9,10-Anthracenedione, 1,4,5,8-tetrakis[(4-butylphenyl)amino]-

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:

2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to OECD TG 407, EPA OPPTS 870.3050 and in accordance with the Principles of Good Laboratory Practice (GLP).

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

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)

Deviations:

yes

Remarks:

Minor deviations (eg. single instance of extension of the 12 hour light/dark cycle and a missing right adrenal gland from one animal at neropsy - omitted from statistical analysis) which did not have an impact on the quality and outcome of the study

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS Guideline 870.3050

Deviations:

yes

Remarks:

same as above

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

445-710-5

EC Name:

-

Cas Number:

108313-21-9

Molecular formula:

C54H60N4O2

IUPAC Name:

1,4,5,8-tetrakis[(4-butylphenyl)amino]-9,10-dihydroanthracene-9,10-dione

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Spectrace MD-710 Marker [solvent stripped]/1,4,5,8-Tetra (4'-n-butylphenylamino) Anthraquinone (TBPAAQ)
- Substance type: very dark green to black powder
- Physical state: solid
- Analytical purity: > 97% active ingredient
- Lot/batch No.: Lot 20070406
- Expiration date of the lot/batch: March 2010
- Stability under test conditions: expected to be stable for the duration of testing
- Storage condition of test material: room temperature

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, North Carolina
- Age at study initiation: approximately 7 weeks
- Weight at study initiation: 220 g to 260 g for males and 164 g to 208 g for females
- Housing: individually in clean, stainless steel, wire-mesh cages suspended above cage-board
- Diet (e.g. ad libitum): in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996). PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002 (meal)
- Water (e.g. ad libitum): Reverse osmosis-treated (on-site) drinking water, delivered by an automatic watering system, and the basal diet were provided ad libitum throughout the study, except during the period of fasting prior to blood collection when food, but not water, was withheld.
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 70.1°F to 71.5°F (21.2°C to 22.0°C)
- Humidity (%): 37.5% to 53.4%
- Air changes (per hr): 10 fresh air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Remarks:

(lot no. 026K0051, exp. dates: 12 June 2012 and 12 July 2012, and lot no. 126K0117, exp. date: 17 July 2008, both manufactured by Sigma Aldrich)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The appropriate amount of test substance for each formulation was weighed into tared, calibrated glass containers. Approximately 70% of the required amount of corn oil was added to each container. The formulation was mixed until uniform using a magnetic stirrer. Corn oil was then added to each container to bring the formulation to the calibration mark, and the formulation was homogenized using a Silverson L4RT or L4RT-A electric homogenizer until a uniform mixture was obtained. The formulation was then mixed using a magnetic stirrer until uniform.
The test substance formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation and stored refrigerated. The daily aliquots were removed from refrigeration, homogenized and stirred at room temperature prior to release for dose administration. The test substance formulations were stirred continuously throughout the preparation, sampling and dose administration procedures.

VEHICLE
- Justification for use and choice of vehicle: Corn oil is a recommended vehicle by various regulatory agencies
- Amount of vehicle (if gavage): 10 ml/kg
- Lot/batch no. (if required): (lot no. 026K0051, exp. dates: 12 June 2012 and 12 July 2012, and lot no. 126K0117, exp. date: 17 July 2008, both manufactured by Sigma Aldrich)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Prior to the initiation of dose administration, quadruplicate samples (1 mL each) for determination of homogeneity, resuspension homogeneity (3 and 10 days) and stability were collected from the top, middle and bottom strata of the 10 and 100 mg/mL dosing formulations. Due to out-of-specification results for the 3-day stability assessment, which was thought to be the result of resuspension technique, additional samples were collected from the formulations prepared for analysis of 6-day resuspension homogeneity and stability. In addition, quadruplicate samples (1 mL each) for concentration analysis were collected from the
middle strata of the dosing formulations prepared for use during study weeks 0 and 3. Two samples from each set of 4 were analyzed; the remaining 2 samples were stored frozen (approximately -65°C to -85°C) as back-up samples. All analyses were conducted by the Analytical Chemistry Department, WIL Research Laboratories, LLC.
The analyzed dosing formulations were found to contain the amount of Spectrace MD-710 Marker [solvent stripped] (85% to 115% of target), were homogeneous (variability for the mean concentration was ≤10% RSD at a concentration within the acceptable limits) and were stable (post-storage concentration was not less than 90% of the pre-storage value) under refrigerated conditions for up to 10 days with the following exception. The 10 mg/mL formulation stored refrigerated for 3 days was determined to be 89.4% of the pre-storage value. The results were thought to be due to the resuspension technique, which was magnetic stirring for at least 15 minutes. Subsequent to the collection and analysis of the 3-day samples, the resuspension technique was amended to included homogenization with an electric homogenizer; consequently, all formulations resuspended after the 3-day time point met test substance stability criteria. No test substance was detected in the vehicle formulation used for administration to the control group (Group 1).

