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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From Jul. 14, 2009 to Aug. 08, 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, followed guideline, GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
according to US FDA and OECD principles of GLP
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material: Toluene-2,5-diamine sulphate (WR 23005, A005) - TSIN: 23005- Substance type: Pure active substance - Physical state: Light purple powder- Storage condition of test material: Room temperature, protected from light on desiccant.

Test animals

Species:
rat
Strain:
other: Sprague-Dawley Crl:CD (SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS- Source: Sprague-Dawley Crl:CD(SD) rats were obtained from Charles River Laboratories, Portage, Michigan, United States- Age at study initiation: Approx 8 wk of age at the time of randomization to groups.- Weight at study initiation: Males - 243-275 g, Females - 177-213 g at time of randomization to groups.- Housing: The animals were housed individually in suspended stainless steel cages during acclimation and while on study. Housing and care were as specified in the USDA Animal Welfare act (9 CFR, Parts 1, 2 and 3) and as described in the Guide for the Care and Use of Laboratory Animals (Washington, D.C.: National Academy Press. NRC; 1996). At least one marble was placed in each animal’s cage starting on Day 2 (males)/Day 1 (females) and for the remainder of the study as a means of environmental enrichment.- Diet: PMI Nutritional International Certified Rodent Chow #5002 (ad libitum) - Water: Reverse osmosis treated and ultraviolet –irradiated municipal tap water (ad libitum) - Acclimation period: Minimum of 9 d prior to first day of dosingResults of analysis of diet (nutrients and environmental contaminants) and water (total dissolved solids, hardness, microbiological content and various potential environmental contaminants) were assessed and did not reveal any findings that would interfere with the conduct or interpretation of the study.ENVIRONMENTAL CONDITIONS- Temperature: 18-22°C (Actual range)- Humidity: 42-66% (Actual range)- Air changes : Ten or more air changes per hour with 100% fresh air.- Photoperiod 12 h light/12 h dark cycleIN-LIFE DATES: From: Jul. 23, 2009 To: Nov. 20, 2009

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
reverse osmosis deionized (RODI) water, pH of 5.0 ± 0.3 (adjusted using 1 N HCl)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: For each dose level, an appropriate amount of the test substance was weighed into a pre-calibrated screw cap jar. and dissolved in pH-adjusted RODI water. The pH was adjusted in two steps to 5.0 ± 0.3 (using sodium hydroxide) . A sufficient quantity of the pH-adjusted RODI water was added to a pre-calibrated mark to achieve the desired concentration and the jar was wrapped in aluminum foil. The final pH was then measured (4.70 to 5.27). - Frequency of preparation of dosing solutions: The dosing formulations were prepared daily and used within 4 h of preparation based on established stability. Each dosing formulation for the test substance groups was covered with a nitrogen blanket following preparation prior to dispensation. VEHICLE- Concentration in vehicle: 0, 0.25, 0.5, 1 and 2 mg/mL- Amount of vehicle: 10 mL/kg (based on most recent body weight measurement)Frequency of preparation of vehicle: The pH-adjusted RODI water was prepared daily, dispensed into amber, glass, screw cap jars with a nitrogen blanket applied.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration, homogeneity and stability of the dose formulations (collected within 8 h of preparation) were determined in samples taken after experimental start.Name of the method: HPLCTime schedule for sample collection:- Determination of concentration: Day 1, 8, 50, 85- Determination of homogeneity: Day 1Dose concentrations analyzed:- Concentration: 0.25, 0.5, 1.0, 2.0 mg/mL- Homogeneity: 0.25, 2.0 mg/mL (preparations from top, middle and bottom were analyzed)- Stability: 0.25, 0.5, 1.0, 2.0 mg/mLConcentration of the dose formulations prepared were within acceptable limits of ± 15% error. The values for homogeneity obtained were 0.3% and 1.3% RSD for the 0.25 mg/mL and 2.0 mg/mL formulations, respectively. Stability of formulations under the storage conditions (room temperature, protected from light, covered with nitrogen) was confirmed for 8 hours.Details are provided in the study report
Duration of treatment / exposure:
91 d
Frequency of treatment:
once daily
Doses / concentrations
Remarks:
Doses / Concentrations:0, 2.5, 5, 10 and 20 mg/kg/dayBasis:actual ingested
No. of animals per sex per dose:
- Assessment of toxicity: 150 animals (15 males and 15 females per dose group) - Assessment of recovery: 20 animals (5 males and 5 females each for control and the highest dose group)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based a previous 13-wk oral (gavage) toxicity study conducted at these dose levels. The study indicated that the highest dose produced some toxic effects, but not excessive lethality that prevented meaningful evaluation. Intermediate dose levels were expected to produce no or minimal toxic effects. The low-dose level was expected to produce no observable indications of toxicity. - Rationale for animal assignment: Animals were randomly assigned to groups by a stratified randomization scheme designed to achieve similar group mean body weights.- Animal assignment for assessment of toxicity: The animals were assigned into following groups:Group 1: Vehicle treated (control)Group 2: 2.5 mg/kg bw/dayGroup 3: 5mg/kg bw/dayGroup 4: 10 mg/kg bw/dayGroup 5: 20 mg/kg bw/day- Animal assignment in satellite groups: Prior to main phase termination, recovery animals were randomly selected with no restrictions on randomization. Additionally, 5 animals per sex in each of Group 1 and 5 were used to assess the recovery.- Post-exposure recovery period in satellite groups: 28 d

