Benzenamine, 3,3'-[[1,1'-biphenyl]-4,4'-diylbis(oxy)]bis-

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The test methods followed the relevant Japanese guidelines and are described in detail together with the results of the study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Ministry of Health and Welfare of Japan - Part II of the Chemical Substance Control Law (1986)

Deviations:

not specified

GLP compliance:

yes

Remarks:

United Kingdom Good Laboratory Practice Regulations (1997)

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

3-{[4'-(3-aminophenoxy)-[1,1'-biphenyl]-4-yl]oxy}aniline

EC Number:

700-102-1

Cas Number:

105112-76-3

Molecular formula:

C24H20N2O2

IUPAC Name:

3-{[4'-(3-aminophenoxy)-[1,1'-biphenyl]-4-yl]oxy}aniline

Details on test material:

- Name of test material (as cited in study report): 4,4'-Bis(3-Aminophenoxy)Biphenyl
- Physical state: fine, cream-coloured powder
- Analytical purity: not reported
- Lot/batch No.: 89-42001
- Expiration date of the lot/batch: not reported

Test animals

Species:

rat

Strain:

other: CD strain

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd
- Age at study initiation: 34 to 41 days
- Weight at study initiation:
- Fasting period before study:
- Housing: Animals were housed five of same sex per cage. The cages used were Type TR18 (Modular Systems and Developments Company Limited, Hereford, England), which were made of stainless steel with a stainless steel mesh lid and floor. The cages were suspended above absorbent crepe paper changed at appropriate intervals.
- Diet (e.g. ad libitum): expanded rodent diet RM1(E) SQR was freely available
- Water (e.g. ad libitum): was freely available via polycarbonate bottles fitted with sipper tubes
- Acclimation period: 13 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25
- Humidity (%): 40 to 70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % w/v methylcellulose in purified water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The m-BP was prepared for administration as a series of graded concentrations in 0.5 % w/v methylcellulose in purified water (obtained by reverse osmosis).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations of the test substance were determined by HPLC in formulations prepared for dosing at one occasion during Weeks 1 and 4 of treatment. Measured concentrations (control, 40, 200, 1000 mg/kg/day) were (not detected, 4.27, 20.7, 99.6) during Week 1 and (not detected, 4.16, 21, 102) during Week 4.

Duration of treatment / exposure:

30 days

Frequency of treatment:

once a day

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
40 mg/kg/day
Basis:
other: by gavage

Doses / concentrations 2

Remarks:

Doses / Concentrations:
200 mg/kg/day
Basis:
other: by gavage

Doses / concentrations 3

Remarks:

Doses / Concentrations:
1000 mg/kg/day
Basis:
other: by gavage

No. of animals per sex per dose:

five females and five males (in the reversibility phase only for highest dose)

Control animals:

yes, plain diet

Details on study design:

- Post-exposure recovery period in satellite groups: A two week recovery period was chosen for animals in the control (without test substance) and the high dose (1000 mg/kg/day) groups

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected at least twice daily. Individual observations of all animals were recorded before and after dosing on each day. The schedule was: Week 1 daily, Weeks 2-4 twice weekly. Cages and cage-trays were inspected daily for evidence of ill-health, such as blood or loose faeces. During acclimatisation and reversibility periods, observations of the animals and cages were recorded at least once per day.
- Cage side observations checked in table [No.?] were included.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
weekly

BODY WEIGHT: Yes
- Time schedule for examinations:
during acclimatisation, on the day of experiment start, at weekly intervals throughout treatment and reversibility periods and before necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on Day 30 of treatment (before dosing)
- Anaesthetic used for blood collection: Yes (halothane/nitrous oxide)
- Animals fasted: Yes (overnight)
- How many animals: all main study animals (30)
- Parameters checked in table [No.?] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on Day 30 of treatment (before dosing)
- Animals fasted: Yes (overnight)
- How many animals: all main study animals (30)
- Parameters checked in table [No.?] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: on Day 30 of treatment (overnight)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [No.?] were examined.

NEUROBEHAVIOURAL EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:


OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 1a and table 1b)
HISTOPATHOLOGY: Yes (see table 2a and table 2b)

Statistics:

The significance of inter-group differences in haematology, blood chemistry and urinalysis (volume, specific gravity and pH only) was assessed by Student's t-test using a pooled error variance. Statistical significances for eosinophil, basophil, monocyte and large unstained cell counts are not reported as these data are normally distributed.
For organ weights and bodyweight changes, homogeneity of variance was tested using Bartlett's test. Whenever this was found to be statistically significant a Behrens-Fisher test was used to perform pairwise comparisons, otherwise a Dunnett's test was used.
Inter-group differences in macrocopic pathology and histopathology were assessed using Fisher's Exact test.
Unless stated, group mean values or incidences for the treated groups were not significantly different from those of the Controls (p>0.05).
Because of the small gnumber of animals in each group, the results of these tests cannot be considered definitive and are used merely as a guide in the interpretation of the results.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
No deaths and no signs attributable to treatment with 3,3'-[biphenyl-4,4'-diylbis(oxy)]dianiline

BODY WEIGHT AND WEIGHT GAIN
Compared with the Controls there was a lower weight gain in males receiving 40 mg/kg/day and a higher weight gain seen in females receiving 40 or 200 mg/kg/day. In the absence of any dosage-relationship, these differences were considered to represent normal biological variation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Total food consumption during the treatment period and reversibility phase was similar for Controls and treated animals.

