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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 November 2013 to 26 February 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
September 1998
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Version / remarks:
August 1998
Deviations:
no
Qualifier:
according to
Guideline:
other: 4. Japanese Chemical Substances Control Law 1973, Notification of Mar. 31 2012 by MHLW (0331 No.7), METI (No. 5) and MOE (No. 110331009).
Version / remarks:
March 2012
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
Batch number: A1314401
CAS number : 116-37-0
EC number : 204-137-9

To the best of knowledge, the sample used are representative to the boundary composition shared and agreed by each registrant.

Test animals

Species:
rat
Strain:
other: Wistar (Han)
Sex:
male/female
Details on test animals and environmental conditions:
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Young adult animals (approx. 6 weeks old).
- Weight at study initiation: Body weight variation was within +/- 20% of the sex mean (males: 153 grams; females: 130 grams).
- Housing: Group housing of 5 animals per cage in labeled Macrolon cages.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days.

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

IN-LIFE DATES: From: 26 November 2013 to 26 February 2014

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing, and were homogenized to visually acceptable levels. Adjustment was made for specific gravity of the vehicle. No correction was made for the purity of the test substance.

VEHICLE
- Justification for use and choice of vehicle: The same vehicle was used in the Combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test of LIN10001 4, 4’- Isopropylidenediphenol propoxylated (BPA+2PO) in rats by oral gavage (project 496519).

DOSE VOLUME:
5 ml/kg body weight. Actual dose volumes were calculated according to the latest body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted according to a validated method (project 495511). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations, in Weeks 1, 6 and 13). Stability in vehicle over 6 hours at room temperature under normal laboratory light conditions (lowest concentration) and over 8 days in the refrigerator (highest and lowest concentration) was also determined (in Week 1).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration for solutions. 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%.
Duration of treatment / exposure:
90 days
Frequency of treatment:
Once daily, 7 d/w.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 20, 60, 180 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
In a combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test of LIN10001 4, 4’- Isopropylidenediphenol propoxylated (BPA+2PO) in rats by oral gavage (project 496519), Wistar Han rats were dosed by oral gavage at 125, 250 and 500 mg/kg. 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 was 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.
Positive control:
Not required.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS:
- Time schedule: At least twice daily. The time of death was recorded as precisely as possible.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: At least once daily from start of treatment onwards, detailed clinical observations were made in all animals immediately after dosing (based on the absence of a peak effect of occurrence of clinical signs in the dose range finding study (project 496584) conducted for the Combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test range finding study (project 496519). Once prior to start of treatment and at weekly intervals, this was also performed outside the home cage in a standard arena. The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity.

BODY WEIGHT:
- Time schedule for examinations: Weekly.

FOOD CONSUMPTION:
- Time schedule for examinations: Weekly.

WATER CONSUMPTION: no

OPHTHALMOSCOPIC EXAMINATION:
- Time schedule for examinations: at pretest : all animals, at Week 13: Groups 1 and 4.

HAEMATOLOGY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: yes
- How many animals: all animals
- Parameters checked: According to test guidelines

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: yes
- How many animals: all animals
- Parameters checked: According to test guidelines

URINALYSIS: no

NEUROBEHAVIOURAL EXAMINATION:
- Time schedule for examinations: During Week 12-13 of treatment
- Dose groups that were examined: Groups 1 and 4
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex and grip strength, motor activity test.

OTHER:
Estrous cycle determination: All females had a daily lavage from 05 February 2014 (Day 72) up to and including 25 February 2014 (Day 92) to determine the stage of estrous.
Sacrifice and pathology:
GROSS PATHOLOGY:
- All animals were fasted overnight with a maximum of 24 hours prior to planned necropsy, but water was provided. Animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetised and subsequently exsanguinated.
- Dose groups that were examined: all groups
- Tissues/organs checked: According to test guidelines

ORGAN WEIGHTS:
Organs checked according to test guidelines

HISTOPATHOLOGY:
According to test guidelines
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.
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.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Based on subjective appraisal.
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
MORTALITY
One male at 180 mg/kg was found dead approximately 3 hours after dosing on Day 84, and one female at 180 mg/kg was found dead approximately 1 hour after dosing on Day 52. The female showed lethargy on the day of death, and no relevant clinical signs were shown by the male. Body weight gain was normal for these animals prior to death. At necropsy, both animals showed watery-clear fluid in the thoracic cavity. Histopathology revealed no cause of death. No further mortality occurred.

CLINICAL SIGNS
No toxicologically relevant clinical signs were recorded. One surviving female at 180 mg/kg showed hunched posture during the second half of the treatment period. As this clinical sign was not shown by other animals of the same dose group, this was considered to be of no toxicological relevance. Salivation seen after dosing among animals at 20, 60 and 180 mg/kg was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign may be related to taste of the test substance. Other incidental findings that were noted included scabs, rales, alopecia, chromodacryorrhoea and thickening of the genital region. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological significance.

