2,2`-[6-(phenylamino)-heteromonocycle-2,4-diyl]diphenol

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From August 8th, 2007 to Jannuary 14th, 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

July 27, 1995

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Directive 96 / 54 / EC, B. 7. "Repeated dose (28 days) toxicity (oral)

Version / remarks:

September 30, 1996

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2,2`-[6-(phenylamino)-1,3,5-triazine-2,4-diyl]diphenol

Cas Number:

1248-66-4

Molecular formula:

C21H16N4O2

IUPAC Name:

2,2`-[6-(phenylamino)-1,3,5-triazine-2,4-diyl]diphenol

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd
- Age at delivery: 6 weeks
- Weight at study initiation: Body weight range (at acclimatization): Males: 137.7 to 160.4 g (Mean: 149.0 ± 6.9 g). Females: 111.3 g to 130.6 g (Mean: 118.7 ± 5.2 g).
- Housing: in groups of five in Makrolon type-4 cages with wire mesh tops.
- Diet: pelleted standard Kliba Nafag 3433 rat mainenance diet ad libitum.
- Water: community tap water from Itingen ad libitum.
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70%
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light/ 12 hour dark

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on oral exposure:

The dose formulations were prepared weekly. The test item was weighed into a glass beaker on a tared Mettler balance and the vehicle (PEG 300) added (weight:volume). The mixtures were stirred using a magnetic stirrer and stored at room temperature (15 - 25 °C). Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

The following doses were administered:
Group 1: 0 mg/kg/day
Group 2: 50 mg/kg/day
Group 3: 200 mg/kg/day
Group 4: 1000 mg/kg/day
The dose levels were selected based on a previous dose range finding toxicity study in Wistar rats, using dose levels of 200, 600, and1000 mg/kg/day.

The dose volume was 5 ml/kg body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

HPLC
On the first treatment day and once during week 3 a sample of the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of homogeneity and concentration. An additional sample of the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken after the end of in life phase. A sample of about 2 g of each concentration was taken five hours and 7 days after commencement of dosing (first treatment day) and on an additional time point after end of in life phase to confirm stability. Additional dose formulation preparation and analysis were performed after end of in life because samples prepared on day 1 needed to be reanalyzed due to technical problems but were inadvertently discarded. The aliquots for analysis of dose formulations were frozen (-20 ± 5 °C) and delivered to RCC Ltd, Itingen /Switzerland and stored there at -20 ± 5 °C until analysis.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily; 7 days/week for 4 weeks;

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
50 mg/kg body weight/day
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
200 mg/kg body weight/day
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
1000 mg/kg body weight/day
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

Doses are based on dose-range-finding

Examinations

Observations and examinations performed and frequency:

Viability/Mortality, General Cageside Observations/ Detailed Clinical Observations (weekly), Grip strenght, Locomotor activity, food consumption, body weight, hematology, clinical biochemistry, urinalysis, organ weight, macroscopic findings, microscopic findings.

Sacrifice and pathology:

HISTOPATHOLOGY: Yes
Gross Pathology: yes

Statistics:

Dunnett-test
Steel-test
Fisher`s exact-test
Armitage/Cochran Trend Test

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:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

One animal (male, 50 mg/kg(day) died spontaneously on treatment day 5. This spontaneously death was not considered to be test-related.

No test item-related clinical signs were observed.

Inreased locomotor activity in both sexes at 1000 mg/kg/day.

Absolute and relative food consumption and body weight was not influenced by the treatment during the treatment and recovery period.

No test item-related alterations in the mean fore- and handlimb grip strenght were recorded in rats at any dose levels.
No test item-related findings in hematology were recorded.

No test item-related changes in clinical biochemistry and urinalysis.

Increased mean thymus/body weight ratio and mean thymus/brain weight ratio was recorded in males at 1000 mg/kg/day at the end of the recovery period. As there were no histopathological correlates this finding was considered to be incidental. No further findings were recorded.

No test item-related macroscopic lesions were observed.

Minimal hypertrophy of the zona fasciculata was recorded in male animals and slight hypertrophy of the zona fasciculata in one female. Minimal hypertrophy of the zona glomerulosa in one male animal.
Minimal increased erythropoiesis was recorded in female animals; after a 14-day-treatment-free recovery period none of these changes were present

Effect levels

1

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: Increased locomotor activity in both sexes at 1000 mg/kg/day

2

Dose descriptor:

LOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: Increased locomotor activity in both sexes at 1000 mg/kg/day

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

not subject of classification and labeling

Executive summary:

Oral administration of test item to Wistar rats of both sexes at doses of 50, 200 and 1000 mg/kg/day, for 28 days resulted in a statistically significant increase in total locomotor activity was observed in males and females at 1000 mg/kg/day. A minimal increased erythropoiesis in the bone marrow was recorded in female animals of group 4. Histopthological findings were reported in the adrenals (minimal hypertrophy of the zona fasciculata in male animals of groups 2, 3 and 4, and slight hypertrophy of the zona fasciculata in one female animal of group 4, minimal hypertrophy of the zona glomerulosa in one male animal of group 2) and an increase in mean adrenal weight, mean adrenal to terminal body weight ratio, and mean adrenal to brain weight ratio in males at 50, 200 and 1000 mg/kg/day. These findings in adrenals were considered to be stress-related and not to be test item-related. After a 14 day treatment-free recovery period none of these histopathological changes were present.
A no-observed-effect-level (NOEL) could not be established within this study.
Due to the increased locomotor activity in both sexes at 1000 mg/kg/day the no-adverse-effect-level (NOAEL) of test item was established at 200 mg/kg/day.