Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

A screening test according to OECD 422 (repeated dose/reproductive/developmental toxicity study) in Sprague-Dawley rats exposed by oral route is available and reports a NOAEL of 40 mg/kg bw/d for general toxicity and a NOAEL of 200 mg/kg bw/d for reproductive toxicity (the highest dose level tested).

In this study, treatment up to 200 mg/kg bw/d did not affect the reproductive performance of the parental animals.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was conducted in accordance with OECD Test guideline No 422 and with the principles of GLP
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Principles of method if other than guideline:
Study was in accordance with the range-finding assay described in OECD method 422.
Males were treated for 42 days, with a satellite high dose group retained for a further 15 days to demonstrate recovery after treatment.
The females were treated for 14 days prior to mating and then through gestation to lactation day 4. A high dose satellite recovery group of females was also retained for fifteen days following completion of treatment.
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
- Purity: 99.96%
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Crj:CD(SD)IGS rats used but no details provided regarding supplier
- Age at study initiation: (P) x wks; (F1) x wks No data
- Weight at study initiation: (P) Males: x-x g; Females: x-x g; (F1) Males: x-x g; Females: x-x g No data
- Fasting period before study: No data
- Housing: No data

- Diet (e.g. ad libitum): no data
- Water (e.g. ad libitum): no data
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): No data
- Humidity (%): No data
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): No data

IN-LIFE DATES: From: not stated To: not stated
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% sodium carboxymethylcellulose solution
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water):0.5% sodium carboxymethylcellulose solution used as suspending agent

Details on mating procedure:
- M/F ratio per cage: 1:1.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No information provided
Duration of treatment / exposure:
Males were dosed for 42 days prior to mating; the females were treated for 14 days prior to mating and then until day 4 of lactation. The female satellite animals were treated or 42 days. The recovery period for males and satellite females was 14 days. The males were terminated on day 43 of treatment (day 15 of recovery) and the females were terminated on day 5 of lactation, satellite females on day 15 of recovery and the offspring were terminated 4 days after birth.
Frequency of treatment:
Once daily for up to 42 days
Details on study schedule:
As a screening study this investigation was limited to the parental generation and F1 offspring only. The repeated adminstration phase for males (42 days) and females (14 days) prior to mating provides some subacute toxicity data and the treatment of females through gestation gives some reproductive and developmental toxicity information.
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
8 mg/kg bw/day (actual dose received)
Dose / conc.:
40 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
twelve; an additional five rats per sex were allocated to the satellite groups for retention through the recovery phase.
Control animals:
yes, concurrent vehicle
Details on study design:
No further details provided
Positive control:
Not applicable
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at weekly intervals - assessment in homecage, then in observers hands and then outside the homecage
Assessments included posture, sleeping, locomotion,vocalisations, tremors and convulsions, response to capture and handling, salivation, grading of heart beat, body temperature, exophthalmus and pupil size, exhibition of any discoloration of fur,skin or lacrimation. Outside the cage the assessment of posture, grooming, vocalisations, occurrence of straub tail, gait, tremor, convulsion pilo-erection and palpebral opening; exploration and respiratory rate and any exhibiion of stereotypy or bizarre behaviour.

BODY WEIGHT: recorded on days 1, 7, 14, 21, 28, 35 and 42 of treatment and days 1, 7, and 14 of recovery for males.
Females were weighed on days 1, 7, and 14 of treatment; days 0, 7, 14 and 20 of pregnancy and days 0 and 4 of lactation

Food consumption was recorded for the males and for the satellite females (treated at 200 mg/kg) on days 1-2, 7-8, 14-15, 29-30, 35-36 and 41-42 and then days 6-7 and 13-14 of the recovery phase.


Urinalysis was performed on Day 31 and 32 for males and females respectively
Necropsy was completed on termination of treatment or after completion of the recovery phase or day 5 of lactation

The reproductive performance of the parents was recorded for the twelve pairings per treatment group; the number of pairs copulating provided the copulation index and the number of pregnant females was used to calculate the fertility index. the mean number of days from pairing to copulation was also calculated

Oestrous cyclicity (parental animals):
All females examined. The number of rats showing a 4 day cycle in the pre-treatment phase was recorded, together with number with 5-day cycle. Mean cycle duration was calculated
Sperm parameters (parental animals):
no data
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring on day 0 of lactation:
number of pups, stillbirths, live births, birth index and live birth index

