2, 3, and 2 non pregnant animals in the same respective groups. There were no abortions or premature births. There were no differences between control and treated animals in the numbers of corpora lutea, implantation sites, viable or dead fetuses, early or late resorptions, or in pre- or post-implantation loss.
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Abnormalities:
effects observed, treatment-related
Description (incidence and severity):
Body weight, body weight gain, food consumption, histopathological
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Fetal body weight was unaffected with treatment up to 250 mg/kg. Mean (combined) fetal weights
were 3.6, 3.5, 3.5 and 3.5 grams for the control, 40, 100 and 250 mg/kg groups, respectively.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There were no differences in the sex ratio of fetuses between control and treated animals.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There were no effects of treatment up to 250 mg/kg on litter size.
Mean litter sizes were 11.9, 11.5, 11.6 and 12.2 fetuses in the control, 40, 100 and 250 mg/kg groups,
respectively.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
There were no external malformations or variations seen for any fetus in any group.
Skeletal malformations:
no effects observed
Description (incidence and severity):
Bent limb bones, a skeletal malformation, were seen in 2(1) and 3(2) fetuses (litters) in the control and
250 mg/kg groups, respectively. This corresponds to and incidence of 0.9% in the control group and
1.0% in the 250 mg/kg group. As this occurred at the same frequency for high dose and control
animals, the bent limb bones were not attributable to treatment.
The incidence of reduced ossification of the vertebral centra (1.8%) and 7th cervical rudimentary ribs
(4.4%) was statistically significantly higher than for controls (0% for both findings). The higher
incidence was not considered toxicologically relevant, however, as both findings remained within the
range of historical control data.
Other variations included 14th rudimentary ribs, reduced ossification of the skull or vertebral arches,
bent ribs, ossified cervical centrum #1, unossified sternebrae #1, 2, 3, 4, 5 and/or 6, unossified hyoid
or vertebral centra, malaligned (slight or moderate) sternebrae, partially fused zygomatic arch, caudal
shift of the pelvic girdle and 14th full ribs. These variations were not considered to be treatment related
because they occurred at similar frequencies in the control and high dose group and/or remained
within the historical control data range.
Visceral malformations:
no effects observed
Description (incidence and severity):
There were no treatment related effects on fetal visceral morphology with treatment up to 250 mg/kg.
Visceral malformations were seen for one fetus in the 40 and 250 mg/kg groups. A small eye was
seen for one fetus (A032-09) at 40 mg/kg and hydrocephaly was seen for one fetus (A079-14) at 250
mg/kg. There were no visceral malformations in the control and 100 mg/kg groups.
Visceral variations noted for control and/or treated groups included small supernumerary liver lobe,
liver appendix, supernumerary artery, partially undescended thymus horn(s), dilated and/or convoluted
ureters, and retroesophageal right subclavian.
These variations were not considered to be treatment related because they occurred at similar
frequencies in the control and treatment groups, without dose response relationship and/or remained
within the historical control data range.
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
FETAL FINDINGS
LITTER SIZE
There were no effects of treatment up to 250 mg/kg on litter size. Mean litter sizes were 11.9, 11.5, 11.6 and 12.2 fetuses in the control, 40, 100 and 250 mg/kg groups, respectively.

SEX RATIO
There were no differences in the sex ratio of fetuses between control and treated animals.

FETAL BODY WEIGHT
Fetal body weight was unaffected with treatment up to 250 mg/kg. Mean (combined) fetal weights were 3.6, 3.5, 3.5 and 3.5 grams for the control, 40, 100 and 250 mg/kg groups, respectively.

