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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:
18 November 2016 to 30 August 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study performed according to the OECD Test Guideline No. 408.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
21 Nov 2016 - 12 December 2016
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This 14-days Dose Range Finding (DRF) phase was to select dose levels for the 13-week main study phase
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 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
This study was a 14-days Dose Range Finding (DRF) assay conducted in order to select dose levels for the main study.
Principles of method if other than guideline:
The test item 4,4'-biphenyldiol, was administered by daily gavage at dose levels of 20, 60 and 150 mg/kg/day to groups of 3 or 6 Wistar rats for 14 days.
Morbidity/mortality, clinical signs, body weight and food consumption were monitored.
All animals were killed at the end of the treatment period and necropsied. The kidneys and liver were weighed, were fixed and preserved at necropsy for all animals. Kidneys and liver of all animals were examined histopathologically.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
> See the main final 13-week Toxicity study.
> Rationale for species selection: one of the rodent species acceptable to the regulatory agencies.
> Historical control data for the strain are available at the Test Facility.
Sex:
male/female
Details on test animals or test system and environmental conditions:
See the main final 13-week Toxicity study.
Route of administration:
oral: gavage
Details on route of administration:
See the main final 13-week Toxicity study.
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% [w/v] Carboxymethylcellulose 300-600 centipoises in water for injection.
Details on oral exposure:
> See the main final 13-week Toxicity study.
> Frequency of formulation preparation: at least weekly.
> Formulation concentrations: 20, 60 and 150 mg/mL.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
> Duration of the Dosing Period: 14 days.
Frequency of treatment:
Treatment once daily.
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Group 1: Low dose
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Group 2: Intermediate dose
Dose / conc.:
750 mg/kg bw/day (nominal)
Remarks:
Group 3: High dose
No. of animals per sex per dose:
group 1: Females x 3
group 2: Females x 3
group 3: Males x 3 and Females x 3
Control animals:
no
Details on study design:
Rationale for dose selection: the doses have been selected based on the information provided by the Sponsor on preliminary and "equivalent or similar to" OECD422 studies.
In the preliminary study, oral administration of the test item for 18 days in male and female rats at 1000 mg/kg/day induced suppression of body weight gain, increased liver and kidney weight and premature death of the animals. In the "equivalent or similar to" OECD422 study, oral administration (gavage) of the test item to rats at 8, 40 and 200 mg/kg/day was only associated with calcium oxalate-like urinary crystal sediment (probably attributable to precipitation of the test item) at 40 mg/kg/day in females only and at 200 mg/kg/day in both
sexes. At necropsy, the following hepatic changes were also noted at 200 mg/kg/day in males only: increased relative weight, darkening, enlarged appearance histopathologically
associated with centrilobular hepatocyte hypertrophy and reduction in occurrence of periportal fatty change.
Consequently, in the current DRF study, the high dose of 750 mg/kg/day is selected in order to elicit adverse clinical signs with limited risk of premature death. The lower doses of 100 and 300 mg/kg/day are selected to further characterise dose-relationships for any potential test item-related effects in the rat. Only females will be administered at all dose levels, assuming they are generally more sensitive than males to toxicity-related clinical signs.
Nevertheless, males will also be administered at the highest dose, taking into account the hepatic effects noted only in males at the high dose level in the previous "equivalent or similar to" OECD422 study.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
All animals gained weight throughout the dosing period and, at study termination, all body weight gains were considered satisfactory.
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Minor and dose-unrelated intergroup differences in liver and kidney weights in females were observed and were considered to be unrelated to treatment.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At necropsy of the 750-mg/kg/day groups, the surface of the kidneys was mottled in 3/3 males and pale foci were observed in the kidneys from 1/3 female. These macroscopic abnormalities correlated with the test item-related bilateral retrograde nephropathy.
There were no test item-related gross observations in females at 100 or 300 mg/kg/day.