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily once for 28 days

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
100 mg/kg/day
Basis:
other: nominal concentration

Doses / concentrations 2

Remarks:

Doses / Concentrations:
300 mg/kg/day
Basis:
other: nominal concentration

Doses / concentrations 3

Remarks:

Doses / Concentrations:
1000 mg/kg/day
Basis:
other: nominal concentration

No. of animals per sex per dose:

10 male + 10 female rats/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on the results of the 14-day dose range-finding toxicity study.
- Rationale for animal assignment: random

Positive control:

not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, once in the morning and once in the afternoon for mortality and moribundity. Clinical examinations were performed twice daily fro all animals, at the time of dosing and approximately 1-2 hours after dosing.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to the scheduled necropsy
- Anaesthetic used for blood collection: Yes (inhalation of isoflurane)
- Animals fasted: Yes
- How many animals: all animals
- Parameters examined: Total leukocyte count (White Cells), Erythrocyte count (Red Cells), Hemoglobin, Hematocrit, Mean corpuscular volume (MCV), Mean corpuscular hemoglobin, (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (Platelet), Prothrombin time (ProTime), Activated partial thromboplastin time (APTT), Reticulocyte count - Percent (Reticulocyte) Absolute (Retic Absolute), Differential leukocyte count - Percent and absolute - Neutrophil, Lymphocyte, Monocyte, Eosinophil, Basophil, Large unstained cell, Platelet estimate, Red cell morphology - (RBC Morphology) and coagulation time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to necropsy
- Animals fasted: Yes
- How many animals: all animals
- Parameters examined: Albumin, Total protein, Globulin [by calculation], Albumin/globulin ratio (A/G Ratio) [by calculation], Total bilirubin (Total Bili), Urea nitrogen, Creatinine, Alkaline phosphatase (AlkalinePhos’tse), Alanine aminotransferase (Alanine Transfer), Aspartate aminotransferase (AspartatTransfer), Gamma glutamyltransferase (GlutamylTransfer), Glucose, Total cholesterol (Cholesterol), Calcium, Chloride, Phosphorus, Potassium, Sodium, Triglycerides (Triglyceride)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: study week 3
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity / grip strength / motor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Other examinations:

The following organs were weighed from all animals at the scheduled necropsy: Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries (with oviducts), Spleen, Testes, Thymus and Uterus. Paired organs were weighed together and organ-to-final-body-weight ratios were calculated

Statistics:

Standard statistical methods were employed

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:

not examined

Behaviour (functional findings):

no effects observed

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:

CLINICAL SIGNS AND MORTALITY - All animals survived to the scheduled necropsy. There were no adverse test substance-related clinical observations. Test substance-related clinical observations were noted in all test substance-treated groups and consisted of low incidences of wet/dry green material on various body surfaces (mouth, ventral neck, dorsal trunk and head, and anogenital region). These findings did not occur in a dose-responsive manner. The coloration was attributed to the physical properties of the test substance formulations and was characterized as staining, which is not an adverse effect. All other clinical findings in the test substance-treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose-related manner and/or were common findings for laboratory rats of this age and strain.

BODY WEIGHT AND WEIGHT GAIN - Body weights were unaffected by test substance administration. There were no statistically significant differences when the control and test substance-treated groups were compared.

FOOD CONSUMPTION - There were no test substance-related effects on food consumption. There were no statistically significant differences when the control and test substance-treated groups were compared.

HAEMATOLOGY - Hematology and coagulation parameters were unaffected by test substance administration. The only statistically significant (p<0.05) differences from the control group consisted of higher platelet values for the 100 and 1000 mg/kg/day group males. These group mean differences were not considered to be test substance-related because the values were not observed in a dose-related manner, were observed in males only and were of a magnitude that would be considered to be toxicologically unimportant.

CLINICAL CHEMISTRY - There were no test substance-related effects on serum chemistry parameters. However, some statistically significant (p<0.05) differences were observed when the control and test substance-treated groups were compared. These findings included: lower A/G ratio for 300 mg/kg/day group males, higher globulin for 1000 mg/kg/day group females and lower alkaline phosphatase for 1000 mg/kg/day group females. These group mean differences were not considered to be test substance-related because the values did not show a dose-related response, involved a single sex, were of a magnitude that would be considered toxicologically unimportant and/or involved a change in a direction of no known biological importance.