Bodyweights recorded on day 0 and 4 of lactation

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

Necropsy of live pups surviving to lactation day 4- examination of external and visceral changes
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals following day 42 of treatment or on day 15 of the recovery phase.
- Maternal animals: All surviving animals on day 5 of lactation or on day 15 for the satellite animals in the recovery phase.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in the attached Table were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 4 days of age.
- These animals were subjected to macroscopic postmortem examinations for external and visceral changes
Statistics:
No data
Reproductive indices:
The reproductive performance of the parents was recorded for the twelve pairings per treatment group; the number of pairs copulating provided the copulation index and the number of pregnant females was used to calculate the fertility index. the mean number of days from pairing to copulation was also calculated
Offspring viability indices:
Number of newborn, delivery index, number of live newborn, birth index and live bith index calculated on day 0 of lactation. On day 4 of lactation the number of live pups, viability index and sex ratio were calculated.
Clinical signs:
no effects observed
Description (incidence and severity):
daily observations showed no adverse reactions; detailed clinical assessment revealed no treatment related changes
Body weight and weight changes:
no effects observed
Description (incidence and severity):
bodyweights for males and females showed no significant differences from controls at any timepoint. Food consumption showed no significant treatment related differences
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
bodyweights for males and females showed no significant differences from controls at any timepoint. Food consumption showed no significant treatment related differences
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
increased liver weight in males at 200 mg/kg bw/d
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
liver pathology in males at 200 mg/kg bw/d
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
liver histopathology in males at 200 mg/kg bw.d
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
No significant effects recorded
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Oral administration of 4,4'-biphenyldiol at dose levels of 8, 40 or 200 mg/kg bw/day did not cause death or a moribund condition in any animals. None of the scores obtained during detailed clinical observations differed in a biologically significant manner between the control and the compound-treated groups.
No apparent changes were observed in general clinical conditions, except that urine in the 200 mg/kg-treated group became cloudy with elapse of time after excretion. Urinalysis was performed on treatment days 31 and 32 in male and female rats, respectively; calcium oxalate-like urinary crystal sediments were found in males given 200 mg/kg and in females given 40 or 200 mg/kg. Turbidity was enhanced at dose levels of 40 or 200 mg/kg, and urinary specific gravity was decreased in these females. These changes in urine parameters were not found when urinalysis was performed 11 days after cessation of the treatment (on day 11 of recovery) in either sex for the satellite animals.
Body weight, weight gain and food consumption were not affected by treatment at any dose level in either sex. No animals showed any abnormality in functional parameters after the final treatment.
At necropsy on termination of the treatment, no apparent effects of the compound were found on haematological or blood-biochemical examination at any dose level of the compound in either sex.
No effects of the compound were apparent in the female organs, including their weights or macroscopic or microscopic findings.
In contrast, 200 mg/kg of the compound exerted effects on male livers, with significant increase in relative weight, darkening or enlargement of the macroscopic appearance, development of centrilobular hepatocyte hypertrophy and reduction in occurrence of periportal fatty change. However, no animals showed any abnormality in functional observations on day 14 of the recovery period. At necropsy on day 15 of recovery, changes in the liver were not found in males given 200 mg/kg. In females given 200 mg/kg for 42 days and killed for necropsy on day 15 of recovery (satellite group), there were similarly no abnormal findings.

The treatment did not affect parameters of reproductive performance, such as the estrous cycle, ovulation, mating, implantation, delivery or lactational condition at any dose level. No adverse effects of the compound were observed on viability, morphology or growth of offspring.
Key result
Dose descriptor:
NOAEL
Remarks:
General toxicity
Effect level:
40 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Liver effects at 200 mg/kg bw/d: urinary turbidity at 40 and 200 mg/kg bw/d is not considered to be of toxicological significance
Key result
Dose descriptor:
NOEL
Remarks:
Reproductive toxicity
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No evidence of reproductive toxicity was seen in this study at the highest dose level tested of 200 mg/kg bw/d
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings:
not specified
There were no adverse effects of administration on offspring viability, morphology or growth.
Key result
Dose descriptor:
NOEL
Remarks:
Developmental toxicity
Generation:
F1
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No evidence of developmental toxicity was seen in this study at the highest dose level tested of 200 mg/kg bw/d
Key result
Reproductive effects observed:
no

The no observed adverse effect level (NOAEL) for repeat dose toxicity of 4,4'- biphenyldiol is considered to be 40 mg/kg bw/d in both sexes. The NOAEL for reproductive toxicity is considered to be 200 mg/kg bw/d, in the absence of any relevant effects.

Summary of oestrous cycle data for females treated with 4,4’-biphenyldiol

 

4,4’-biphenyldiol

0 (0.5% CMC Na solution, 5 mL/kg bw

8 mg/kg bw/day

40 mg/kg bw/day

200 mg/kg bw/day

Number of females examined

12

12

12

12

Number of females with 4-day cycle in pre-treatment period

12

12

12

12

Number of females in treatment period with 4-day cycle or
5 day cycle


12
0


11
1


12
0


12
0

Mean length of oestrous cycle (days)

4.0

4.0

4.0

4.0

Number of vaginal oestrous during mating phase

1.0

1.0

1.0

1.0

 

Reproductive performance of rats treated with 4,4’-biphenyldiol

 

4,4’-biphenyldiol

0 (0.5% CMC Na solution, 5 mL/kg bw

8 mg/kg bw/day

40 mg/kg bw/day

200 mg/kg bw/day

Number of pairs examined

12

12

12

12

Number of pairs copulated

11

11

12

12

Copulation index

91.7

91.7

100

100

Number of pregnant females

11

11

10

12

Fertility index

100

100

83.3

100

Mean number of pairing days to copulation

2.5 ± 1.1

2.6 ± 1.2

2.7 ± 0.8

2.7 ± 1.2

 

Development of pups up to day 4 of lactation

 

4,4’-biphenyldiol

0 (0.5% CMC Na solution, 5 mL/kg bw

8 mg/kg bw/day

40 mg/kg bw/day

200 mg/kg bw/day

Number of pregnant females

12

12

10

12

Number of pregnant females with live newborns

11

11

10

12

Gestation index

100

100

100

100

Gestation length in days

22.4 ± 0.5

22.4 ± 0.5

22.0 ± 0.0

22.4 ± 0.5

Number of corpora lutea

15.9 ± 1.6

16.5 ± 1.1

16.2 ± 1.2

15.2 ± 1.5

Number of implantations

15.5 ± 1.7

14.3 ± 3.6

16.0 ± 1.6

14.7 ± 1.72

Implantation index

97.1 ± 4.3

86.9 ± 21.6

98.7 ± 4.2

96.7 ± 5.3

 

 

 

 

 

Day 0 of lactation

 

 

 

 

Number of newborns

14.5 ± 2.3

13.8 ± 4.0

15.1 ± 1.7

14.0 ± 1.8

Delivery index

93.2 ± 1.1

94.6 ± 12.2

94.4 ± 4.8

95.5 ± 5.2

Number of live newborns

14.3 ± 2.3

13.8 ± 4.0

14.6 ± 1.6

12.8 ± 2.9

Birth index

92.0 ± 7.6

94.6 ± 12.2

91.5 ± 8.1

88.8 ± 20.1

Live birth index

98.7 ± 3.0

100.0 ± 0.0

97.0 ± 7.5

93.3 ± 20.8

Sex ratio on Day 0

54.8 ± 14.5

49.4 ± 17.4

52.3 ± 15.1

51.6 ± 14.7

 