No macroscopic abnormalities were observed in the liver.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
At 750 mg/kg/day, test item-related bilateral retrograde nephropathy, generally associated with tubular casts, was observed in the kidneys from 3/3 males and 2/3 females, with a greater severity in males (moderate or marked versus minimal or slight in females).
> The three males given 750 mg/kg/day had moderate or marked bilateral retrograde nephropathy characterised by multifocal tubular dilatation and basophilia in the papilla, medulla and cortex, accompanied by inflammatory cell infiltration (mainly acute type) and variable degrees of interstitial fibrosis. Tubular cell degeneration may also be observed as scattered foci.
> In females given 750 mg/kg/day, the incidence and severity of the renal findings were lower than in males. Minimal or slight retrograde nephropathy was observed in two females. No treatment-related histopathological renal changes were
observed in the third female.
> Tubular casts with presence of eosinophilic radiating material were also noted in dilated tubules in the papilla in both sexes.

There were no test item-related histologic changes in females at 100 or 300 mg/kg/day.

No treatment-related changes were observed in the liver in both sexes.
Key result
Dose descriptor:
other: Maximum Tolerated Dose (MTD)
Effect level:
< 750 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
other: Maximum Tolerated Dose (MTD)
Effect level:
> 750 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No test item related effects considered adverse in females
Conclusions:
Under the defined experimental conditions, the test item 4,4'-biphenyldiol was daily administered by oral route to Wistar rats at doses of 100 and 300 mg/kg/day in females and 750 mg/kg/day in both sexes, for 14 days. There were no test item-related antemortem changes. Histopathological examination showed test item-related retrograde nephropathy in both sexes at 750 mg/kg/day; based on the incidence and severity of this finding, it was considered adverse in males only. Therefore, the MTD in the present DRF Phase was <750 mg/kg/day in males and >750 mg/kg/day in females.
Executive summary:

The test item 4,4'-biphenyldiol, was administered by daily gavage at dose levels of 100, 300 and 750 mg/kg/day to groups of 3 or 6 Wistar rats for 14 days. the doses were selected based on the data of an OECD 422 study (Japanese NIHS). Parameters monitored included morbidity/mortality, clinical signs, body weight and food consumption. All animals were killed at the end of the treatment period and necropsied. The kidneys and liver were weighed, were fixed and preserved at necropsy for all animals. Kidneys and liver of all animals were examined histopathologically. The test item 4,4'-biphenyldiol did not induce antemortem changes at all dose levels. Histopathological examination showed test item-related retrograde nephropathy in both sexes at 750 mg/kg/day; this effect was considered adverse in males only, based on the incidence and severity of this finding.

Therefore, the Maximum Tolerated Dose (MTD) in the present DRF phase was < 750 mg/kg/day in males and > 750 mg/kg/day in females.

Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
03 March 2017 - 07 April 2017
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This 28-days Dose Range Finding (DRF) phase was to select dose levels for the 13-week main study phase.
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 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
This study was a 28-days Dose Range Finding (DRF) assay conducted in order to select dose levels for the main study.
Principles of method if other than guideline:
The test item 4,4'-biphenyldiol, was administered by daily gavage at dose levels of 30, 125 and 500 mg/kg/day to groups of 10 Wistar rats (5 males and 5 females per group) for 28 days.
Morbidity/mortality, clinical signs, body weight and food consumption were monitored.
Blood sampling for clinical laboratory determinations and urine collection were performed after Week 4.
All animals were killed at the end of the treatment period and necropsied. Selected organs were weighed. Organ/tissue samples were fixed and preserved at necropsy for all animals.
Selected organs/tissues from group 1 and 4 animals killed at the end of the treatment period were examined histopathologically.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
> See the main final 13-week Toxicity study.
> Rationale for species selection: one of the rodent species acceptable to the regulatory agencies.
> Historical control data for the strain are available at the Test Facility.
Sex:
male/female
Details on test animals or test system and environmental conditions:
See the main final 13-week Toxicity study.
Route of administration:
oral: gavage
Details on route of administration:
See the main final 13-week Toxicity study.
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% [w/v] Carboxymethylcellulose 300-600 centipoises in water for injection.
Details on oral exposure:
> See the main final 13-week Toxicity study.
> Frequency of formulation preparation: at least weekly.
> Formulation concentrations: 6, 25 and 100 mg/mL.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
Duration of the Dosing Period: 28 days.
Frequency of treatment:
Treatment: Once daily.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1: Control
Dose / conc.:
30 mg/kg bw/day (nominal)
Remarks:
Group 2: Low dose
Dose / conc.:
125 mg/kg bw/day (nominal)
Remarks:
Group 3: Intermediate dose
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
Group 4: High dose
No. of animals per sex per dose:
5 animals/group/sex
Control animals:
yes, concurrent vehicle
Details on study design:
Rationale for dose selection: In the first 14-days DRF phase, there were no clinical signs noted after a 14 day-administration period at 100, 300 and 750 mg/kg/day in females and at 750 mg/kg/day in males. However, at 750 mg/kg/day, there were treatment-related kidney changes in both sex, similar to those observed in pregnant female rats given 500 or 750 mg/kg/day which were sacrificed prematurely due to poor health in the dose-range finding study (CRL Reference no. AB21564).).
Consequently, for the second 28-days DRF phase, the doses of 30, 150 and 500 mg/kg/day were selected in order to define dose-relationships for any potential test item-related effects in the rat of both sexes after a 28-day treatment period.
The objective was to select to better select the dose levels of the main 13-week study.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Slight piloerection was noted in female no. 59 given 500 mg/kg/day on Day 4 +3h post dose.
Since this was observed in only one animal and at only one occasion, it was considered incidental.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A lower mean body weight gain was noted after 28 days in males given 500 mg/kg/day (-8% compared to controls) and in females given 125 and 500 mg/kg/day (respectively -17% and -17% compared to controls).
Owing to their low magnitude, these variations were considered non adverse.
Food consumption and compound intake (if feeding study):
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
No test item related effects on hematology and coagulation parameters were noted at 30 and 125 mg/kg/day.
At 500 mg/kg/day, mean activated partial thromboplastin time (APTT) was respectively +15% and +13% longer than controls in males and females. In males only, mean prothrombin time was +13% longer than controls. In females only, mean fibrinogen concentration was +16% higher than controls.
A test item-related effect could not be excluded. However, these variations were considered non adverse since values remained within or close to background
data and had no histological correlate.
All other variations in hematology and coagulation parameters, including those that were statistically significant, were of low magnitude and were therefore considered not to be toxicologically relevant.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
No test item related effects on serum clinical chemistry parameters were noted at 30 and 125 mg/kg/day.
At 500 mg/kg/day, mean cholesterol concentration was -43 % lower than controls in males only, below the background control data range. In females only, a slightly elevated chloride concentration was noted (+3% compared to controls), above the background control data range, and alkaline phosphatase activity was 1.8-fold higher than in controls, within background data.
Although the above-mentioned variations were noted in only one sex, a test item-related effect could not be excluded. However, they were not considered adverse.
All other variations in serum clinical chemistry parameters, including those that were statistically significant, were of low magnitude and were therefore considered not to be toxicologically relevant.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
In males only, a lower pH was noted at all dose levels compared to controls, more marked at 500 mg/kg/day. A slightly higher mean specific gravity was noted at all dose levels, more marked and associated with a lower mean urine volume at 500 mg/kg/day. All urinalysis variations remained within background data, had no histological correlate and were therefore considered non adverse.
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Key result
Dose descriptor:
other: Highest dose suitable for the main 13-week study.
Effect level:
ca. 500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Conclusions:
Under the defined experimental conditions, a daily oral administration of the test item 4,4'-biphenyldiol to Wistar rats at doses of 30, 125 and 500 mg/kg/day for 28 days did not induce any adverse findings. Test item related effects consisted of lower body weight gain at 125 mg/kg in females only and 500 mg/kg in both sexes, slight hematology and clinical pathology changes at 500 mg/kg/day only and of urinalysis changes from 30 mg/kg/day.
There were no treatment-related macroscopic, organ weight or histopathological changes at any dose level.
Based on these findings, and because in the first 14-days DRF phase, the dose level of 750 mg/kg/day induced adverse effects on kidneys, which would be likely to lead to premature death in a long-term study, the dose level of 500 mg/kg/day is considered as a suitable highest dose in the main 13-week study.
Executive summary:

The test item 4,4'-biphenyldiol, was administered by daily gavage at dose levels of 30, 125 and 500 mg/kg/day to groups of 10 Wistar rats (5 males and 5 females per group) for 28 days. Parameters monitored included morbidity/mortality, clinical signs, body weight and food consumption. Blood sampling for clinical laboratory determinations and urine collection were performed after Week 4. All animals were killed at the end of the treatment period and necropsied. Selected organs were weighed. Organ/tissue samples were fixed and preserved at necropsy for all animals. Selected organs/tissues from group 1 and 4 animals killed at the end of the treatment period were examined histopathologically.