FETAL MORPHOLOGICAL EXAMINATIONS
EXTERNAL MALFORMATIONS AND VARIATIONS
The numbers of fetuses (litters) available for external and visceral morphological evaluations were 214(18), 229(20), 221(19) and 243(20) in the control, 40, 100 and 250 mg/kg groups, respectively. Soft tissue cephalic examination was done for approximately half of the fetuses of all groups and skeletal examinations were done for all the fetuses of Groups 1 and 4. Malformations were observed in 2(1), 1(1)*, 0(0)* and 4(3) fetuses (litters) in the control, 40, 100 and 250 mg/kg groups, respectively. (* Note: For Group 2 and 3 treated at 40 and 100 mg/kg, these numbers of malformations were based on external, visceral and soft tissue cephalic examination; no skeletal examination had been performed for these groups.)
There were no external malformations or variations seen for any fetus in any group.

VISCERAL MALFORMATIONS AND VARIATIONS
There were no treatment related effects on fetal visceral morphology with treatment up to 250 mg/kg. Visceral malformations were seen for one fetus in the 40 and 250 mg/kg groups. A small eye was seen for one fetus (A032-09) at 40 mg/kg and hydrocephaly was seen for one fetus at 250 mg/kg. There were no visceral malformations in the control and 100 mg/kg groups. Visceral variations noted for control and/or treated groups included small supernumerary liver lobe, liver appendix, supernumerary artery, partially undescended thymus horn(s), dilated and/or convoluted ureters, and retroesophageal right subclavian. These variations were not considered to be treatment related because they occurred at similar frequencies in the control and treatment groups, without dose response relationship and/or remained within the historical control data range.

SKELETAL MALFORMATIONS AND VARIATIONS
There were no treatment related effects on fetal skeletal morphology up to 250 mg/kg. Bent limb bones, a skeletal malformation, were seen in 2(1) and 3(2) fetuses (litters) in the control and 250 mg/kg groups, respectively. This corresponds to and incidence of 0.9% in the control group and 1.0% in the 250 mg/kg group. As this occurred at the same frequency for high dose and control animals, the bent limb bones were not attributable to treatment. The incidence of reduced ossification of the vertebral centra (1.8%) and 7th cervical rudimentary ribs (4.4%) was statistically significantly higher than for controls (0% for both findings). The higher incidence was not considered toxicologically relevant, however, as both findings remained within the range of historical control data. Other variations included 14th rudimentary ribs, reduced ossification of the skull or vertebral arches, bent ribs, ossified cervical centrum #1, unossified sternebrae #1, 2, 3, 4, 5 and/or 6, unossified hyoid or vertebral centra, malaligned (slight or moderate) sternebrae, partially fused zygomatic arch, caudal shift of the pelvic girdle and 14th full ribs. These variations were not considered to be treatment related because they occurred at similar frequencies in the control and high dose group and/or remained within the historical control data range.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity No developmental effects observed up to 250 mg/kg bw/day (highest dose tested)
Abnormalities:
no effects observed
Developmental effects observed:
no

FORMULATION ANALYSIS: The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). No test substance was detected in the Group 1 formulation. The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation= 10%). Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 6 hours.

BODY WEIGHTS (GRAM)SUMMARY FEMALES

F0 -GENERATION                                                                                                                                                                                  

 

 

GROUP1

CONTROL

GROUP2

40MG/KG

GROUP3

100MG/KG

GROUP4

250MG/KG

 

POSTCOITUM

DAY0

 

 

MEAN

 

 

225

 

 

222

 

 

223

 

 

223

 

ST.DEV.

9.5

8.8

12.3

9.5

 

N

18

20

19

20

DAY3

MEAN

234

233

235

234

 

ST.DEV.

11.3

10.3

12.7

10.2

 

N

18

20

19

20

DAY6

MEAN

243

241

244

243

 

ST.DEV.

11.6

10.6

10.8

9.4

 

N

18

20

19

20

DAY9

MEAN

251

248

251

247

 

ST.DEV.

11.9

11.1

11.6

10.4

 

N

18

20

19

20

DAY12

MEAN

263

260

262

253*

 

ST.DEV.