 

 

 

 

Day 4 of lactation

 

 

 

 

Number of live pups

13.9 ± 2.1

13.6 ± 4.0

14.5 ± 1.5

12.4 ± 4.2

Viability index

97.7 ± 4.0

98.8 ± 2.6

99.4 ± 2.0

91.7 ± 28.9

Sex ratio on Day 4

54.4 ± 14.9

49.3 ± 17.9

52.1 ± 15.9

50.8 ± 15.2

 

Pup bodyweights up to Day 4 of lactation

 

4,4’-biphenyldiol

0 (0.5% CMC Na solution, 5 mL/kg bw

8 mg/kg bw/day

40 mg/kg bw/day

200 mg/kg bw/day

Day 0 of lactation

 

 

 

 

Number of live newborns

 

 

 

 

Male

7.7 ± 2.1

6.9 ± 3.0

7.6 ± 2.1

6.5 ± 2.2

Female

6.5 ± 3.0

6.9 ± 3.3

7.0 ± 2.6

6.3 ± 2.7

Bodyweight of live newborn (g)

 

 

 

 

Male

6.8 ± 0.8

6.9 ± 0.8

6.5 ± 0.5

6.6 ± 0.8

Female

6.6 ± 0.6

6.6 ± 0.7

6.0 ± 0.4

6.2 ± 0.8

 

 

 

 

 

Day 4 of lactation

 

 

 

 

Number of live pups

 

 

 

 

Male

7.5 ± 2.3

6.8± 3.1

7.5± 2.2

6.8± 2.0

Female

6.4 ± 2.2

6.8± 3.2

7.0 ± 2.6

6.7± 2.5

Bodyweight of pups (g)

 

 

 

 

Male

10.6 ± 1.7

10.7 ± 1.1

10.2 ± 1.1

10.8 ± 1.0

Female

10.3 ± 1.4

10.4 ± 1.4

9.8 ± 1.0

10.0 ± 0.9

 

Pup morphology findings

 

4,4’-biphenyldiol

0 (0.5% CMC Na solution, 5 mL/kg bw

8 mg/kg bw/day

40 mg/kg bw/day

200 mg/kg bw/day

Dead pups

 

 

 

 

Number of dead pups examined

3

1

6

12

External examinations

3

1

6

12

Visceral examinations

0

0

5

1

Number of pups with external changes

0

0

0

0

Number of pups with visceral changes

0

0

0

0

 

 

 

 

 

Live pups

 

 

 

 

At birth (day 0)

 

 

 

 

Number of newborns examined

157

152

146

154

Number of newborns with
external changes


0


0


0


0

At necropsy (day 4 of lactation)

 

 

 

 

Number of pups examined

153

150

145

149

Number of pups with external changes

0

0

0

0

Number of pups with visceral changes

0

0

0

0

 

Conclusions:
The treatment up to 200 mg/kg bw/d did not affect the reproductive performance of parental animals or the early development of their offspring. The no observed adverse effect dose level (NOAEL) for repeat dose toxicity of 4,4'-biphenyldiol is considered to be 40 mg/kg/day for both sexes of animals. The NOAEL for reproductive toxicity is considered to be 200 mg/kg bw/d, in the absence of any findings.
Executive summary:

A combined repeat dose and reproductive/developmental toxicity screening test was conducted in rats according to the OECD Test Guideline 422 with 4,4'-biphenyldiol. No effects on fertillity or reproductive parameters (oestrus cyclicity, mating, implantation or lactation) were observed at any dose level. In the absence of any effects on reproductive parameters, a NOAEL for reproductive toxicity of 200 mg/kg bw/d can be determined. A NOAEL for general toxicity of 40 mg/kg bw/d can be determined based on liver effects at the highest dose level. Effects of treatment on urinalysis parameters seen at 40 and 200 mg/kg bw/d are not considered to be of toxicological significance.

 

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
200 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
A recent, guideline-compliant screening study is available.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Description of key information

A screening test according to OECD 422 (repeated dose/reproductive/developmental toxicity study) in Sprague-Dawley rats exposed by oral route is available and reports a NOAEL of 40 mg/kg bw/d for general toxicity and a NOAEL of 200 mg/kg bw/d for develomental toxicity (the highest dose level tested).

In this study, treatment up to 200 mg/kg bw/d did not affect the the developmental parameters assessed.

A developmental toxicity study according to OECD 414 (prenatal developmental toxicity study) in Wistar pregnant rats exposed by the oral route is available.

Firstly a Dose Range Finding study was conducted at 250, 500 and 750 mg/kg/day. Severe toxicity was observed in one and all females given 500 and 750 mg/kg/day, respectively. Therefore in the main experiment, the substance was administered by daily gavage at dose levels of 25, 75 and 250 mg/kg/day to groups of 22 mated female Wistar rats from G 6 to G 20 of gestation inclusive.

A control group received the same volume (5 mL/kg) of the vehicle (0.5 % [w/v] Carboxymethylcellulose 300-600 centipoises in water for injection).

A transient reduced body weight gain at 75 mg/kg/day and persistent reduced body weight gain and food consumption at 250 mg/kg/day were observed.

Slightly lower mean placental and foetal weights were seen at the highest group (250 mg/kg bw/d) in the presence of maternal toxicity. These findings were likely related to the maternal toxicity rather than a direct effect of the test item.

Therefore a NOAEL of 75 mg/kg bw/d for maternal toxicity and a NOAEL of 250 mg/kg bw/d for embryo-foetal toxicity (the highest dose level tested) were reported.