NEUROBEHAVIOUR -
Home cage observations - Home cage observations were unaffected by test substance administration. There were no statistically significant differences when the test substance-treated males and females were compared to the control group at the study week 3 evaluation.
Handling observations - Handling observations were unaffected by test substance administration. There were no statistically significant differences when the test substance-treated males and females were compared to the control group at the study week 3 evaluation.
Open field observations - Open field observations were unaffected by test substance administration. There were no statistically significant differences when the test substance-treated males and females were compared to the control group at the study week 3 evaluation.
Sensory observations - Sensory observations were unaffected by test substance administration. There were no statistically significant differences when the test substance-treated males and females were compared to the control group at the study week 3 evaluation.
Neuromuscular observations - Neuromuscular observations were unaffected by test substance administration. The 300 mg/kg/day group females were noted to have a significantly higher mean hindlimb footsplay value when compared to the control group; however, the higher hindlimb footsplay noted in the 300 mg/kg/day group females was not considered to be related to test substance administration because a dose-related response was not observed. There were no other statistically significant differences when the test substance-treated males and females were compared to the control group at the study week 3 evaluation.
Physiological observations - Physiological observations were unaffected by test substance administration. There were no statistically significant differences when the test substance-treated males and females were compared to the control group at the study week 3 evaluation.
Locomotor activity - Locomotor activity patterns (total and ambulatory activity counts) were unaffected by test substance administration. Values obtained from the 4 epochs evaluated (0-15 minutes, 16-30 minutes, 31-45 minutes and 46-60 minutes) and the overall 60-minute test session were similar to the concurrent control values. No remarkable shifts in the pattern of habituation occurred in any of the test substance-treated groups when the animals were evaluated.

ORGAN WEIGHTS - Organ weights were unaffected by test substance administration. There were no statistically significant differences when the control and test substance-treated groups were compared.

GROSS PATHOLOGY - Test substance-related macroscopic findings at the scheduled necropsy included green discoloration and/or dark green contents of various segments of the gastrointestinal tract (stomach, jejunum, ileum, cecum and colon). These findings were not observed in a dose responsive manner and were attributed to the color of the test substance formulations and were not considered to be adverse.

HISTOPATHOLOGY - No test substance-related microscopic changes were observed in any of the rats administered 1000 mg/kg/day of Spectrace MD-710 Marker [solvent stripped]. Incidental microscopic findings consisted of foci of mononuclear inflammatory cell infiltrations in the coagulating gland, epididymides, heart, kidneys, lacrimal glands, larynx, liver and trachea. These observations occurred sporadically and were not attributed to the test substance. The data support the conclusion that test substance administration had no effect on the occurrence of this common, spontaneously-occurring lesion in young rats. Other microscopic changes observed in the designated organs and tissues were typical of those that occur spontaneously in laboratory rats of this age and strain and were not test substance-related.

Effect levels

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, the no-observed-effect-level (NOEL) for oral (gavage) administration of Spectrace MD-710 Marker [solvent stripped] to Crl:CD(SD) rats over a period of 28 days was 1000 mg/kg/day.

Executive summary:

The objective of this study was to evaluate the possible toxic effects of the test substance, Spectrace MD-710 Marker [solvent stripped], also known as 1,4,5,8-tetra (4’-n-butylphenylamino) anthraquinone, or TBPAAQ, when administered orally (by gavage) to rats for 28 days. This study included evaluation of potential neurotoxicity by functional observational battery (FOB) and locomotor

activity (MA) assessment.

Spectrace MD-710 Marker [solvent stripped] in the corn oil vehicle was administered orally by gavage once daily for 28 consecutive days to 3 groups (Groups 2-4) of Crl:CD(SD) rats. Dosage levels were 100, 300 and 1000 mg/kg/day. A concurrent control group (Group 1) received the vehicle on a comparable regimen. The dose volume was 10 mL/kg for all groups. Each group consisted of 10 animals/sex. Study day 0 was designated as the first day of dosing.

All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed at the time of dosing and approximately 1-2 hours after dosing. Detailed physical examinations were performed weekly. Individual body weights and food consumption were recorded approximately weekly. Functional observational battery (FOB) and locomotor activity data were recorded for all animals during study week 3. Clinical pathology evaluations (hematology and serum chemistry) were conducted for all animals on the day of the scheduled necropsy (study day 28). Complete necropsies were conducted on all animals, and selected organs were weighed at the scheduled necropsy. Selected tissues were examined microscopically from selected animals.

All animals survived to the scheduled necropsy. Body weights and food consumption were unaffected by test substance administration. There were no test substance-related functional observational battery or locomotor activity findings or effects on hematology, coagulation and serum chemistry parameters. Organ weights were unaffected by test substance administration, and there were no test substance-related histopathologic findings.

Clinical observations of green material on various body surfaces and macroscopic observations of green discoloration and/or green contents of various segments of the gastrointestinal tract were noted in all test substance-treated groups. These findings were attributed to the presence of Spectrace MD-710 Marker [solvent stripped] and were not an adverse effect.

Daily oral (gavage) administration of Spectrace® MD-710 Marker [solvent stripped] to Crl:CD(SD) rats at dosage levels of 100, 300 and 1000 mg/kg/day for 28 days was well tolerated. There were no effects on body weights, food consumption, hematology or serum chemistry parameters, organ weights or histopathology. There were no indications of neurotoxicity following FOB and locomotor activity assessment. Clinical and macroscopic observations of green material were noted on various body surfaces and in or on various segments of the gastrointestinal tract. These observations were attributed to the presence of Spectrace MD-710 Marker [solvent stripped] and were not an adverse effect. Based on the results of this study, the no-observed-effect-level (NOEL) for oral (gavage) administration of Spectrace MD-710 Marker [solvent stripped] to Crl:CD(SD) rats was 1000 mg/kg/day.