BODY WEIGHT AND WEIGHT GAIN
Males at 180 mg/kg showed an apparent slightly lower body weight (gain) over the treatment period, being statistically significant for body weight gain in Week 2 only. Body weight (gain) among other male and female dose groups was similar to control levels.

FOOD CONSUMPTION
Food consumption before or after correction for body weight remained similar to the control level over the study period.

OPHTHALMOSCOPIC EXAMINATION
No toxicologically significant ophthalmology findings were noted. Incidental ophthalmology findings at pretest and/or in Week 13 consisted of (focal) corneal oedema or opacity, haemorrhage from the hyaloid vessel, pinpoint corneal opacities, and haemorrhagic discharge. The nature and incidence of these findings were within the range considered to be normal for rats of this age and strain.

HAEMATOLOGY
The following statistically significant changes in haematology parameters distinguished treated animals from control animals:
- Lower red blood cell counts in males and females at 180 mg/kg, and in females also at 60 mg/kg,
- Lower haemoglobin level in males and females at 180 mg/kg, and in females also at 60 mg/kg,
- Lower haematocrit level in females at 60 and 180 mg/kg,
- Higher mean corpuscular volume (MCV) in males at 180 mg/kg,
- Higher mean corpuscular haemoglobin (MCH) level in males at 180 mg/kg.
Means of the above parameters remained within the range considered normal for rats of this age and strain, except for the higher mean corpuscular volume which only slightly exceeded this normal range.
Any other statistically significant changes in haematology parameters were considered to be of no toxicological significance as they occurred in the absence of a dose-related trend and remained within the range considered normal for rats of this age and strain. The statistically significantly lower Prothrombin time (PT) of males at 60 and 180 mg/kg was considered to be of no toxicological relevance since the opposite effect (i.e. an increase) would be expected in case of target organ toxicity.

CLINICAL CHEMISTRY
The following statistically significant changes in clinical biochemistry parameters distinguished treated animals from control animals:
- Higher alanine aminotransferase activity (ALAT) in males at 180 mg/kg,
- Higher urea level in females at 60 and 180 mg/kg,
- Higher cholesterol level in males and females at 180 mg/kg,
- Lower potassium level in males at 60 and 180 mg/kg,
- Higher calcium level in females at 180 mg/kg,
- Higher inorganic phosphate level in males at 180 mg/kg.
Only cholesterol levels of males clearly exceeded the range considered normal for rats of this age and strain.
Any other statistically significant changes in clinical biochemistry parameters were considered to be of no toxicological significance as they occurred in the absence of a dose-related trend and remained within the range considered normal for rats of this age and strain.

NEUROBEHAVIOUR
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals. Motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with high activity in the first interval that decreased over the duration of the test period.

ORGAN WEIGHTS
The following (statistically significant) changes in absolute organ weights and relative organ weights (organ to body weight ratio) were considered to be related to treatment:
- Higher liver to body weight ratio in males at 180 mg/kg (relative weight approximately 21% higher than controls; absolute liver weight also slightly higher but not statistically significant),
- Higher kidney to body weight ratio in males at 180 mg/kg (relative weight approximately 13% higher than controls; absolute kidney weight also slightly higher but not statistically significant),
- Higher adrenal to body weight ratio in males at 180 mg/kg (relative weight approximately 25% higher than controls)
- Lower heart to body weight ratio in females at 180 mg/kg (relative weight approximately 10% lower than controls),
- Trend towards higher ovary weight among female dose groups (relative weight at 180 mg/kg approximately 24% higher than controls).
The statistically significantly higher prostate weight at 60 mg/kg was considered to be of no toxicological significance as this change occurred in the absence of a dose-related trend and remained within the range considered normal for rats of this age and strain.

GROSS PATHOLOGY
Necropsy did not reveal any toxicologically relevant alterations. The incidence of necropsy findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, did not show a dose-related incidence trend and/or had no treatment-related histopathological correlates. These necropsy findings were therefore considered to be of no toxicological relevance.

HISTOPATHOLOGY
The following histopathological changes were considered to be related to treatment:
- Interstitial cell hypertrophy in the interstitial gland of the ovaries in 6/10 females at 60 mg/kg (6, minimal) and in 7/10 females at 180 mg/kg (5 minimal, 2 slight).
- Centrilobular hepatocellular hypertrophy of the liver in 7/10 males (including the unscheduled death at Day 84) at 180 mg/kg (4 minimal, 3 slight).
Spermatogenic staging profiles were normal for all animals assessed. All other microscopic findings were within the range of background pathology encountered in Wistar rats of this age and strain and occurred at similar incidences and severity in both control and treated rats.

