4,4'-Isopropylidenediphenol, propoxylated

Endpoint summary

Description of key information

Investigation of the repeated-dose toxicity of the registered substance 4,4'-Isopropylidenediphenol, propoxylated (thereafter referred to as BPA PO) was performed in several steps.

Based on the uses and exposure to the registered substance and its physicochemical properties (BPA PO has a vapour pressure << 1 Pa), the oral route was considered as the most relevant route of exposure for the purpose of the repeated-dose toxicity testing in accordance with Annexes VIII to X of REACH.

 

1.           Short-term repeated-dose toxicity

Short-term (28-day) repeated-dose toxicity was performed as part of studies conducted in accordance with the OECD Testing Guideline 422 on BPA 2PO and Grade 5 of the registered substance.

 

1.1 Short-term repeated-dose toxicity study conducted on BPA 2PO

The test substance was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 125, 250 and 500 mg/kg bw/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.

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 bw/d.

No toxicologically significant changes were noted in functional observations, and no toxicologically relevant effects were seen in any parameter at 125 mg/kg bw/d.

In conclusion, treatment with 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.

 

1.2 Short-term toxicity study conducted on Grade 5 of BPA PO

The test substance was administered by daily oral gavage to male and female Sprague-Dawley rats at dose levels of 30, 120 and 500 mg/kg bw/day. Males were exposed for 42 days. The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 42-53 days).

One female rat at 500 mg/kg bw/d died on day 39 (day 22 of pregnancy).Incidence of oestrous cycle disorder increased at 500 mg/kg bw/d. Transient salivation was observed at 120 and 500 mg/kg bw/d for both sexes. Emaciation was observed for both sexes at 500 mg/kg bw/d. Incidence of ptosis, decrease in locomotor activity and decrease in body weight gain was observed for male at 500 mg/kg bw/d. Some haematological changes observed at 500 mg/kg bw/d in males as well as in the 500 mg/kg bw/d male and female satellite groups. Neutrophil count was significantly lower in males from the high-dose group at the end of recovery period compared to control. Significant decrease in total protein was observed in male animals at 250 and 500 mg/kg, along with a significant decrease in albumin for male at 500 mg/kg bw/d and significant increase in total cholesterol for both sexes. During the urinalysis significantly higher levels of potassium levels in 50males from the high-dose group on week 2 of the recovery period. Absolute and relative weight of liver increased significantly for males at 500 mg/kg bw/d and relative weight increased for females at 500 mg/kg bw/d. Dilatation of lacteal in small intestine was observed at 500 mg/kg bw/d for both sexes. Hypertrophy of centrilobular hepatocyte was observed at 500 mg/kg bw/d for both sexes.

Clinical chemistry changes and adaptive liver changes at 500 mg/kg bw/d were not considered as adverse.

In conclusion, treatment with Grade 5 of BPA PO resulted in high rate of oestrus cycle disorder at 500 mg/kg bw/d.

 

2. Subchronic repeated-dose toxicity

Investigation of the subchronic (90-day) repeated-dose toxicity of the registered substance was requested by the evaluating Competent Authority (eCA).

A concern for systemic toxicity was identified in the OECD 422 study conducted with Grade 5 of BPA PO based on dilatation of lacteals, effects on the small intestine, and high rate of oestrus cycle disorder. This test substance was therefore considered as more relevant than BPA 2PO for the purpose of subsequent repeated-dose toxicity testing.

However Grade 5 of BPA PO is regarded as a polymer under REACH, so it was a ruled out as a test substance for the purpose of the subchronic repeated-dose toxicity testing. The outcome of the Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening studies suggested that potential endocrine disruption properties could be related to a higher degree of propoxylation and constituents with higher chain-length. Therefore, Grade 4 of BPA PO was deemed as the most relevant test substance for this study by the eCA.

The objective of this study was to evaluate the potential toxicity of the test item following daily oral administration (gavage) to rats for 13 weeks. On completion of the treatment period, designated animals were held for a 4‑week treatment‑free period in order to evaluate the reversibility of any findings.

