3-Pyridinecarbonitrile, 4-methyl-2,6-bis[(4-methylphenyl)amino]-5-[2-[2-(trifluoromethyl)phenyl]diazenyl]-

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:

2003-08-28 to 2004-02-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline and GLP-compliant study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

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:

1995

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

RCC Ltd.

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

444-370-5

EC Name:

-

Cas Number:

669005-94-1

Molecular formula:

C28 H23 F3 N6

IUPAC Name:

4-methyl-2,6-bis[(4-methylphenyl)amino]-5-{2-[2-(trifluoromethyl)phenyl]diazen-1-yl}pyridine-3-carbonitrile

Details on test material:

- Description: orange solid
- Expiration date: 01 Oktober 2005
- Sorage conditions: at room temperature (18-25 °C) in the original container away from direct sunlight
- Stability of the test item: Stable under storage conditions

Test animals

Species:

rat

Strain:

other: HanBrl:WIST (SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd.
- Age at study initiation: 6 weeks
- Weight at study initiation: males: 133.5 - 159.1 g (mean 144.7 grams); females: 117.3 - 130.2 g (mean 122.3 grams)
- Housing: in groups of five, in Macolon type-4 cages
- Diet: Pelleted standard Provimi Kliba 3433, ad libitum
- Water: Community tap-water was available ad libitum
- Acclimatization period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 3 °C
- Humidity: 30-70 %
- Air changes: 10-15 air changes per hour
- Photoperiod: 12 hours fluorescent light / 12 hours dark, music during the light period.

In-Life phase: 28 August 2003 to 23 October 2003

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

300

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item formulations were prepared weekly.
The test item was weighed into a glass beaker on a tared Mettler balance and the vehicle added. The mixtures were prepared using a magnetic stirrer and stored at room temperature (17-23°C).
Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration, homogeneity and stability (after 2 hours and 7 days) of the dose formulations were determined in samples taken after experimental start. Concentration and homogeneity of the dose formulations were determined in samples taken during week 3 of the treatment.
The analyses were performed by RCC Ltd using an HPLC method.

Duration of treatment / exposure:

28 days (duration of recovery 14 days)

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 50, 200, 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

Groups 1 and 4: 10 males; 10 females
Groups 2 and 3: 5 males; 5 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based upon the results of a non-GLP 5-day dose range- finding study (RCC Study Number 850315) in which the test item was administered by gavage to 2 rats per group and sex.

Positive control:

Not required

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily on days 1-3; as well as once daily on days 4-28 and once daily during days 29-42 (recovery)


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once weekly (weeks 1-3)

BODY WEIGHT: Yes
- Time schedule for examinations: weekly during pretest, treatment and recovery

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 4, 6 weeks
- Anaesthetic used for blood collection: Yes (under light isoflurane anesthesia)
- Animals fasted: Yes
-Parameters examined: See Table 1

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 4, 6 weeks
- Animals fasted: Yes
-Parameters examined: See Table 2


URINALYSIS:
- Time schedule for collection of urine: after 4, 6 weeks
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: Volume (18 hours), specific gravity (relative density), Color, Appearance, pH, Nitrite, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Erythrocytes, Leukocytes


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during week 4
- Battery of functions tested: grip strength / motor activity

Sacrifice and pathology:

ORGAN WEIGHTS: yes (See table 3)
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes (see table 4)

Statistics:

The following statistical methods were used to analyze the grip strength, locomotor activity, body weight, organ weights and ratios:
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- Fisher's exact-test was applied to the macroscopic findings.
The following statistical methods were used for statistical analysis of clinical laboratory data:
-Quantitative data were analyzed by a one-way analysis of variance (ANOVA) when the variances were considered homogeneous according to Bartlett.
Alternatively, if the variances are considered to be heterogenous (p<0.05), a non-parametric Kruskal-Wallis test was used. Treated groups were compared to the control groups using Dunnett's test if the ANOVA was significant at the 5% level and by Dunn's test in the case of a significant Kruskal-Wallis test (p<0.05).
- Ordinal data were analyzed using the Kruskal-Wallis test. If this test was significant (p<0.05), comparisons were made between the control group and each of the treatment groups using Dunn's test.

Results and discussion

Results of examinations

Details on results:

CLINICAL SIGNS AND MORTALITY
All animals survived until scheduled necropsy.

BODY WEIGHT AND WEIGHT GAIN
There were no test item-related effects on the mean body weights or mean body weight gain during the treatment or recovery periods when compared with the controls.

HAEMATOLOGY:
The haematology parameters of the test item-treated males were unaffected.

CLINICAL CHEMISTRY
There were no test- item-related effects on clinical biochemistry parameters.

URINALYSIS
The urinalysis parameters of the test item-treated males and females were unaffected.

NEUROBEHAVIOUR
Grip Strength: There were no test item-related changes in the mean fore- and hindlimb grip strength.
Locomotor Activity: There were no test item-related changes in the mean locomotor activity.

ORGAN WEIGHTS
The mean absolute and relative organ weights were generally unaffected by the treatment with the test item after 4 weeks and no late effects were noted after the recovery period.

MACROSCOPIC / MICROSCOPIC FINDINGS
There were no test item-related macroscopic findings with the exception of pink mucosal staining of the cecum. This finding was considered to be a typical change following oral ingestion of dyestuffs.
There were no test item-related microscopic changes.

Effect levels

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Test item-related findings were restricted to passive findings (discoloration of the feces at all dose levels and discoloration at 1000 mg/kg bw/day).

Target system / organ toxicity

Critical effects observed:

not specified