FOOD EFFICIENCY
The food conversion efficiency of Control and treated animals was essentially the same.

HAEMATOLOGY
Haematological investigations on Day 30 of treatment revealed many changes in comparison with the Controls, the majority of which were confined to animals receiving 1000 mg/kg/day.
Packed cell volumes were slightly reduced and haemoglobin concentrations and red blood cell counts were reduced in animals receiving 1000 mg/kg/day. Haemoglobin concentrations were also reduced in females receiving 200 mg/kg/day. Mean cell haemoglobin concentrations were low in females receiving 200 or 1000 mg/kg/day, mean cell haemoglobins were slightly high in males receiving 1000 mg/kg/day and mean cell volumes were high in animals receiving 1000 mg/kg/day. Examination of blood smears indicated polychromasia in one male receiving 1000 mg/kg/day and in four females receiving 1000 mg/kg/day.
Animals receiving 1000 mg/kg/day had higher lymphocyte and neutrophil counts than the Controls, whilst animals receiving 200 mg/kg/day also had high neutrophil counts. As a result of this, total leucocyte count was raised in animals receiving 1000 mg/kg/day, with a similar but less marked effect being present in animals receiving 200 mg/kg/day.
Of the canges detected at the end of the treatment period, only increased mean cell volumes in females previously given 1000 mg/kg/day were still present at Day 16 of the reversibility period. In contrast to the findings on Day 30, packed cell volumes and haemoglobin concentrations were higher in females that previously received 1000 mg/kg/day than in Controls.

CLINICAL CHEMISTRY
There were no changes in plasma on Day 30 that could be attributed with any confidence to treatment with m-BP.

URINALYSIS
The composition of the urine on Day 30 of treatment and on Day 16 of the reversibility phase was unaffected by treatment with m-BP.

ORGAN WEIGHTS
After 30 days of treatment, absolute and bodyweight-related spleen weights in males given 200 or 1000 mg/kg/day and in females given 1000 mg/kg/day were higher than those of the Controls. These changes had recovered by the end of the 16 day reversibility period.

GROSS PATHOLOGY
The spleen of one female given 1000 mg/kg/day was considered swollen after 30 days of treatment.
There were no other macroscopic findings after 30 days of treatment or after 16 days of reversibility which were attributed to treatment with m-BP. The findings in animals on this study were of the types commonly seen in rats of this age and occurred with the expected frequency.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no microscopic findings that were considered to be related to treatment with m-BP. The findings in animals on this study were of the types commonly seen in rats of this age and occurred with the expected frequency.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)


HISTORICAL CONTROL DATA (if applicable)


OTHER FINDINGS

Effect levels

Dose descriptor:

NOAEL

Effect level:

40 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: overall effects A dosage of 200 mg/kg/day or more was associated with an increase in lymphocyte and neutrophil numbers. These changes were fully reversible.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

It is concluded that oral administration of the test substance 3,3-(4,4-biphenylenedioxy)dianiline to CD rats for 30 days at a dosage of 1000 mg/kg/day was associated with a slight macrocytic anaemia and at 200 mg/kg/day or more with an increase in lymphocyte and neutrophil numbers. These changes were fully reversible. The no-observed-effect level (NOEL) in this study was 40 mg/kg/day.

Executive summary:

A study was performed to test the effects of repeated doses of the test substance 3,3-(4,4-biphenylenedioxy)dianiline to CD rats for 28 days in accordance with the requirements of the Japanese Ministry of Health and Welfare for Japan - Part II of the Chemical Substance Control Law (1986).

Groups of five male and five female CD rats received the test substance orally, by gavage, at dosage of 40, 200 or 1000 mg/kg bw/day for 30 days. A similarly constituted group received the vehicle only and served as a control. A further five males and five females were assigned to the control and high dosage group and were treated for 30 days, followed by a 16 day period without treatment for reversibility studies. There were no signs related to treatment and no animals died. Body weight gain, food consumption and food conversion efficiency were not adversely affected by treatment. On Day 30, haematologic effects were seen in female rats treated with 200 mg/kg bw/day and higher and in male rats treated with 1000 mg/kg bw/day. Lymphocyte counts were increased in animals receiving 1000 mg/kg bw/day and neutrophil counts were increased in animals receiving 200 or 1000 mg/kg bw/day. Spleen weights were increased in males that received 200 or 1000 mg/kg bw/day and females that received 1000 mg/kg bw/day. Macroscopic examination on Day 30 indicated a swollen spleen in one female given 1000 mg/kg bw/day. There were no microscopic findings that were attributed to the treatment. It is concluded that oral administration of 3,3'-[biphenyl-4,4'-diylbis(oxy)]dianiline to CD rats for 30 days at dosage of 1000 mg/kg bw/day was associated with a slight macrocytic anaemia and with an increase in lymphocyte and neutrophil numbers at 200 mg/kg bw/day or higher. These changes were fully reversible. Therefore, the no-observed-effect level (NOEL) in this study was 40 mg/kg bw/day.