The test item 4,4'-biphenyldiol did not induce any adverse findings at all dose levels. Test item related effects consisted of lower body weight gain at 125 mg/kg in females only and 500 mg/kg in both sexes, slight hematology and clinical pathology changes at 500 mg/kg/day only and of urinalysis changes from 30 mg/kg/day. There were no treatment-related macroscopic, organ weight or histopathological changes at any dose level.

Based on these findings, and because in the first 14-days DRF phase, the dose level of 750 mg/kg/day induced adverse effects on kidneys, which would be likely to lead to premature death in a longterm study, the dose level of 500 mg/kg/day was considered as a suitable highest dose in the main 13-week study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
September 1998
Qualifier:
according to guideline
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
May 2008
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Version / remarks:
August 1998
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
4,4'-biphenyldiol
Cas Number:
92-88-6
IUPAC Name:
4,4'-biphenyldiol
Test material form:
solid: crystalline
Details on test material:
Biphenyl-4,4’-diol
CAS 92-88-6

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Rat Wistar: Crl: WI (Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Supplier: Charles River Laboratories France, Domaine des Oncins, 69210 Saint-Germain-Nuelles, France.
- Age at study initiation: approximately 6 weeks.
- Weight at study initiation:
males: 151.7 to 191.2 g
females: 141.2 to 189.0 g.
- Housing: One air-conditioned room in a barrier protected unit. Animals were housed in groups of 5 of the same sex and dose group in polycarbonate cages with sawdust bedding, in compliance with European Regulations (Directive 2010/63/EU).
- Diet: ad libitum
- Water: ad libitum
- Acclimatisation period: 7 days between animal arrival and the start of treatment.

DETAILS OF FOOD AND WATER QUALITY:
- Food: complete diet sterilised by irradiation and analysed for a predefined list of chemical and bacteriological contaminants.
Each batch of diet is supplied with a certificate of analysis which is verified and authorized for release by a veterinarian.
- Water: softened and filtered (0.2 µm) mains drinking water.
Water is analysed twice a year for chemical and bacterial contaminants by Laboratoire Santé Environnement Hygiène de Lyon, France.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 °C (target range)
- Humidity (%): 35 to 70 % (target range)
- Air changes (per hr): At least 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light (artificial)/12 hours dark (except when required for technical acts).

IN-LIFE DATES: From: 30 August 2017 (Day 1 of treatment) To: 30 November 2017 (Last necropsy)

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The oral route was selected as this is a potential route of human exposure during manufacture, handling or use of the test substance as specified in the applicable guidelines.
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5 % [w/v] Carboxymethylcellulose 300-600 centipoises in water for injection
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
- The test item was prepared as a suspension in the vehicle at concentrations of 6, 25 and 100 mg/mL
- Rate of preparation: At least weekly
- Storage of formulations: refrigerated (between +2 and +8 °C)
- Stability of the test item in the vehicle: 11 days at room temperature (between +15 and +25 °C) or refrigerated (between +2 and +8 °C), and 26 days when stored frozen (between -15 and -25 °C)
- Homogeneity of the test item in the vehicle: homogeneity at 1 and 200 mg/mL was validated

- VEHICLE
- Vehicle: 0.5% [w/v] Carboxymethylcellulose 300-600 centipoises in water for injection.
- Concentration in vehicle: 6, 25 and 100 mg/mL

- ADMINISTRATION
- Volume of administration: 5 mL/kg/day
- Individual dose volumes were adjusted weekly using the latest body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test item 4,4'-biphenyldiol was quantified in dosing formulations generated during the study.
This assay was performed by UPLC-UV according to a validated analytical method in a Test site. This method was specific, linear in the calibration range, precise and accurate.
Concentrations of 4,4'-biphenyldiol were determined in dosing formulations from four validated series of analysis. They were validated by the assay of quality control samples dispatched throughout the formulation samples.