OTHER FINDINGS
Estrous cycle determination: Estrous cycle length was normal for all examined females at 20, 60 and 180 mg/kg. One control female showed an extended estrus cycle length, the incidence of which is within the range considered normal for rats of this age and strain.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
>= 180 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Accuracy of preparation: The concentrations analysed in 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 Group 1 formulations.

Homogeneity: Formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation = 10%).

Stability: The Group 2 formulation was stable when stored at room temperature under normal laboratory light conditions for at least 6 hours. Formulations of Group 2 and Group 4 were stable when stored in a refrigerator for at least 8 days.

Applicant's summary and conclusion

Conclusions:
In this oecd408 study with rats, the NOAEL was determined to be at least 180 mg/kg bw/day.

Executive summary:

An OECD 408 study (Sub-chronic toxicity study (90-day), oral route (gavage) in rats) was performed with the 4,4’-Isopropylidenediphenol, propoxylated (BPA+2PO) at the dose-levels of 0, 20, 60 and 180 mg/kg bw/day with approval of ECHA for the 2010 Reach registration dossier. The test substance, formulated in polyethylene glycol 400, was administered daily for at least 90 days by oral gavage to SPF-bred Wistar rats. One control group and three treated groups were tested, each consisting of 10 males and 10 females. Chemical analyses of formulations preparations were conducted during the study to assess accuracy,

homogeneity and stability over 6 hours. The following parameters were evaluated: clinical signs daily; functional observation tests in Week 12; body weight and food consumption weekly; ophthalmoscopy at pretest and in Week 13; estrous cycle determination; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues including spermatogenic staging profiles assessment.

 

Formulation analyses confirmed that formulations of test substance in polyethylene glycol 400 were prepared accurately and homogenously, and were stable over at least 6 hours.

Two animals given180 mg/kg (one male and one female) were found dead between after dosing on Day 84 and 52, respectively. Histopathology revealed no cause of death but the presence of watery-clear fluid in the thoracic cavity in both of these animals suggest that these deaths were due to a gavage incident.

A number of changes in red blood cell parameters were noted in both sexes at 60 and 180 mg/kg, including lower red blood cell counts, lower haemoglobin and haematocrit level, and higher mean corpuscular volume and mean corpuscular haemoglobin. Means remained essentially within the range considered normal for rats of this age and strain, and no morphological correlates indicative of increased breakdown or production of red blood cells were noted. Therefore, these haematological changes were considered not indicative of an adverse effect on red blood cell turn over. The higher liver to body weight ratio in males at 180 mg/kg corresponded with the centrilobular hepatocellular hypertrophy of the liver observed in most at 180 mg/kg (up to slight degree). Given the low severity of liver hypertrophy, absence of morphological liver findings, and only slight potentially supportive clinical biochemistry changes (higher cholesterol and higher alanine aminotransferase activity in males), these findings were considered adaptive and not adverse in nature. The higher cholesterol levels of females at 180 mg/kg occurred without any apparent morphological correlates.

Other clinical biochemistry changes at 180 mg/kg included lower potassium level (males), higher urea and calcium level (females), and higher inorganic phosphate level (males). At 60 mg/kg, clinical biochemistry changes were confined to lower potassium level (males) and higher urea level (females). As these changes were slight in nature and also occurred in the absence of any supportive morphological changes, these were considered not to be adverse in nature.

Reproductive parameters assessed in this study revealed no adverse changes. The trend towards higher ovary weight among female dose groups was considered to be related to the interstitial cell hypertrophy in the interstitial gland of the ovaries in most females at 60 and 180 mg/kg (up to slight degree). Ovaries are steroid synthezing organs and play a major role in the endocrine system and reproductive system. The main precursor for the production of all steroid hormone synthesis is cholesterol (or cholesterylesters), that can be stored intracellular as lipid droplets in the ovary. The histologic morphology of the recorded interstitial cell hypertrophy, with fine vacuolar appearance is suggestive for an exaggerated lipid storage which can lead to increased organ weight. Since estrous cycle length was normal for all examined females at 20, 60 and 180 mg/kg and the interstitial gland hypertrophy was of a low severity, this finding was considered to be non-adverse.

Spermatogenic staging profiles were normal for all animals assessed.

No histopathological correlates were found for the higher adrenal and kidney to body weight ratio in males and lower heart weight in females at 180 mg/kg. Therefore, these changes were considered not to be of toxicological relevance.

No relevant clinical signs were noted during treatment. Functional observation tests revealed no abnormalities, and ophthalmoscopy showed no treatment-related changes. The slightly lower body weight gain of males at 180 mg/kg was only minor in nature and well within the range considered normal for rats of this age and strain, and was therefore considered to be of no toxicological relevance.

No changes in food intake occurred.

From the results presented above, a No Observed Adverse Effect Level (NOAEL) for 4,4’-Isopropylidenediphenol, propoxylated (BPA+2PO) of at least 180 mg/kg was established.