13.4

10.6

12.3

13.9

 

N

18

20

19

20

DAY15

MEAN

277

274

277

264*

 

ST.DEV.

14.9

12.4

12.4

16.1

 

N

18

20

19

20

DAY 17

MEAN

298

292

296

280**

 

ST.DEV.

14.6

14.3

14.9

18.3

 

N

18

20

19

20

DAY20

MEAN

336

330

333

321

 

ST.DEV.

19.7

17.3

22.9

22.4

 

N

18

20

19

20

 

*/**Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Explanations for excluded data are listed in the tables of the individual values

Food consumption( g/animal/day)

SUMMARY FEMALES

F0-GENERATION                                                                                                                                                                                   

 

 

GROUP1

CONTROL

GROUP2

40MG/KG

GROUP3

100MG/KG

GROUP4

250MG/KG

POSTCOITUM

DAYS0-3

 

MEAN

 

19

 

19

 

18

 

19

 

ST.DEV.

2.0

2.2

2.1

1.9

 

N

18

20

17

20

DAYS3-6

MEAN

20

20

21

21

 

ST.DEV.

2.1

2.3

2.2

1.6

 

N

18

20

19

20

DAYS6-9

MEAN

20

19

19

16**

 

ST.DEV.

2.1

2.6

2.4

2.7

 

N

18

20

19

20

DAYS9-12

MEAN

22

20

20*

17**

 

ST.DEV.

1.8

2.5

2.2

3.4

 

N

18

20

19

20

DAYS12-15

MEAN

23

22

22

19**

 

ST.DEV.

1.8

2.7

2.2

4.5

 

N

18

20

19

20

DAYS15-17

MEAN

24

24

24

21

 

ST.DEV.

4.3

2.5

2.5

4.7

 

N

18

20

19

20

DAYS17-20

MEAN

25

25

25

24

 

ST.DEV.

3.2

2.4

2.9

3.8

 

N

18

20

19

20

MEANOFMEANS

22

21

21

20

 */** Dunnett-test based on pooled variance significantat 5% (*) or 1% (**) level

Explanations for excluded data are listed in the tables of the individual values

Organ weights females

F0 generation

 

 

GROUP1

CONTROL

GROUP2

40MG/KG

GROUP3

100MG/KG

GROUP4

250MG/KG

 

DAY20POST-COITUM

BODYW.

 

 

MEAN

 

 

332

 

 

327

 

 

329

 

 

317

(GRAM)

ST.DEV

20

17

22

22

 

N

17

20

19

20

LIVER

MEAN

15.56

15.13

15.44

15.29

(GRAM)

ST.DEV

1.28

1.66

1.63

1.60

 

N

18

20

19

20

THYMUS

MEAN

0.286

0.336*

0.277

0.239*

(GRAM)

ST.DEV

0.054

0.067

0.055

0.058

 

N

18

20

19

20

KIDNEYS

MEAN

1.82

1.75

1.79

1.75

(GRAM)

ST.DEV

0.21

0.13

0.14

0.13

 

N

18

20

19

20

*/**Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Explanations for excluded data are listed in the tables of the individual values

Summary of maternal survival and pregnancy status

 

Dose Group

1

2

3

4

 

No.

%

No.

%

No.

%

No.

%

Females on study

22

 

22

 

22

 

22

 

Females that aborted or delivered

0

0.0

0

0.0

0

0.0

0

0.0

Females that died

0

0.0

0

0.0

0

0.0

0

0.0

Females that aborted

0

0.0

0

0.0

0

0.0

0

0.0

Non gravid

0

0.0

0

0.0

0

0.0

0

0.0

gravid

0

0.0

0

0.0

0

0.0

0

0.0

 

0

0.0

0

0.0

0

0.0

0

0.0

Females that were euthanized

0

0.0

0

0.0

0

0.0

0

0.0

Non gravid

0

0.0

0

0.0

0

0.0

0

0.0

Gravid

0

0.0

0

0.0

0

0.0

0

0.0

 