> Preparation of 4,4'-biphenyldiol stock solutions, working solurions and calibration standards:
- The test item was accurately weighed and dissolved into a flask by hand shaking in methanol/isopropanol (4/1). The solution was sonicated for 2 minutes and the volume was adjusted to 5.00 mL with methanol/isopropanol (4/1) giving a parent stock solution at 1000 µg/mL. The solution was then transferred in an amber glass vial and stored protected from light for 8 days at 5±4°C.
- A 200 µg/mL working solution was prepared daily by dilution of the stock solution in methanol/isopropanol (4/1).
- The calibration standards and the quality control samples were prepared daily by dilution of a 200 µg/mL working solution in methanol/isopropanol (4/1). Then they were mixed for 10 sec using a vortex mixer, transferred in vials and centrifuged for 3 min at 4000 rpm and 4°C.

> Preparation and analysis of the study samples:
- 4 samples of 1 g each were taken from each formulation including the control, used on the first day of treatment, on a suitable day during Week 4 and during the last week of treatment. The samples were stored frozen (between -15 and -25 °C) before shipment to the Test Site. One set of samples was dispatched to the Test Site for analysis. They were stored and analysed within the stability period as defined in the validation study.
- Results of achieved concentrations should be within the range of ±15 %.
- Before analysis, the frozen study samples were left to thaw at room temperature and were diluted in methanol/isopropanol (4/1). Each diluted sample was mixed for 10 sec, transferred in a vial and centrifuged for 3 min at 4000 rpm and 4°C.
Duration of treatment / exposure:
At least 91 days.
The first day of dosing is designated as Day 1. At the end of the treatment period, the animals were sacrificed on the day after the last day of treatment.
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 (Control vehicle) - Formulation concentration: 0 mg/mL
Dose / conc.:
30 mg/kg bw/day (nominal)
Remarks:
Group 2 (Low dose) - Formulation concentration: 6 mg/mL
Dose / conc.:
125 mg/kg bw/day (nominal)
Remarks:
Group 3 (Intermediate dose) - Formulation concentration: 25 mg/mL
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
Group 4 (High dose) - Formulation concentration: 100 mg/mL
No. of animals per sex per dose:
10 per sex and per group
Control animals:
yes, concurrent vehicle
Details on study design:
Additional tests were performed, which are not part of recommended tests in the OCDE 408 guideline.
These tests include oestrus cycle determination and sperm analysis:
> Daily vaginal smears were sampled from all females during the last 4 weeks of treatment and the day of necropsy to determine Oestrus cycles.
> Sperm analysis was performed at necropsy in males from groups 1 and 4.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed at least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals were observed daily during the study.
To detect any clinical signs or reactions to treatment, the animals were observed before and at least once after dosing.
A full clinical examination was performed weekly out of the cage.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed at the time of randomisation, prior to the first dosing and then once weekly.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption was measured weekly for each cage of animals during the treatment period and reported in g/animal/day.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: All animals pretest, all animals in groups 1 and 4 during Week 13 (Day 86).

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: All animals, on Day 92/93 for males/females
- Anaesthetic used for blood collection: Blood was withdrawn from the retro-orbital sinus under isoflurane anaesthesia
- Animals fasted: Yes (animals fasted overnight)
- Standard haematology, coagulation and clinical chemistry parameters were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: All animals, on Day 92/93 for males/females from animals fasted overnight.
- Metabolism cages used for collection of urine: Yes (Urine was collected in individual metabolism cages for approximately 16 hours from animals deprived of food and water but receiving 20 mL/kg of tap water by gavage before the beginning of the collection period).
- Standard parameters were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: On Days 91/92 for males/females.
- Dose groups that were examined: 5 first animals/sex/group
- Battery of functions tested: auditory reflex / pupillary reflex / righting reflex / fore- and hind-limb grip strenght / locomotor activity in an open field test

IMMUNOLOGY: No

ESTROUS CYCLES: Yes
- Animals examined: All females
- Frequency:
Daily vaginal smears were performed to determine the stage of oestrus during the last 4 weeks of treatment.
On the day of scheduled necropsy, a vaginal smear was taken to determine the stage of the oestrous cycle and allow correlation with histopathology of female reproductive organs.