In this study, treatment up to 250 mg/kg bw/d was considered not to affect the prenatal embryo-foetal development.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was conducted in accordance with OECD Test guideline No 422 and with the principles of GLP
Qualifier:
equivalent or similar to
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
- Purity: 99.96%
Species:
rat
Strain:
Crj: CD(SD)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Crj:CD(SD)IGS rats used but no details provided regarding supplier
- Age at study initiation: (P) x wks; (F1) x wks No data
- Weight at study initiation: (P) Males: x-x g; Females: x-x g; (F1) Males: x-x g; Females: x-x g No data
- Fasting period before study: No data
- Housing: No data

- Diet (e.g. ad libitum):
- Water (e.g. ad libitum):
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): No data
- Humidity (%): No data
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): No data

IN-LIFE DATES: From: not stated To: not stated
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% sodium carboxymethylcellulose solution
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water):0.5% sodium carboxymethylcellulose solution used as suspending agent
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No information provided
Details on mating procedure:
- M/F ratio per cage: 1:1.
Duration of treatment / exposure:
Males were dosed for 42 days prior to mating; the females were treated for 14 days prior to mating and then until day 4 of lactation. The female satellite animals were treated or 42 days. The recovery period for males and satellite females was 14 days. The males were terminated on day 43 of treatment (day 15 of recovery) and the females were terminated on day 5 of lactation, satellite females on day 15 of recovery and the offspring were terminated 4 days after birth.
Frequency of treatment:
Once daily for up to 42 days
Duration of test:
6 weeks
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
8 mg/kg bw/day (actual dose received)
Dose / conc.:
40 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
twelve; an additional five rats per sex were allocated to the satellite groups for retention through the recovery phase.
Control animals:
yes, concurrent vehicle
Details on study design:
No further details provided.
As a screening study this investigation was limited to the parental generation and F1 offspring only. The repeated adminstration phase for males (42 days) and females (14 days) prior to mating provides some subacute toxicity data and the treatment of females through gestation gives some reproductive and developmental toxicity information.

The males and females were dosed for up to 42 days by oral gavage and this subacute exposure was used to address the short-term toxicology endpoints. Clinical signs, bodyweight gain, food consumption haematology, clinical chemistry and urinalysis parameters were recorded together with a limited functional observation battery, organ weights and macroscopic and microscopic examinations at termination.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at weekly intervals - assessment in homecage, then in observers hands and then outside the homecage
Assessments included posture, sleeping, locomotion,vocalisations, tremors and convulsions, response to capture and handling, salivation, grading of heart beat, body temperature, exophthalmus and pupil size, exhibition of any discoloration of fur,skin or lacrimation. Outside the cage the assessment of posture, grooming, vocalisations, occurrence of straub tail, gait, tremor, convulsion pilo-erection and palpebral opening; exploration and respiratory rate and any exhibiion of stereotypy or bizarre behaviour.

A functional assessment was completed on the final day of treatment to assess various reflex responses.

BODY WEIGHT: recorded on days 1, 7, 14, 21, 28, 35 and 42 of treatment and days 1, 7, and 14 of recovery for males.
Females were weighed on days 1, 7, and 14 of treatment; days 0, 7, 14 and 20 of pregnancy and days 0 and 4 of lactation
Bodyweights and bodyweight gains are reported.

Food consumption was recorded for the males and for the satellite females (treated at 200 mg/kg) on days 1-2, 7-8, 14-15, 29-30, 35-36 and 41-42 and then days 6-7 and 13-14 of the recovery phase.

Urinalysis was performed on Day 31 and 32 for males and females respectively. Parameters assessed included colour, turbidity, pH, protein, glucose, ketone and bilirubin levels, occult blood, urobilinogen and presence in urinary sediment of crystals by incidence and type/shape and urinary specific gravity.

Clinical pathology in the form of standard haematology and biochemical parameters were assessed on Day 43 of treatment and on Day 15 of the recovery phase for male rats and on Day 5 of lactation for the females.

Organ weights were recorded on Day 43 of treatment and on Day 15 of the recovery phase for male rats and on Day 5 of lactation for the females.


Necropsy was completed on termination of treatment or after completion of the recovery phase or on day 5 of lactation. Macroscpic findings were recorded at each scheduled necropsy. Histopathological findings were documented for tissues sampled following each of the scheduled necropsy events. The list of tissues examined was in accordance with the test guideline.

The reproductive performance of the parents was recorded for the twelve pairings per treatment group; the number of pairs copulating provided the copulation index and the number of pregnant females was used to calculate the fertility index. the mean number of days from pairing to copulation was also calculated

All females examined for oestrous cyclicity . The number of rats showing a 4 day cycle in the pre-treatment phase was recorded, together with number with 5-day cycle. Mean cycle duration was calculated
Ovaries and uterine content:
No data
Fetal examinations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring on day 0 of lactation:
number of pups, stillbirths, live births, birth index and live birth index

Bodyweights recorded on day 0 and 4 of lactation

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

Necropsy of live pups surviving to lactation day 4- examination of external and visceral changes
Statistics:
No data
Indices:
Number of newborn, delivery index, number of live newborn, birth index and live bith index calculated on day 0 of lactation. On day 4 of lactation the number of live pups, viability index and sex ratio were calculated.
Historical control data:
No data
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
Oral administration of 4,4'-biphenyldiol at dose levels of 8, 40 or 200 mg/kg bw/day did not cause death or a moribund condition in any animals. None of the scores obtained during detailed clinical observations differed in a biologically significant manner between the control and the compound-treated groups.
No apparent changes were observed in general clinical conditions, except that urine in the 200 mg/kg-treated group became cloudy with elapse of time after excretion. Urinalysis was performed on treatment days 31 and 32 in male and female rats, respectively, calcium oxalate-like urinary crystal sediments were found in males given 200 mg/kg and in females given 40 or 200 mg/kg. Turbidity was enhanced at dose levels of 40 or 200 mg/kg, and urinary specific gravity was decreased in these females. These changes in urine parameters were not found when urinalysis was performed 11 days after cessation of the treatment (on day 11 of recovery) in either sex for the satellite animals.
Body weight, weight gain and food consumption were not affected by treatment at any dose level in either sex. No animals showed any abnormality in functional parameters after the final treatment.
At necropsy on termination of the treatment, no apparent effects of the compound were found on haematological or blood-biochemical examination at any dose level of the compound in either sex.

The reproductive and developmental toxicity indices evaluated in this screening study indicated no adverse effects on the dams. No effects were apparent in the female oestrous cycle either during the pre-treatment, mating or treament phases. The reproductive indices included the numbers of pairings examined, copulating and the number of resulting pregnant females and the length of pairing time required before copulation. There were no changes between control and treated groups.
Key result
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
External malformations:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There were no adverse effects of administration on offspring viability, morphology or growth.
Pup development indices were evaluated to assess the duration of gestation, numbers of corpora lutea and implantations, the numbers of pups born, numbers of live born and sex ratio for the liveborn pups and survival and viability of pups to day 4 of lactation was also evaluated . None of the comparisons between treated and control groups revelaed any significant changes in these indices or any statistically significant effects. Similarly no effects were apparent when newborn or neonate (lactation day 4) pup weights were assessed.

Morphological evaluations for external and visceral changes showed no effects on the live pups at birth or those that survived to lactation Day 4. The dead pups examined (3 controls and 1, 6 or 12 pups in the 8, 40 or 200 mg/kg bw/day groups) also had no external and visceral changes .

Key result
Dose descriptor:
NOAEL
Effect level:
>= 200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effect observed
Key result
Developmental effects observed:
no

Combined repeat dose and reproductive/developmental toxicity screening test for 4,4’-biphenyldiol

 

4,4’-biphenyldiol mg/kg bw/day

 

0

8

40

200

No. of females examined

12

12

12

12

No. of females showing 4-day cycle in pre-treatment phase

12

12

12

12

No. of females showing 4-day cycle in treatment phase

12

11

12

12

No. of females showing 4 and 5-day cycle in treatment phase

0

1

0

0

Mean length of oestrous cycle in days

4.0±0.0

4.0±0.1

4.0±0.0

4.0±0.0

Number of vaginal oestrous during mating period

1.0±0.0

1.0±0.0

1.0±0.0

1.0±0.0

 

 

4,4’-biphenyldiol mg/kg bw/day

 

0

8

40

200

No. of pairs examined

12

12

12

12

No. of pairs copulated

11

11

12

12

Copulation index

91.7

91.7

100.0

100.0

No. of pregnant females

11

11

10

12

Fertility index

100.0

100.0

83.3

100.0

Number of pairing days
 to copulation

2.5±1.1

2.6±1.2

2.7±0.8

2.7±1.2

 

  Development of pups up to Day 4 of lactation

 

4,4’-biphenyldiol mg/kg bw/day

 

0

8

40

200

No. of pregnant females

11

11

10

12

No. of pregnant females with live neonates

11

11

10

12

Gestation index

100

100

100

100

Gestation length in days

22.4±0.5

22.4±0.5

22.0±0.0

22.4±0.5

Number of corpora lutea

15.9±1.6

16.5±1.1

16.2±1.2

15.2±1.5

Number of implantations

15.5±1.7

14.3±3.6

16.0±1.6

14.7±1.7

Implantation Index

97.1±4.3

86.9±21.6

98.7±4.2

96.7±5.3

 

 

 

 

 

Day 0 of lactation

 

 

 

 

Number of newborn

14.5±2.3

13.8±4.0

15.1±1.7

14.0±1.8

Delivery index

93.2±6.9

94.6±12.2

94.4±4.8

95.5±5.2

Number of live newborn

14.3±2.3

13.8±4.0

14.6±1.6

12.8±2.9

Birth index

92.0±7.6

94.6±12.2

91.5±8.1

88.8±20.1

Live birth index

98.7±3.0

100.0±0.0

97.0±7.5

93.3±20.8

Sex ratio on day 0

54.8±14.5

49.4±17.4

52.5±15.1

51.6±14.7

 

 

 

 

 

Day 4 of lactation

 

 

 

 

Number of live pups

13.9±2.1

13.6±4.0

14.5±1.5

12.4±4.2

Viability index

97.7±4.0

98.8±2.6

99.4±2.0

91.7±28.9

 

 

 

 

 

Sex ratio on day 4

54.4±14.9

49.3±17.9

52.1±15.9

50.8±15.2


The vehicle control was 5 ml/kg of 0.5% CMC Na solution.
Gestation index
      (number of pregnant females with live newborns/number of pregnant females) x 100,%
Implantation index
 (number of implantations/number of corpora lutea) x 100,%
Delivery index
        (number of newborns/number of implantations) x 100,%
Birth index
             (number of live newborns/number of implantations) x 100,%
Live birth index
      (number of live newborns/number of newborns) x 100,%
sex ratio (day 0)
     (number of male live newborns / number of live newborns) x 100,%
viability index
        (number of live pups on day 4 of lactation/ number of live newborns) x 100,%
sex ratio (day 4)
     (number of male live pups on day 4 of lactation / number of live pups on day 4 of lactation) x 100,%

Bodyweight of pups up to Day 4 of lactation

 

4,4’-biphenyldiol mg/kg bw/day

 

0

8

40

200

Day 0 of lactation

 

 

 

 

Number of live newborn

 

 

 

 

male

7.7±2.1

6.9±3.0

7.6±2.1

6.5±2.2

female

6.5±2.5

6.9±3.3

7.0±2.6

6.3±2.7

 

 

 

 

 

Birthweight of live newborn

 

 

 

 

Males

6.8±0.8

6.9±0.8

6.5±0.5

6.6±.0.8

females

6.6±0.6

6.6±0.7

6.0±0.4

6.2±0.8

 

 

 

 

 

Day 4 of lactation

 

 

 

 

Number of live pups

 

 

 

 