 

 

 

 

 

 

 

 

Females examined at scheduled necropsy

22

100.0

22

100.0

22

100.0

22

100.0

Non gravid

4

18.2

2

9.1

3

13.6

2

9.1

Gravid

18

81.8

20

90.9

19

86.4

20

90.9

With resorptions only

0

0.0

0

0.0

0

0.0

0

0.0

With viable fetuses

18

100.0

20

100.0

19

100.0

20

100.0

 

 

 

 

 

 

 

 

 

Total females gravid

18

81.8

20

90.9

19

86.4

20

90.9

 

1-0 mg/kg                          2-40 mg/kg                       3-100 mg/kg                     4-250 mg/kg

 

 

Conclusions:
In an oral OECD 414 prenatal developmental toxicity study with rats, the maternal NOAEL was determined to be 100 mg/kg bw/day, while the developmental NOAEL was established as 250 mg/kg.
Executive summary:

A prenatal developmental toxicity study (OECD 414) was performed in rats by oral gavage with 4,4’-Isopropylidenediphenol, propoxylated (BPA+2PO).

Eighty-eight mated female Wistar Han rats were assigned to four dose groups. The test item was administered once daily by oral gavage from Days 6 to 19 post-coitum at doses of 40, 100 and 250 mg/kg. The rats of the control group received the vehicle, polyethylene glycol 400, alone. Females were checked daily for the presence of clinical signs. Food consumption and body weight were determined daily during treatment and at periodic intervals in the other periods. All animals surviving to Day 20 post-coitum were subjected to an examination post-mortemand external, thoracic and abdominal macroscopic findings and weights of the kidneys, liver and thymus were recorded.

A microscopic examination of the thymus was also performed for selected females.

A laparohysterectomy was performed on each surviving female of the groups. The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and corrected body weights (changes) were calculated. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations. All live fetuses were euthanized. One half of the fetuses were decapitated and the heads were fixed in Bouin’s fixative, all fetuses were dissected and examined for visceral anomalies. All fetuses of control and high-dose groups were subsequently fixed in 96% aqueous ethanol and stained with Alizarin Red S for skeletal examinations.

Formulations prepared on one day during treatment were analyzed for accuracy, homogeneity and stability.

 Accuracy, homogeneity and stability of formulations were demonstrated by analyses.

Maternal toxicity was noted at 250 mg/kg and consisted of clinical signs (piloerection, salivation for several animals, and laboured respiration, rales and hunched posture at lower frequencies), and lower body weights, body weight gains and food consumption through most of the treatment period. No developmental toxicity was observed in the 40, 100 and 250 mg/kg groups.

 

 Based on the results in this prenatal developmental toxicity study the maternal No Observed Adverse Effect Level (NOAEL) for 4,4’-Isopropylidenediphenol, propoxylated (BPA+2PO) was established as being 100 mg/kg and the developmental NOAEL was established as 250 mg/kg.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
75 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Quality of whole database:
GLP study which meets the Annex IX chapter 8.7.2 requirements.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

 Prenatal developmental toxicity study in rats

A prenatal developmental toxicity study (OECD 414) was performed in rats by oral gavage with 1,1'-isopropylidenebis(p-phenyleneoxy)dipropan-2-ol. Eighty-eight mated female Wistar Han rats were assigned to four dose groups. The test item was administered once daily by oral gavage from Days 6 to 19 post-coitum at doses of 40, 100 and 250mg/kg. The rats of the control group received the vehicle, polyethylene glycol 400, alone. Females were checked daily for the presence of clinical signs. Food consumption and body weight were determined daily during treatment and at periodic intervals in the other periods. All animals surviving to Day 20 post-coitum were subjected to an examinationpost-mortemand external, thoracic and abdominal macroscopic findings and weights of the kidneys, liver and thymus were recorded. A microscopic examination of the thymus was also performed for selected females. A laparohysterectomy was performed on each surviving female of the groups. The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, totalimplantations and corpora lutea were recorded. Gravid uterine weights were recorded, and corrected body weights (changes) were calculated. The fetuses were weighed, sexed and examined forexternal, visceral and skeletal malformations and developmental variations. All live fetuses wereeuthanized. One half of the fetuses were decapitated and the heads were fixed in Bouin’s fixative, all fetuses were dissected and examined for visceral anomalies. All fetuses of control and high-dose groups were subsequently fixed in 96% aqueous ethanol and stained with Alizarin Red S for skeletal examinations.