SPERM ANALYSIS: Yes
- Animals examined and frequency: All animals in groups 1 and 4 at necropsy.
- Sperm analysis was performed using automated equipment (Hamilton Thorne Research IVOS). The left cauda epididymis was sampled and used for the assessment of caudal sperm reserves, sperm motility and progressive motility. Sperm counts were performed using the left testis following removal of the tunica albuginea.
- Sperm morphology was evaluated (at least 200 spermatozoids per sample, where possible). The numbers of spermatozoids with each type of abnormality was recorded.
- In the case of an abnormality of the left epididymis or testis, the right organs may be used for sperm analysis.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
The following parameters were analysed statistically on each occasion for males and females separately:
body weights and body weight gains
haematology, coagulation and serum clinical chemistry parameters, urine volume, specific gravity and urine pH
terminal body weights, absolute and relative organ weights.
Statistical analyses were performed by the Provantis data acquisition system as follows:
The best transformation for the data (none, log or rank) was determined depending upon the kurtosis of the data, the probability of the Bartlett's test for homogeneity of the variances and the similarity of the group sizes. Non- or log-transformed data were analysed by parametric methods. Rank transformed data were analysed using non-parametric methods. The homogeneity of means was assessed by analysis of variance (ANOVA).
Data were then analysed to test for a dose-related trend to detect the lowest dose at which there was a significant effect, based on the Williams test for parametric data or Shirley's test for non-parametric data.
If no trend was found but the means were not homogeneous, the data were analysed by a stepwise parametric or non-parametric Dunnett's test to look for significant differences from the control group.
Data from functional test (fore- and hind-limb grip strength and locomotor activity in an open field test) and from sperm analysis was analysed using a SAS software package. Levene’s test was used to test the equality of variance across groups and Shapiro-Wilk's test was used to assess the normality of the data distribution in each group. Data with homogeneous variances and normal distribution in all groups were analysed using Anova followed by Dunnett’s test. Data showing non-homogeneous variances or a non-normal distribution in at least one group will be analysed using Kruskal-Wallis test followed by the Wilcoxon's rank sum test.
Microsoft Excel® 2003 was employed to present certain results and perform associated calculations.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Observed clinical signs consisted of chromodacryorrhea in one group 4 male from Day 68 to Day 92 and in one group 2 female from Day 68 to Day 69 and from Day 91 to Day 93, soft distended abdomen in group 3 female from Day 41 to Day 42, slight piloerection in two group 4 females on a single occasion (Day 88), exophthalmos and tilted head in a group 3 male on a single occasion (D51). They were considered incidental since they were only observed sporadically in very few animals. Scabs, sores and localised hairloss were noted in treated and control animals and were also considered incidental.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
An overall lower mean body weight gain was noted in rats given the highest dose (500 mg/kg/day) compared to controls (-13 % and -10 % in males and females, respectively), without obvious impact on mean body weight. At the end of the treatment period, mean body weight from group 4 males and females was -4 % lower than controls.
Food consumption and compound intake (if feeding study):
no effects observed
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The ophthalmological findings observed in Week 13 consisted of corneal opacities noted as punctiform or linear and persistent pupillary membrane. They were considered to be incidental since they were already noted pretest and/or in the control group. Moreover, all these ocular findings are known to be observed in the laboratory rat [1] as congenital, spontaneous or common changes, and can evolve with time.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
There were no test item related variations in haematology parameters in males, at any dose levels.
In females given 500 mg/kg/day, increased mean total white blood cell counts was noted compared to controls (+34 %), due to elevated mean neutrophil counts (+120 %), above historical background data. Higher mean fibrinogen concentration was also noted in females at 500 mg/kg/day (+53 %), above historical background data. The increases in neutrophils counts and fibrinogen concentration correlated with adverse retrograde nephropathy and interstitial inflammatory cell infiltration noted at the histopathological examination.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The following test item-related changes were noted:
- lower mean cholesterol concentration noted at 30, 125 and 500 mg/kg/day in males only, with dose-dependency (-11 %, -26 % and -47 % from controls, respectively). Mean values at 500 mg/kg/day were below historical background data.
- slightly higher mean inorganic phosphorus concentration in males (+9 %) and females (+27 %) given 500 mg/kg/day, above historical background data in females only.
- higher mean calcium (+5 %), urea (+22 %), triglyceride (+55 %), total protein (+6 %) and globulin (+11 %) concentrations at 500 mg/kg/day in females only.
The increases in electrolytes, urea, protein and globulin concentration, mostly seen in females at 500 mg/kg/day, correlate with histopathological findings in kidneys, considered adverse in females only.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
In treated animals, the following variations were noted at the end of the treatment period compared to controls:
- Lower mean pH in males at 500 mg/kg/day (7.50 vs 8.65 in controls) and in females at 125 and 500 mg/kg/day (respectively 6.95 and 6.10 vs 7.44 in controls).
- Minimally higher mean specific gravity in males and females from 125 mg/kg/day.
- Higher ketone content in males given 500 mg/kg/day.
- Higher turbidity and crystals content in females given 500 mg/kg/day. The nature of crystals was not determined by additional analysis.
Variations of urinalysis parameters correlate with adverse pathology findings on the kidneys.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant effects on hearing ability, pupillary reflex, static righting reflex and grip strength were observed, with the exception of a lower mean grip strength (anterior and posterior) in females given 500 mg/kg/day. Because these lower force values had no repercussions on motor activity (open field session), they were considered non adverse.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
In both sexes, the group mean absolute weight of the kidneys in all treated groups was higher than in control group. The differences from the control group were generally dose-related and were marginal, although statistically significant at the high dose in both sexes. This was considered likely related to the treatment-related histopathological changes observed in the kidney.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related macroscopic changes were observed in the kidneys in females treated at 500 mg/kg/day and consisted of bilateral renal enlargement and/or presence of pale foci.
At microscopic evaluation, this correlated with treatment-related retrograde nephropathy.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related retrograde nephropathy and tubular casts were observed in the kidneys in most females (minimal to marked) and in a few males (minimal) treated at 500 mg/kg/day. In female, theses renal changes were considered adverse in view of their severity and correlated with the enlarged appearance and/or pale foci noted macroscopically in several females.
There were no treatment-related histopathological changes in the kidneys in males and females treated at 30 or 125 mg/kg/day.
Other effects:
no effects observed
Description (incidence and severity):
- There were no test item-related effects on sperm analysis, at any dose levels. Absence of sperm or very low sperm count was noted in 2/10 animals in the control group and 1/10 animals in group 4 (500 mg/kg/day).
- There were no test item-related effects on oestrus cycles, at any dose levels.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
125 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (nominal)
System:
urinary
Organ:
kidney
Treatment related:
yes
Relevant for humans:
yes