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes - Time schedule: At least once daily.MORTALITY/MORBUNDITY: Yes- Time schedule: Twice dailyDETAILED CLINICAL OBSERVATIONS: Yes- Time schedule: Once prior to initiation of treatment and weekly during the treatment and recovery periods for each sex on Day 1, 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, 85, 91, 98, 105, 112 and 119. A final detailed clinical observation was performed for each animal on the day of scheduled euthanasia.BODY WEIGHT: Yes - Time schedule for examinations: Individual body weights were recorded on the day of randomization (Day -1 for males and Day -2 for females) and weekly throughout the treatment and recovery periods (Day 1, 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, 85, 91, 98, 105, 112 and 119). A final fasted body weight was recorded on the day of scheduled euthanasia for calculation of organ-to-body weight ratios.FOOD CONSUMPTION: Time schedule for examinations: Food consumption measurements were recorded weekly throughout the treatment and recovery periods (Day 1, 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, 85, 91, 98, 105, 112 and 119). 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 reportedOPHTHALMOSCOPIC EXAMINATION: Yes- Time schedule for examinations: Prior to in-life initiation (Day-3 males/Day-4 females) and during the last week of the treatment period (Day 86 males/Day 85 females). The ophthalmic examinations were not performed on the same days that FOB evaluations were scheduled.- Method of examination: The ocular examinations were conducted using a hand-held slit lamp and indirect ophthalmoscope. A short-acting mydriatic solution was used to dilate the eyes and facilitate the indirect ocular examinations. - Dose groups that were examined: All groupsCLINICAL PATHOLOGYAnimals were fasted overnight prior to clinical pathology sample collections, but had ad libitum access to water. Hematology, blood coagulation, Clinical chemistry, Urinalysis were assessed on Day 92 (groups for assessment of toxicity) and Day 120 (groups for assessment of recovery) or prior to euthanasia (unscheduled euthanasia animals).Haematology: Yes- Collection of Blood samples: Blood samples (target volume of 2.0 mL/sample) were collected from the vena cava (at gross necropsy) into tubes containing K2EDTA as the anticoagulant.- How many animals: All animals- Parameters examined: Red blood cell count, hemoglobin concentration, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin concentration, mean corpuscular hemoglobin, reticulocyte count (absolute), platelet count, white blood cell count, neutrophil count (segmented neutrophils), lymphocyte count, monocyte count, eosinophil count, basophil count, large unstained cells, other cells (as appropriate)Blood Coagulation: Yes- Collection of blood samples: Blood samples were collected (target volume of 1.8 mL/sample) from the vena cava (at gross necropsy) into tubes containing sodium citrate as the anticoagulant. The blood samples were processed to obtain plasma - How many animals: All animals- Parameters examined: Activated partial thromboplastin time, prothrombin time.Clinical Chemistry: Yes - Collection of Blood samples: Blood samples (target volume of 2 mL/sample) were collected from the vena cava (at gross necropsy) into serum separator tubes- How many animals: All animals- Parameters examined: Alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma glutamyltransferase, creatinine phosphokinase, total bilirubin, urea nitrogen, creatinine, calcium, phosphorus, total protein, albumin, globulin, albumin/globulin ratio, glucose, cholesterol, triglycerides, sodium, potassium, chloride.Urinalysis: Yes- Time schedule for collection of urine: Day 92 (groups for assessment of toxicity) and Day 120 (groups for assessment of recovery)- Metabolism cages used for collection of urine: Yes; the animals were individually housed in stainless steel urine collection cages and urine was collected by cage pan drainage overnight. - Parameters examined: Color, clarity, specific gravity, microscopic evaluation of urine sediment, total volume, pH, protein, glucose, bilirubin, ketones, nitrites, leukocytes, blood and urobilinogen.NEUROBEHAVIOURAL EXAMINATION: Yes- Time schedule for examinations: Full Functional observational battery: Once prior to in-life initiation (males: Day 7; females: Day 8) and once two days prior to cessation of treatment (males: Day 89; females: Day 90).Abbreviated functional observational battery: Day 26 and 54- Dose groups that were examined: All dose groups- Battery of functions tested: Abbreviated FOB assessment: Home cage, removal from home cage, and open field evaluation. The evaluations were performed blind to the observer Full FOB assessment: Home cage, removal from home cage, open field evaluation, manipulative tests, and motor activity. The evaluations were performed blind to the observer.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes- Time schedule for examinations: Surviving animals were euthanized (on Day 92 (groups for assessment of toxicity) and Day 120 (groups for assessment of recovery)- Method of euthanasia:Isoflurane inhalation followed by exsanguination. - How many animals: All animals- Examinations: The necropsy examination included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues. HISTOPATHOLOGY: Yes- Time schedule for tissue collection: Tissues were collected after euthanasia on Day 92 (groups for assessment of toxicity) and Day 120 (groups for assessment of recovery.