Formulations prepared on one day during treatment were analyzed for accuracy, homogeneity andstability.  Accuracy, homogeneity and stability of formulations were demonstrated by analyses.

Maternal toxicity was noted at 250 mg/kg and consisted of clinical signs (piloerection, salivation forseveral animals, and laboured respiration, rales and hunched posture at lower frequencies), and lower body weights, body weight gains and food consumption through most of the treatment period. No developmental toxicity was observed in the 40, 100 and 250 mg/kg groups.

Based on the results in this prenatal developmental toxicity study the maternal No Observed Adverse Effect Level (NOAEL) for 1,1'-isopropylidenebis(p-phenyleneoxy)dipropan-2-ol was established as being 100 mg/kg and the developmental NOAEL was established as 250 mg/kg.

 

Prenatal developmental toxicity study in rabbits

A prenatal developmental toxicity study (OECD 414) was performed in rabbits by oral gavage with 1,1'-isopropylidenebis(p-phenyleneoxy)dipropan-2-ol. The objectives of this study were todetermine the potential of the substance to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given orally by gavage to time-mated female New Zealand White rabbits from Day 6 to 28 post-coitum, inclusive. In addition, theNo Observed Adverse Effect Levels(NOAELs) for maternal toxicity and developmental toxicity were evaluated.

The dose levels in this study were selected to be 0 (0.5% aqueous carboxymethyl cellulose with 1.25% Tween 80), 8, 25 and 75 mg/kg/day, based on the results of a dose range finder.

The following parameters and end points were evaluated in this study for the F0-generation: mortality/moribundity, clinical signs, body weights, food consumption, gross necropsy findings, number of corpora lutea, (gravid)uterine weight and uterine contents. In addition, the following parameters were determined for the F1-generation: the number of fetuses, early and late resorptions, total implantations, fetal body weights, sex ratio, and external visceral and skeletal malformations and developmental variations.

 

No mortality occurred during the study period that was considered to be related to treatment with the test item. Treatment at 75 mg/kg resulted in slight body weight loss (average weight loss of 1%) on post-coitum Day 9 and significantly reduced food consumption over post-coitum Days 6-13 (relative food consumption about 38% lower than controls). As during the remaining of the treatment period, body weight and food consumption values returned back to normal, these effects were transient and therefore considered not to be adverse.

No maternal toxicity was observed in the 8 and 25 mg/kg groups.

No developmental toxicity was observedin the 8, 25 and 75 mg/kg groups.

In conclusion, based on the results in this prenatal developmental toxicity study in rabbit a maternal and developmental No Observed Adverse Effect Level (NOAEL) for 1,1’-isopropylidenebis(p-phenyleneoxy)dipropan-2-ol of at least 75 mg/kg was established.

The high dose of 75 mg/kg was considered to be the highest tolerated dose, as in the dose range finder treatment at 100 mg/kg resulted in mortality of one female, piloerection noted for all females, body weight loss with an average of -4% on post-coitum Days 9 and 13, and significantly reduced food consumption of 70% over post-coitum Days 6 to 20.

Justification for classification or non-classification

Based on the results available,1,1'-isopropylidenebis(p-phenyleneoxy)dipropan-2 -ol does not have to be classified currently, and has no obligatory labelling requirement for reproduction and developmental toxicity according to the:

- Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2007),

- Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures.