1,4-bis[(2-ethyl-6-methylphenyl)amino]anthraquinone

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 24 September 1997 to 18 November 1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

When comparing to the OECD TG 407 (1995 version), the following deviations were reported: - sensory reactivity to stimuli and functional observations were not conducted - gross necropsy of testes was not performed - histopathological examinations were not performed on all the organs listed in the OECD TG.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

1995

Deviations:

no

Principles of method if other than guideline:

Reinblau RLW Tr. was administered by oral gavage to male and female SD rats for 28 days at dose levels of 0, 8, 40, 200, and 1000 mg/kg to assess its toxicity and reversibility.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1,4-bis[(2-ethyl-6-methylphenyl)amino]anthraquinone

EC Number:

255-460-7

EC Name:

1,4-bis[(2-ethyl-6-methylphenyl)amino]anthraquinone

Cas Number:

41611-76-1

Molecular formula:

C32H30N2O2

IUPAC Name:

1,4-bis[(2-ethyl-6-methylphenyl)amino]-9,10-dihydroanthracene-9,10-dione

Details on test material:

Reinblau RLW Tr. (CAS No. 41611-76-1, Lot No. 25C, Purity: 99.7%) was provided by Bayer Co., Ltd. and used in this study.

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: n/a
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: the stability of the lot used in this study was confirmed but the Sponsor using IR analysis
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: n/a
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: The stability and homogeneity of the test substance in the dosing formulations were confirmed between 0.4 and 100 mg/mL for 8 days in a refrigerator before the start of dosing.
- Reactivity of the test material with the incubation material used (e.g. plastic ware): n/a

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): n/a
- Preliminary purification step (if any): n/a
- Final concentration of a dissolved solid, stock liquid or gel: n/a
- Final preparation of a solid (e.g. stock crystals ground to fine powder using a mortar and pestle): n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material) n/a

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Remarks:

Crj: CD(SD) IGS

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles Liver Laboratories Japan Ltd. on September 24, 1997
- Females (if applicable) nulliparous and non-pregnant: yes; 48 females; 48 males.
- Age at study initiation: 5 weeks
- Weight at study initiation:146 g to 184 g for males and 119 g to 146 g for females.
- Fasting period before study: none
- Housing: polycarbonate cages (265W x 426D x 200H mm, Tokiwa Kagaku Kikai Co., Ltd.) with sterilized hardwood chip for experimental animals (Beta chip, Charles River Laboratories Japan Ltd.). After grouping, two animals were housed in a cage, which was placed on stainless steels racks (Tokiwa Kagaku Kikai Co., Ltd.), and the location of the cages was rotated once a week. Replaced once a week
- Diet (e.g. ad libitum): pellet diet ad libitum (MF, Oriental Yeast Co., Ltd.)
- Water (e.g. ad libitum): ad libitum, tap water passed through 5 µm filter and irradiated with UV light. Replaced once a week.
- Acclimation period: 13 days.

DETAILS OF FOOD AND WATER QUALITY: it was confirmed that the contaminants such as residual pesticides in the diet and bedding were meet the specifications of the standard operating procedure (SOP) of the test facility. In addition, the tap water was periodically analyzed based on the Water Supply Act, and was confirmed to meet the specification of the SOP of the test facility.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2°C (intended value)
- Humidity (%): 55±15%(intended value)
- Air changes (per hr):ventilation was about 12 times per hour
- Photoperiod (hrs dark / hrs light):12/12 (light from 7:00 to 19:00).

IN-LIFE DATES: From: 24 September 1997 To: 4 November 1997.

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

Oral route was selected according to the guideline.

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

CMC-Na solutions

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The dosing volume was set at 10 mL/kg, and the individual dose volume was calculated on the basis of the most recent body weight.

VEHICLE
- Justification for use and choice of vehicle (if other than water): n/a
- Concentration in vehicle: 0.5% (test item suspended with motor and agate)
- Amount of vehicle (if gavage): n/a
- Lot/batch no. (if required): Iwai Chemicals Company Ltd.; Lot Nos. 121208 and 100709
- Purity:n/a

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability and homogeneity for 8 days of the test substance in the dosing formulations at 0.4 and 100 mg/mL were confirmed by the HPLC method.
The dosing formulations of initial preparation were analyzed and confirmed that the mean concentrations were within the nominal concentration ±10%.

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily in the morning

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

8 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

40 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

200 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Control + 200 + 1000 mg/kg bw/day: 12 / sex (incl. 6/sex recovery animals).
8 and 40 mg/kg bw/day: 6 / sex.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: From the results of a preliminary study, the highest dose in this study was set at 1000 mg/kg, and then, doses of 200, 40, and 8 mg/kg were also set by common ratio of 4.
- Rationale for animal assignment (if not random): random
- Fasting period before blood sampling for clinical biochemistry: no
- Rationale for selecting satellite groups: control and high dose groups
- Post-exposure recovery period in satellite groups: 14 days
- Section schedule rationale (if not random): not applicable
- Dose range finding studies: In a preliminary 2-week repeated dose study (dose level: 0, 100, 500, and 1000 mg/kg bw/day; 3 males and 3 females per group), there were no changes attributable to the test substance treatment in clinical sign, body weight, haematology, organ weight, and necropsy.
- Other: none

Examinations

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed twice a day (before and after dosing) during the dosing period, and once in the morning during the other periods.

