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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
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

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2004

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
No further details.
Specific details on test material used for the study:
- Purity: 99.96%

Test animals

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

Administration / exposure

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.
Doses / concentrationsopen allclose all
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

Examinations

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.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

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 / performance (P0)

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

Details on results (P0)

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.

Effect levels (P0)

open allclose all
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

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings:
not specified

Details on results (F1)

There were no adverse effects of administration on offspring viability, morphology or growth.

Effect levels (F1)

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

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

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

 

Applicant's summary and conclusion

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.