Two groups of 10 male and 10 female (low- and mid-doses) and one group of 15 male and 15 female (high-dose) Sprague-Dawley rats were treated daily by the oral route (gavage) with the test item, at dose-levels of 30, 120 or 500 mg/kg bw/d for 13 weeks. The test item was administered as an emulsion in the vehicle (olive oil) under a constant dosage-volume of 5 mL/kg/day.

Another group of 15 male and 15 female Sprague-Dawley rats received the vehicle alone under the same experimental conditions and acted as a control group. The actual test item concentrations in the dose formulations prepared for use in Weeks 1, 4, 8 and 12 were determined using a HPLC with UV detection analytical method. The animals were observed daily for mortality and clinical signs. Detailed clinical examinations were performed weekly and a Functional Observation Battery (FOB) was conducted in Week 12. Body weight was recorded once pre-test, on the first day of treatment and then at least once a week. Food consumption was recorded once a week.

Ophthalmology examinations were performed pre-test on all animals and at the end of the treatment period on all control- and high-dose animals. Haematology and blood biochemistry investigations were performed at the end of the treatment and treatment-free periods. On completion of the treatment or treatment-free periods, the animals were sacrificed and a full macroscopic post-mortem examination was performed. Designated organs were weighed and selected tissue specimens were preserved. A microscopic examination was performed on selected tissues for the control- and high-dose animals sacrificed at the end of the treatment period. A microscopic examination was also performed on selected tissues for the low- and intermediate-dose animals sacrificed at the end of the treatment period and for the control- and high‑dose animals sacrificed at the end of the treatment-free period.

At 500 mg/kg bw/d, the test item induced adverse effects in males on the clinical condition, body weight and food consumption leading to premature sacrifice for ethical reasons. Slight to moderate changes in clinical pathology were also noted in both sexes. There were markedly higher liver weights in males and females that correlated with gross enlargement and microscopic hepatocellular hypertrophy, higher adrenal gland weights in males and females, correlated with the microscopic vacuolation of the adrenal cortex, higher kidney weights in females and decreased thymus weights in males correlated with the lymphoid atrophy. At gross examination, there were test item-related enlarged liver in 6/7 males that correlated with the hepatocellular hypertrophy. The black discoloration and the red discoloration correlated with microscopic centrilobular degeneration/necrosis and hemorrhage, respectively. The small seminal vesicles seen in 1/7 males correlated with acinar cell atrophy and decreased secretory content while the thickening the stomach from in 2/7 males treated at 500 mg/kg bw/d correlated with the mineralization and increased regeneration in the gland. At microscopic examination, there were adverse findings in the liver, urinary bladder and stomach from males treated at 500 mg/kg bw/d and non‑adverse findings in the pituitary gland, mandibular lymph nodes, mesenteric lymph nodes, mesenteric artery, thyroid glands, parathyroid, heart, lungs, adrenals, kidneys, jejunum, GALT, prostate and seminal vesicles, sternum, bone marrow, ovaries, vagina and thymus in males and/or, to a lesser extent, in females treated at 500 mg/kg bw/d. At the end of the treatment-free period, there were kidney and adrenal weight differences while there were no thymus or liver weights differences considered to be related to the test item administration. There were no test item-related gross findings while there were microscopic non-adverse findings in the liver (in males only), mesenteric lymph nodes, mesenteric artery, thyroid glands, heart (in males only), adrenals (in females only), kidneys, jejunum, sternum, bone marrow, and ovaries. These observations were considered to be non-adverse and suggested an incomplete reversibility of test item-related changes.

 At 120 mg/kg bw/d, the test item induced lower body weight and food consumption in males and slight changes in blood biochemistry parameters in both sexes. There were non-adverse findings in the liver, adrenals, kidneys, jejunum, GALT, prostate and seminal vesicles and sternum. 

 At 30 mg/kg bw/d, the test item induced lower body weight in males and slight increase in cholesterol level in both sexes. There were non-adverse findings in the liver, prostate and seminal vesicles.