Males

7.5±2.3

6.8±3.1

7.5±2.2

6.8±2.0

females

6.4±2.2

6.8±3.2

7.0±2.6

6.7±2.5

Birthweight of pups

 

 

 

 

Males

10.6±1.7

10.7±1.1

10.2±1.1

10.8±1.0

females

10.3±1.4

10.4±1.4

9.8±1.0

10.0±0.9

Morphological findings of pups

 

4,4’-biphenyldiol mg/kg bw/day

 

0

8

40

200

Dead pups

 

 

 

 

Number of dead pups examined

3

1

6

12

External observations

3

1

6

12

Visceral observations

0

0

5

1

No. of pups with external changes

0

0

0

0

No. of pups with visceral changes

0

0

0

0

 

 

 

 

 

 

 

 

 

 

Live pups at birth (Day 0)

 

 

 

 

Number of live neonates examined

157

152

146

154

Number with external changes

0

0

0

0

At necropsy on Day 4

 

 

 

 

Number of pups examined

153

150

145

149

No. of pups with external changes

0

0

0

0

No. of pups with visceral changes

0

0

0

0



Conclusions:
Treatment with up to 200 mg/kg bw/d did not affect early offspring development. The no observed adverse effect level (NOAEL) for repeat dose toxicity of 4,4'-biphenyldiol is considered to be 40 mg/kg bw/d. In the absence of any effects on the parameters measured. the NOAEL for developmental toxicity is considered to be 200 mg/kg/ bw/d.
Executive summary:

A combined repeat dose and reproductive/developmental toxicity screening test was conducted in rats according to the OECD Test Guideline 422 with 4,4'-biphenyldiol. No effects on developmental parameters were observed at any dose level. In the absence of any effects on developmental parameters, a NOAEL for developmental toxicity of 200 mg/kg bw/d can be determined. A NOAEL for general toxicity of 40 mg/kg bw/d can be determined based on liver effects at the highest dose level. Effects of treatment on urinalysis parameters seen at 40 and 200 mg/kg bw/d are not considered to be of toxicological significance.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 November 2016 - 6 April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study performed according to the OECD Test Guideline No. 414.
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
January 2001
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
August 1998
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
2008
GLP compliance:
yes
Species:
rat
Strain:
Wistar
Remarks:
Crl: WI (Han)
Details on test animals and environmental conditions:
TEST ANIMALS
- Number of animals in the study: 88 virgin mated females. The females were mated at the supplier with a documented day of mating. They were received at the Test Facility on day 1 of gestation (G0).
- Source: Charles River Laboratories France, Domaine des Oncins, 69210 Saint-Germain-Nuelles, France.
- Age at mating: 10 to 13 weeks.
- Weight at mating: 197 to 255 g.
- Acclimatisation period before study: 6 days between animal arrival and the start of treatment
- Housing: One air-conditioned room in a barrier protected unit. Females were individually housed in plastic cages meeting European directive 2010/63/EU requirements.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 6 days between animal arrival and the start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): >35 %
- Air changes (per hr): At least 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light (artificial)/12 hours dark

IN-LIFE DATES: From: 28 February to 03 March 2017 (G0 Animal arrivals) To: 24 March 2017 (last caesarean)
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5 % [w/v] Carboxymethylcellulose 300-600 centipoises in water for injection
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- Rate of preparation: Weekly
- Storage of formulations (including vehicle control): Refrigerated (between +2 and +8 °C).
- Homogeneity of the test item in the vehicle: The formulations at concentrations of 1 and 200 mg/mL in the vehicle are homogeneous (based on the analytical results in a specific study).
- Stability of the test item in the vehicle: 11 days at room temperature (between +17 and +27 °C) and refrigerated (5±4 °C), and 26 days frozen (between -15 and -25 °C) (based on the analytical results of a specific study).

VEHICLE
- Concentration in vehicle: 5, 15 and 50 mg/mL
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of formulations:
4x1 g samples were taken from each formulation, including the vehicle, on a single occasion during the treatment of the main study phase only.
The samples were stored frozen (between -25 and -15 °C).
The first set of samples (2x1 g) was dispatched on dry ice to the Test Site for analysis.
Details on mating procedure:
The females were mated at the supplier with a documented day of mating. They were received at the Test Facility on day 1 of gestation (G0).
Duration of treatment / exposure:
From G 6 to G 20 inclusive
Frequency of treatment:
Once daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group number: 1 (Control)
Dose / conc.:
25 mg/kg bw/day (nominal)
Remarks:
Group number: 2 (Low dose)
Dose / conc.:
75 mg/kg bw/day (nominal)
Remarks:
Group number: 3 (Intermediate dose)
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
Group number: 4 (High dose)
No. of animals per sex per dose:
22
Control animals:
yes, concurrent vehicle
Details on study design:
- Group 1 animals (Control) received the vehicle (0.5 % [w/v] Carboxymethylcellulose).
- Volume of administration: 5 mL/kg/day. Individual dose volumes were adjusted using the latest body weight.
- Rationale for the dose selection: the doses for the main study phase were selected based on the DRF phase where oral (gavage) administration of the test item in the female rat from G 6 to G 20 was above the maximum tolerated dose (i.e. severe body weight loss, negligible food consumption, poor clinical condition and renal changes) at 500 and 750 mg/kg/day. Dose of 250 mg/kg/day was associated with reduced body weight change and food consumption throughout the dosing period.
Consequently, in the main phase, a high dose of 250 mg/kg/day was selected in order to obtain acceptable maternal toxicity. The low and intermediate doses selected were 25 and 75 mg/kg/day in order to identify a possible No Observed Adverse Effect Level.
Maternal examinations:
OBSERVATIONS:
- Morbidity/mortality: All animals were observed at least twice daily.
- Clinical signs: The animals were observed daily during the study. To detect any abnormalities in appearance, behaviour or other signs of reaction to treatment, the animals were observed before and twice after dosing. A full clinical examination was performed on each weighing day.
- Body weight: All animals were weighed individually on G 0, G 6, G 9, G 12, G 15, G 18 and G 21.
- Food consumption: Individual food consumption was measured for the periods G 0 to G 6, G 6 to G 9, G 9 to G 12, G 12 to G 15, G 15 to G 18 and G 18 to G 21.

