Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

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:
other: The study has been performed according to OECD and/or ED guidelines and according to GLP principles.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): LIN10001 4,4’-Isopropylidenediphenol, propoxylated (BPA+2PO)
- Substance type: White powder with lumps (determined at NOTOX)
- Physical state: solid (powder)
- Analytical purity: >95% in BPA 2 PO
- Lot/batch No.: A101323
- Expiration date of the lot/batch: 09 May 2015 (retest date)
- Stability under test conditions: Stable
- Storage condition of test material: At room temperature in the dark
- Other:
pH : 7 (solution in water 5%)
Stability at higher temperatures: Not indicated

Test animals

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

Administration / exposure

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.
Doses / concentrations
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

Examinations

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

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
effects observed, treatment-related
Reproductive performance:
effects observed, treatment-related

Details on results (P0)

MORTALITY (PARENTAL ANIMALS)
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.

CLINICAL SIGNS (PARENTAL ANIMALS)
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.


BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
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 (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.


REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
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 (PARENTAL ANIMALS)
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.


ORGAN WEIGHTS (PARENTAL ANIMALS)
At 500 mg/kg, significantly lower terminal body weights and absolute heart, thymus (absolute and relative), spleen, prostate and seminal vesicle (absolute and relative) weights were observed for males, as well as significantly higher relative brain, liver, kidneys and testes weights. For females, significantly higher liver (absolute and relative) and kidneys (relative only) were observed.
For animals at 250 mg/kg, significantly lower terminal body weights were seen for males and significantly lower absolute thymus weights were seen for both sexes (relative thymus weights were also significantly lower for females).
At 125 mg/kg, significantly lower absolute heart (males only), absolute and relative thyroid (females only) and higher relative testes weights were observed. These changes were not considered to be toxicologically relevant because they occurred in the absence of a dose response relationship, were not supported by corroborating evidence from the histological examination, and remained within in the range of available historical control data considered normal for animals of this age and strain.

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.

GROSS PATHOLOGY (PARENTAL ANIMALS)
For the animals that were found dead or killed in extremis at 500 mg/kg, the following macroscopic findings were observed: emaciation, isolated reddish focus/foci on the stomach glandular mucosa, isolated dark red foci on the left lateral lobe and right median lobe of the liver, thickened limiting ridge of the stomach, dark red discoloration of the liver, reddish discoloration of the mesenteric lymph node, many grey-white foci on the kidneys, enlarged kidneys, liver, iliac lymph nodes, and/or adrenal glands, reduced size of the thymus, spleen, seminal vesicles and/or prostate, pale and red-brown discoloration of the left kidney and/or the left kidney papilla, GI-tract distended with gas, and/or beginning autolysis.
No macroscopic findings were noted for two animals that were also killed in extremis.
At 250 mg/kg, findings for animals killed in extremis included emaciation, enlarged kidneys and/or liver, pelvic dilation of the right kidney, dilation of the ureter, thickened limiting ridge of the stomach, irregular surface of the stomach glandular mucosa, watery-clear contents of the caecum, enlarged ceacum, reddish contents of the urinary bladder, reduced sized of the seminal vesicles, thymus, and/or spleen.

Several treatment related macroscopic findings were noted for animals at 500 mg/kg that survived to the end of the scheduled treatment period including: emaciated appearance, reduced size of the thymus, prostate, seminal vesicles, and/or pituitary, isolated reddish focus/foci on the stomach glandular mucosa, thickened cranial nerves III-VI, mandibular lymph nodes, and/or the spleen, and/or gelatinous appearance of the salivary glands. Kidney findings included: enlarged appearance or reduced in size, grey-white foci, irregular surface, watery-clear cysts, pelvic dilation, and/or pale discoloration.

An enlarged right mandibular lymph node and an enlarged spleen were noted for a single male and female that survived to the end of the scheduled treatment period at 250 mg/kg, respectively.

There were no toxicologically relevant findings noted up to 125 mg/kg.

At 125 mg/kg, a greenish soft nodule was noted on the tail of the right epididymis for a single male, isolated dark red focus/foci on the lung and tan discoloration of the left clitoral gland were noted for individual animals. At this low incidence and in the absence of any treatment related effects on histopathological findings at this dose level, these findings were not considered to be toxicologically relevant. One female was found with reduced size of, and many watery clear cysts on, the left kidney. At microscopic examination, this animal was found with marked hydronephrosis with moderate atrophy and moderate interstitial lymphocytic inflammation. Because this occurred for a single animal at this dose level, and all histopathological findings fell within the background incidence range, it was not considered a sign of treatment-related toxicity.

Incidental findings included alopecia of various body regions, an enlarged spleen noted for a single control female, pelvic dilation of one kidney (noted for two control males), yellowish soft nodule on the tail of the left epididymis, and a watery clear cyst on the kidney noted for a single control male. The incidence of these findings remained within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend.


HISTOPATHOLOGY (PARENTAL ANIMALS)
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 Group 3 female no. 69, Group 4 female no. 71 and Group 4 male no. 39. 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 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 following 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 had no offspring.

OTHER FINDINGS (PARENTAL ANIMALS):
FUNCTIONAL OBSERVATIONS
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.

HAEMATOLOGY:
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
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.


Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Toxicologically relevant effects on mortality, clinical signs, body weight, food consumption, food efficiency, gross pathology, organ weights and histopathology were seen at 250 and 500 mg/kg.
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:
other: Effects on reproductive performance of parental animals and impaired spermatogenesis.

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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.

Effect levels (F1)

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

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

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