 

Consequently, under the experimental conditions of the study, based on adverse effect observed at 500 mg/kg bw/d in males, the No Observed Adverse Effect Level (NOAEL) after the 90-day treatment period was established at 120 mg/kg bw/d in males and at 500 mg/kg bw/d in female.

 

3.   Endocrine Disruption

An independent scientific review was commissioned to identify evidences of endocrine disruption properties (as defined by the World Health Organization) for BPA PO, based on the studies performed on BPA 2PO and the various grades of the registered substance. The report is attached in Section 13 of the Dossier and a summary provided as part of the endpoint summary for the toxicity to reproduction.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

11 April 2011-21 June 2011

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA, Health Effects Test Guidelines OPPTS 870.3650, Combined Respeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 421

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Crl:WI(Han)

Sex:

male/female

Details on test animals or test system 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 shorlty 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 oral 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
- Dose volume: 5 mL/kg body weight. 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.

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). Females 44 (Group 1) an 68 (Group 3) were not dosed during littering.

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 scheduled necropsy.

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.

-Selection of animals for selected measurements: 5 animals/sex/group* were randomly selected at allocation for functional observations, clinical pathology, macroscopic examination (full list) organ weights (full list) and histopathology. Only females with live offspring were selected.

*With the exception of Group 4, where due to mortality only three females were available for functional observation and locomotor activity. One of these females (no. 75) had a total litter loss before the other measurements were performed, and thus had to be euthanized prior to her scheduled necropsy. As such, data from only two females are available for clinical pathology, macroscopic examination, organ weights and histopathology.

Positive control:

No.

Observations and examinations performed and frequency:

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: Yes
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).

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
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.

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 (Selected 5 animals/sex/group (except for Group 4, where only 2 females could be selected) 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, Seminal 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 testes were processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin (Klinipath, Duiven, The Netherlands).

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 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 Group 4; males that failed to sire and females Group 4; females that failed to deliver healthy pups, were examined.
* Reproductive organs included the cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, and vagina.

Inadvertently, 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 performed 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.

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.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

effects observed, treatment-related

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

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

Histopathological findings: neoplastic:

no effects observed

Details on results:

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 died spontaneously.

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.

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.

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 can be 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 a bit 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.

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.

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 one animal, 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.

MACROSCOPIC EXAMINATION
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.


ORGAN WEIGHTS
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.


HISTOPATHOLOGY
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.

Dose descriptor:

NOAEL

Remarks:

(Parental generation)

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 at 250 and 500 mg/kg.

Critical effects observed:

not specified

Conclusions:

In an OECD 421/422 study with rats, the parental NOAEL was set at 125 mg/kg bw/day based on effects on mortality, clinical signs, body weight, food consumption, gross pathology, organ weights and histopathology from 250 mg/kg bw/day onwards.

Executive summary:

LIN10001 4 4 Isopropylidenediphenol propoxylated 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/d.

No toxicologically significant changes were noted in functional observations, and no toxicologically relevant effects were seen in any parameter at 125 mg/kg/d

In conclusion, treatment with LIN10001 4 4 Isopropylidenediphenol propoxylated 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.

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

Parental NOAEL: 125 mg/kg/day

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

120 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Justification for classification or non-classification

There is no available evidence suggesting that the registered substance can induce significant toxicity in humans following a repeated exposure.

A reliable GLP-compliant subchronic (90-day) repeated-dose toxicity was performed in accordance with the OECD Testing Guideline 408 on Grade 4 of the registered substance. This Grade was selected based on short-term toxicity testing previously performed on BPA 2PO and Grade 5 of BPA PO. This study is deemed as acceptable for the purpose of the classification of the registered substance for toxicity following a repeated exposure, in accordance with Regulation (EC) N°1272/2008.

Based on adverse effect observed at 500 mg/kg bw/d in males, the No Observed Adverse Effect Level (NOAEL) after the 90-day treatment period was established at 120 mg/kg bw/d in males and at 500 mg/kg bw/d in female. Therefore the substance does not require a classification as toxic following a repeated exposure according to the criteria laid down in Section 3.9.2.9 of Regulation (EC) N°1272/2008.