TERMINAL EXAMINATIONS:
- Necropsy: All females were killed on G 21. They were dissected and examined for macroscopic pathological changes. Abnormal organs were sampled and preserved in 10 % neutral formalin but not examined further.
The ovaries and uterus of each female were removed and examined as below.
Ovaries and uterine content:
The ovaries and uterus of each female were removed and examined including examination of the placentae.
The following data were recorded:
- pregnancy status
- number of corpora lutea
- number of implantations
- number and distribution of live foetuses
- number and distribution of embryonic/foetal deaths, classified as follows:
> early: only placenta visible at termination
> late: both placenta and embryonic tissue visible at termination
> dead foetus.
- gravid uterus weight
- individual foetal weights
- individual placental weights
- foetal sex.
Fetal examinations:
- Each foetus was examined for external defects.
- All live foetuses were killed by an oral administration of sodium pentobarbitone.
- Approximately one half of each litter was submitted to fresh visceral examination of the body (abdominal and thoracic cavities) and then processed for skeletal examination. The skeletal examinations were performed following maceration of the soft tissues with aqueous potassium hydroxide, staining of the skeleton with Alizarin red then passage into glycerol.
- The remaining half of the foetuses in each litter was fixed in Harrison’s fluid for subsequent examination of the head only by serial sectioning. The remaining carcass was retained fixed but not examined further in the first instance.
- Soft tissue and skeletal examinations were performed using a binocular microscope.
Statistics:
Statistical analyses were performed by the Provantis data acquisition system, where appropriate, as follows:
- The best transformation for the data (none, log or rank) was determined depending upon
> the normality of the data distribution tested by the Shapiro-Wilk's test
> the homogeneity of the variances across groups tested by the Bartlett's test.
- Non- or log-transformed data were analysed by parametric methods.
- Rank transformed data were analysed using non-parametric methods.
- Data were then analysed to test for a dose-related trend to detect the lowest dose at which there is a significant effect, based on the Williams test for parametric data or the Shirley's test for non-parametric data.
- Homogeneity of means was assessed by analysis of variances (ANOVA) for parametric data or Kruskal-Wallis test for non-parametric data.
- If no trend was found and means were not homogeneous, the data were analysed by parametric or non-parametric Dunnett's test to look for significant differences from the control group.
- The number of resorptions, number of dead foetuses and all litter-based percentages were analysed using non-parametric methods, i.e. Kruskal-Wallis test followed by non-parametric Dunnett’s test if the Kruskal-Wallis is significant.
- Selected incidence data were analysed using a chi2 test for all groups followed by Fisher’s two-tailed test with Bonferroni correction for each treated group versus the control if the chi2 is significant.
Microsoft Excel 2010® was employed to present certain results.
Indices:
For each group, the following parameters were calculated:
- Pre-implantation loss (in %) = [(Number of corpora lutea - Number of implantations) / (Number of corpora lutea)] x 100
- Post-implantation loss (in %) = [(Number of implantations - Number of viable foetuses) (Number of implantations)] x 100
Historical control data:
Historical control data are provided in the study report.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Partly closed eyes were noted for 2 females given 250 mg/kg/day on G 7.
Other clinical signs noted were either associated with pregnancy/parturition (piloerection at the end of the gestation period) or incidental (erythema, localized hairloss, torn claw, scabs and/or sores).
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There was a dose-related lower mean body weight gain for females given 75 and 250 mg/kg/day during the first three days of treatment (G 6 to G 9) compared with the concurrent control.
Thereafter, recovery was noted for females given 75 mg/kg/day and the effect persisted up to the end of treatment (up to G 21) for females given 250 mg/kg/day (-24 % body weight gain between G6 and G21 when compared with control).
As a consequence, absolute terminal mean body weight (including mean carcass weight of the dams after correction for gravid uterus weight) was lower in the 250 mg/kg/day group compared with the concurrent control (-7 %).
This effect on mean body weight gain in the high dose group impacting terminal body weight was considered adverse.

The lower mean body weight gain noted between G 18 and G 21 for females given 25 and 75 mg/kg/day compared with the concurrent control, was considered incidental since values remained comparable with the historical control data.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There was a slightly lower mean food consumption for females given 250 mg/kg/day during the whole treatment period (G 6 to G 21) compared with the concurrent control.
As a consequence to this change, a lower mean body weight gain during the overall dosing period and a lower mean terminal body weight in the high dose group was noted.
This effect was therefore considered adverse.