Any other information on results incl. tables

Analysis of the dosing formulations:

- Samples from day 1: duplicate samples at 100 mg/mL were sent to Test Site and analysed because the first vial sent was broken during sample preparation at the Test Site.

- Samples from Week 13: duplicate samples at 6 and 25 mg/mL were also sent and analysed because first results of analysis were out of the acceptance criteria.

Results of the dosing formulations analysis:

- No 4,4'-biphenyldiol was quantified in the sample of vehicle.

- The concentration of 4,4'-biphenyldiol in test item formulations at 6, 25 and 100 mg/mL (corresponding to the doses of 30, 125 and 500 mg/kg/day, respectively) was comprised in the range of ±15% when compared to the nominal values for 10 of the 12 formulation samples. While all Days 1 and Week 4 results were within acceptance criteria, two formulations used on Week 13 presented a recovery lower than 85%. Indeed, week 13 formulations at 6 and 25 mg/mL showed recovery of respectively 79.5 % and 83.6 %. An investigation was performed on these “out of specification” results but no analytical issues were observed. These formulations were nevertheless controlled again at the Study Director request from duplicates formulation samples and results were confirmed.

Mean recovery for the 6 and 25 mg/mL concentration on Week 13 were respectively 78.7 and 81.2% from nominal concentrations. However, taking into account analytical results from Day 1, Week 4 and Week13, mean recovery during the overall treatment period can be considered to be 89%, 99% and 91%, within acceptance criteria of 85-115%. The deviation from nominal concentration on Week 13 was therefore considered without impact on the overall animal exposure.