BODY WEIGHT: Yes
- Time schedule for examinations: once a week.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
-All feeders in each cages (including diet) were weighed using an electric balance once a week, and mean daily food consumption of each animal was calculated.

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: Not specified

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the scheduled necropsy day (days 29 or 43).
- Anaesthetic used for blood collection: Yes (sodium thiopental)
- Animals fasted: No
- How many animals: all animals
- Parameters checked in table [7.5.1/1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the scheduled necropsy day (days 29 or 43).
- Animals fasted: No
- How many animals: all animals
- Parameters checked in table [7.5.1/1] were examined.

PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: Yes
- Time schedule for collection of urine: day 24
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: No
- Parameters checked in table [7.5.1/1] were examined.

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

OTHER: n/a

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 7.5.1/2)

HISTOPATHOLOGY: Yes (see table 7.5.1/2)
At the results of the examination, since there were no changes suspected to be treatment-related, histopathological examination was not performed in the other groups necropsied at the end of the treatment period and all groups necropsied at the end of the recovery period.

Optional endpoint(s):

Optional endpoints: No

Statistics:

Numerical data were analyzed statistically by the multiple comparison tests.
Numerical data were analyzed statistically by the multiple comparison tests. First, the data were tested by Bartlett’s test for homogeneity of variance. When the variance was homogeneous, one-way analysis of variance (ANOVA) was employed, and when the variance was heterogeneous, the Kruskal-Wallis test was performed. When there was a significant difference among the groups, the Dunnett test or the Dunnett-type multiple comparison test was applied. Categorical data were tested by X test (a*b). When there was a significant difference among the groups, Armitage’s X test was applied to compare between the control group and each dose group.
Statistical analysis was performed on the items listed below. The analysis was not performed on the data obtained in the clinical observation, necropsy, and histopathological examination.
Multiple comparison tests: Xtest:
Body weight, food consumption, hematology, blood chemistry, organ weight
Urinalysis (pH, protein, glucose, ketones, bilirubin, occult blood, urobilinogen)

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

There were no abnormalities in any groups including the control group.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The body weight of animals in the test substance-treated groups was comparable to that in the control group.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The food consumption of animals in the test substance-treated groups was comparable to that in the control group.

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no abnormalities attributable to the test substance treatment at the end of the treatment or recovery periods.
Low values of hemoglobin concentration and hematocrit value were noted in males in the 200 mg/kg bw/day group, and shortening of APTT was noted in females in the 200 mg/kg bw/day group at the end of the recovery period; however, these changes were not considered to be treatment-related because similar changes were not noted at the end of the treatment period or in the 1000 mg/kg bw/day group.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no abnormalities attributable to the test substance treatment at the end of the treatment or recovery periods.
A high value of total bilirubin was noted in females in the 200 mg/kg bw/day group; however, similar change was not noted in the 1000 mg/kg bw/day group, and therefore, this change was not considered to be treatment-related. Moreover, low vales of albumin and sodium were noted in males in the 1000 mg/kg bw/daygroup, and a high value of potassium was noted in males in the 200 and 1000 mg/kg bw/day groups; however, these changes were not considered to be treatment-related because these were slight and within the physiological range, and were not noted at the end of the treatment period.

Endocrine findings:

not examined

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no abnormalities attributable to the test substance treatment at the end of the treatment period.
A high value of protein was noted in males in the 8, 40, and 1000 mg/kg bw/day groups; however it was not considered to be treatment-related because it was within the physiological range.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no significant differences between the control and test substance-treated groups in any organs.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no abnormalities attributable to the test substance treatment.
The following changes were noted; however, since these changes were observed spontaneously in this strain, they were not considered to be treatment-related: erythrocytic extramedullary hematopoiesis in the spleen, eosinophilic inclusion body in the hepatocyte, fatty change of the periportal hepatocyte, microgranuloma and focal necrosis in the liver, cyst and hyaline droplet in the proximal tubular epithelium in the kidney.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no abnormalities attributable to the test substance treatment. Hepatodiaphragmatic nodule in the liver and cyst in the kidney were noted in the test substance-treated group; however, these changes were not considered to be treatment-related because these were often observed as spontaneous changes in this strain, and there was no dose-relation.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Effect levels

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

serum/plasma biochemistry

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

The no-observed-effect-level (NOEL) of Reinblau RLW was considered to be 1000 mg/kg bw/day for male and female rats.

Executive summary:

In a subacute toxicity study performed similarly to OECD TG 407 and in compliance with GLP, Reinblau RLW suspended in CMC-Na solutions was administered to Crl: CD SD IGS rats (6/sex/dose in the control and high dose groups; 6/sex/dose in the other groups) by gavage at dose levels of 0, 8, 40, 200 and 1000 mg/kg bw/day. In addition, 6 rats/sex/dose in the control and 1000 mg/kg bw/day groups were subjected to a recovery period of 14 days.

 

There were no test substance-related toxic changes in clinical sign, body weight, food consumption, hematology, blood chemistry, urinalysis, organ weight, necropsy, or histopathology.

Therefore, the no-observed-effect-level (NOEL) of Reinblau RLW was considered to be 1000 mg/kg bw/day for male and female rats.