The slightly lower mean food consumption noted between G 18 and G 21 for females given 75 mg/kg/day compared with the concurrent control, was considered incidental since values remained comparable with the historical control data.
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No test item-related effect on mean gravid uterus, kidneys and liver weights in any group
Gross pathological findings:
no effects observed
Description (incidence and severity):
No test item-related macroscopic abnormalities in any group
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The pre-implantation data (mean numbers of corpora lutea, implantation sites and the percentage pre-implantation loss) were comparable in all groups.
There was no test item-related effect on embryo-foetal survival; the percentage post-implantation loss was comparable in all groups.
The mean live litter size was comparable in all groups.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
There were slightly lower mean placental and foetal weights in the 250 mg/kg/day group compared with the concurrent control and lower dose groups. However, the mean foetal weight value in the high dose group was comparable with the historical control data (range from 4.9 to 5.1g) and the change was of low magnitude.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There was no test item-related effect on mean foetal sex ratio in any group.
Changes in litter size and weights:
no effects observed
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were 1, 2, 2 and 0 foetus(es) from as many litter(s) with external malformations in the control, 25, 75 and 250 mg/kg/day groups, respectively.
The foetus from the control group (dam no. 106) had a proboscis and one foetus from the 75 mg/kg/day group (dam no. 156) had a microstomia and micrognathia.
One foetus from the 25 mg/kg/day group (dam no. 124) had malrotated hindlimbs, a thread-like tail and an anal atresia. The other foetus from the 25 mg/kg/day group (dam no. 131) had an anasarca.
The last foetus from the 75 mg/kg/day group (dam no. 162) had polydactyly.
These isolated findings, not observed in the 250 mg/kg/day group, were considered incidental.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were 0, 1, 2 and 0 foetus(es) from as many litter(s) with skeletal malformations in the control, 25, 75 and 250 mg/kg/day groups, respectively.
The foetus with the thread-like tail and anal atresia observed externally in the 25 mg/kg/day group (dam no. 124) had a malformed vertebral column.
The foetus with the micrognathia observed externally in the 75 mg/kg/day group (dam no. 156) had a malformed skull.
Polydactyly observed externally in the foetus from the 75 mg/kg/day group (dam no. 162) were confirmed at the skeletal examination (presence of one additional claw).
These isolated findings, not observed in the 250 mg/kg/day group, were considered incidental.
Less severe skeletal anomalies and variations did not suggest any association with the test item and are part of the background of changes noted for this strain of rat.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were 1, 1, 0 and 0 foetus from as many litter with visceral malformations in the control, 25, 75 and 250 mg/kg/day groups, respectively.
The foetus with the anasarca observed externally in the 25 mg/kg/day group (dam no. 131) had malformed great blood vessels. This isolated finding, not observed in the higher dose groups, was considered incidental.
The control foetus with a proboscis observed externally (dam no. 106) had a malformed head.
Less severe visceral anomalies and variations did not suggest any association with the test item and are part of the background of changes noted for this strain of rat.
Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: No evidence of a direct effect of the test item (slightly lower mean placental and foetal weights in the 250 mg/kg/day group, likely related to the maternal toxicity).
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
250 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Conclusions:
Under the experimental conditions of the study (according to the guideline OECD 414), the oral administration of 4,4'-biphenyldiol to the pregnant Wistar rat from G 6 to G 20 at doses of 25, 75 and 250 mg/kg/day was associated with a transient reduced body weight gain at 75 mg/kg/day and persistent reduced body weight gain and food consumption at 250 mg/kg/day. These changes had an impact on the mean terminal body weight in the high dose group and were therefore considered as adverse. The No Observed Adverse Effect Level (NOAEL) for maternal toxicity was therefore considered to be 75 mg/kg/day.

Evidence of embryo-foetal effect was restricted to slightly lower mean placental and foetal weights in the 250 mg/kg/day group. The mean foetal weight in the high dose group was comparable with the historical control data and the change was of low magnitude. As this was not correlated with morphological development changes, these findings were likely related to the maternal toxicity rather than a direct effect of the test item. The NOAEL for embryo-foetal toxicity was therefore considered to be 250 mg/kg/day.
Executive summary:

A GLP compliant-study based on the OECD guidelilne No. 414 was conducted in rat in order to provide information on the effect of the test item, 4,4'-biphenyldiol, on prenatal development.

The test item, 4,4'-biphenyldiol, was administered by daily gavage at dose levels of 25, 75 and 250 mg/kg/day to groups of 22 mated female Wistar rats from G 6 to G 20 of gestation inclusive. A control group received a similar volume (5 mL/kg) of the vehicle 5 % [w/v] Carboxymethylcellulose n water for injection).

Parameters monitored included morbidity/mortality, clinical signs, body weight and food consumption. The females were submitted to a caesarean examination on G 21 and litter parameters were recorded.

At necropsy, the females were examined macroscopically and the gravid uterus, the placenta, the kidneys and the liver were weighed. All live foetuses were

weighed, sexed and examined for external abnormalities. Half of the foetuses were examined internally prior to processing for skeletal examination. The remaining foetuses were preserved for fixed-visceral examination of the head by the modified Wilson-Barrow technique.

There was no unscheduled death in any group.

Partly closed eyes were noted for 2 females given 250 mg/kg/day on G 7 only.

There was a statistically significant lower mean body weight gain and food consumption in the 250 mg/kg/day group during the whole treatment period compared with the control. These changes impacted the mean terminal body weight in the high dose group and were therefore considered as adverse. A slightly lower mean body weight gain was also noted at 75 mg/kg/day during the first three days of treatment (G 6 to G 9) compared with the control.

There were no test item-related effect on macroscopic observations, mean gravid uterus, kidneys and liver weights in any group.

There were 22, 20, 22 and 22 pregnant females in the control, 25, 75 and 250 mg/kg/day groups, respectively, at the terminal caesarean examinations, all of which had viable foetuses.

There were no test item-related effect on embryo-foetal survival and sex ratio in any group.

There were statistically significant slightly lower mean placental and foetal weights in the 250 mg/kg/day group compared with the control. However, the mean foetal weight value in the high dose group was comparable with the historical control and the change was of low magnitude.

There were 1, 2, 2 and 0 malformed foetus(es) from as many litter(s) in the control, 25, 75 and 250 mg/kg/day groups, respectively. None of which were attributable to the test item.

To conclude under the experimental conditions of this study:

The No Observed Adverse Effect Level (NOAEL) for maternal toxicity was therefore considered to be 75 mg/kg/day.

The NOAEL for embryo-foetal toxicity was therefore considered to be 250 mg/kg/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
200 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Two recent, guideline-compliant studies, screening study and prenatal developmental toxicity study, are available.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Justification for selection of oral route:

Oral route is a potential route of human exposure during manufacture, handling or use of the test substance as specified in the applicable guidelines.

Justification for classification or non-classification

No classification for reproductive or developmental toxicity is proposed in the absence of relevant findings in the available data.

A sub-chronic investigation of reproductive performance (OECD 443) is proposed in order to clarify the requirement of classification with respect to reproductive endpoint.