Applicant's summary and conclusion

Conclusions:
The toxicity of the test item 4,4'-biphenyldiol was determinated in the rat following daily oral (gavage) administration for 13 weeks, according to the OECD 408 guidelilne.
The tested dose levels were 30, 125 and 500 mg/kg/day for 13 weeks, and were clinically well-tolerated.
At 30 and 125 mg/kg/day, test item-related findings were limited to decreased cholesterol in males only from 30 mg/kg/day, without histopathological correlate in any organs examined histologically, and decreased urine pH in females at 125 mg/kg/day.
At 500 mg/kg/day, kidney lesions were seen in most females and in a few males and were considered adverse in females in view of their severity. These kidney lesions were associated with changes in clinical haematology, serum chemistry and urine parameters, as well as with lower body weight gain.
Consequently, 125 mg/kg/day was considered to be the No Observed Adverse Effect Level (NOAEL) in this study.
Executive summary:

The objectives of the study were to determine the toxicity of the test item 4,4'-biphenyldiol, in the rat following daily oral (gavage) administration for 13 weeks, according to the OECD 408 guideline and under GLP compliance. The tested dose levels of the main study (30, 125 and 500 mg/kg/day) were selected on the basis of two dose range-finding (DRF) assays: 14 -day repeated dose toxicity study (Ducrocq, 2018) and 28 -day repeated dose toxicity study (Ducrocq, 2018)

Formulations were prepared on a weekly basis as suspension in the vehicle (0.5 % [w/v] Carboxymethylcellulose 300-600 centipoises in water for injection).

Morbidity/mortality checks were performed at least twice daily. Clinical observations were performed daily, before and after administration. A full clinical examination was performed weekly. Individual body weights were recorded weekly. Food consumption was measured weekly for each cage of animals. Ophthalmological examinations were performed pretest and during Week 13. Clinical laboratory determinations (haematology, serum clinical chemistry and urinalysis) were performed on Days 92/93 in males/females respectively. Functional tests were performed on Days 91/92 in males/females respectively.

Additional tests were performed, which are not part of recommended tests in the OCDE 408 guideline. These tests include oestrus cycle determination and sperm analysis. Daily vaginal smears were sampled from all females during the last 4 weeks of treatment and the day of necropsy to determine Oestrus cycles. Sperm analysis was performed at necropsy in males from groups 1 and 4.

All animals were sacrificed at the end of the treatment period and necropsied. Selected organs were weighed. Selected organs/tissues from group 1 (control) and 4 (high dose) animals were examined histopathologically. Target organs identified in group 4 were examined histopathologically in groups 2 and 3.

The concentrations analyzed in all the test item formulations on Day 1 and Week 4 and the high dose formulation on Week 13 were in agreement with target concentrations.

There were no test-item related clinical signs.

There were no test item-related changes in ophthalmology and food consumption.

A lower mean body weight gain was noted in males and females at 500 mg/kg/day, considered non adverse in view of the low magnitude of variations.

There were no test item-related effects on oestrus cycles, sperm analysis and functional test (with the exception of decreased in grip strength without impact on motor activity).

There were test item-related effects on haematology parameters at the end of the dosing period in females given 500 mg/kg/day only, consisting in increased mean total white blood cell and neutrophils and increased mean fibrinogen, correlated with retrograde nephropathy and interstitial inflammatory cell infiltration.

There were test item-related effects on clinical chemistry parameters at the end of the dosing period, consisting in lower mean cholesterol concentration in males from 30 mg/kg/day without histopathological correlate, increased inorganic phosphorus in males and females at 500 mg/kg/day and increased calcium, urea, triglycerides, total protein and globulin in females at 500 mg/kg/day. The increases in electrolytes, urea, protein and globulin concentrations, mostly seen in females at 500 mg/kg/day, correlated with histopathological findings in kidneys.

There were test item-related effects on urine parameters at the end of the dosing period, limited to decrease in pH noted in females at 125 mg/kg/day and in both sexes at 500 mg/kg/day, increased ketone in males at 500 mg/kg/day and increased specific gravity, turbidity and crystals content in females at 500 mg/kg/day.  

At the histopathological examination, treatment-related retrograde nephropathy and tubular casts were observed in the kidneys in most females (minimal to marked) and in a few males (minimal) treated at 500 mg/kg/day. In females, these renal changes were considered adverse in view of their severity and correlated with the enlarged appearance and/or pale foci noted macroscopically in several females. There were no test item-related effects on reproductive organs.

Under the defined experimental conditions, the No Observed Adverse Effect Level (NOAEL) of this study was considered to be 125 mg/kg/day.