- Tissues collected: Adrenal gland (paired), animal identification, aorta, bone (femur), bone (sternum), bone marrow smear (collected from the femur ), brain (cerebrum, cerebellum, brain stem, medulla), cervix, diaphragm, epididymis (paired) esophagus, eye (paired), heart, intestine (cecum), intestine (colon), intestine (duodenum), intestine (ileum with Peyer's patch-examined only if present in the routine section), intestine (jejunum), intestine (rectum) kidney (paired), liver, lung, lymph node (mandibular), lymph node (mesenteric), mammary gland, optic nerve ,sciatic nerve (sciatic), ovary (paired), pancreas, parathyroid gland (examined only if present in the routine section), pituitary gland, prostate gland, salivary gland (paired), seminal vesicle (paired), skeletal muscle (thigh), skin (inguinal), spinal cord (cervical, thoracic, lumbar), spleen, stomach (nonglandular and glandular), testis (paired), thymus, thyroid gland (paired), tongue, trachea, urinary bladder, uterus, vagina and gross lesions/masses.- Preservation of tissue samples: Tissue samples were preserved in 10% neutral buffered formalin except eye and optic nerve (preserved in Davidson’s fixative and then transferred to 10% neutral buffered formalin) and testis (preserved in modified Davidson’s fixative and then transferred to 10% neutral buffered formalin)- Slide Preparation: All tissues and organs collected at necropsy (except animal identification) were trimmed, embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin. - Tissue slides examined microscopically: All tissues collected from the control and highest dose group animals and the gross lesions from all animals in all groups. The skeletal muscle (thigh and diaphragm), tongue, heart, and periocular muscle were identified as possible target tissues in the highest dose animals and, therefore, were processed and examined from the other treatment groups.
Other examinations:
ORGAN WEIGHTS: - Time schedule for measurements: Organ weights were measured at scheduled euthanasia on Day 92 (groups for assessment of toxicity) and Day 120 (groups for assessment of recovery)- Organs weighed: Adrenal gland, brain, epididymis, heart, kidney, liver, lung, ovary, pituitary gland, prostate gland, salivary gland, seminal vesicle, spleen, testes, thymus, thyroid gland with parathyroid gland, and uterus.
Statistics:
Inferential statistical analyses were performed for the toxicity and recovery phase animals using the Compaq Alpha DS10 Computer. The following parameters and end points were analyzed: body weights; body weight changes; food consumption; hematology, coagulation, clinical chemistry, and urinalysis (specific gravity, pH, and total volume); organ weights; and parametric ranked, and count FOB data.Each data set was subjected to a statistical decision tree. Data sets for each interval were initially analyzed for homogeneity of variance using Levene's test followed by the Shapiro-Wilk test for normality. A p<0.001 level of significance was required for each test to reject the null hypothesis.If both Levene's test and the Shapiro-Wilk test were not significant, a single-factor parametric ANOVA was applied, with animal grouping as the factor, using a p<0.05% level of significance. If the parametric ANOVA was significant at p<0.05, Dunnett's test was used to identify statistically significant differences between the control group and each test substance treated group using a minimum significance level of p<0.05.If either Levene's test and/or Shapiro-Wilk test were significant, then the Kruskal-Wallis non-parametric ANOVA was applied, with animal grouping as the factor, using a p<0.05 level of significance. If the non-parametric Kruskal-Wallis ANOVA was significant at p<0.05, Dunn's test was used to identify statistically significant differences between the control group and each test substance treated group using a minimum significance level of p<0.05.Descriptive (categorical) and quanta FOB data was analyzed by Fisher's Exact test. If significance was detected, a group by group comparison was conducted using Chi-Square test.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
elevation in aspartate aminotransferase (AST)in males and females at 20 mg/kg bw/day
Urinalysis findings:
no effects observed
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:
effects observed, treatment-related
Description (incidence and severity):
microscopical finding in the skeletal muscles of thigh, diaphragm, tongue, and periocular muscle at 20 mg/kg bw/day
Details on results:
MORTALITY: No test substance related mortality occurred during the study. On Day 81, one control male was euthanized moribund due to clinical signs of hypothermia, dehydration and lethargy. The cause of death was most likely due to renal dysfunction. Details of gross necropsy findings are available in the study report. CLINICAL SIGNS: No test article-related clinical signs were noted during the study. Sporadic occurrences of overt aggressiveness, vocalization, scabs, hair loss, raised areas, dark material around the facial area, and broken incisors were noted throughout the dose groups. Such clinical signs are typical findings in rats of this age and strain and were not considered to be test article related.Details are available in the study report.BODY WEIGHT AND WEIGHT GAIN: There were no statistically significant differences in mean body weights for the males and females during the study. Few instances of statistically significant differences in mean body weight changes were observed in males and females but these were not considered to be test substance related.Details on individual body weight are available in the study report.FOOD CONSUMPTION: There were no test substance related differences in mean food consumption noted during the study.OPHTHALMOSCOPIC EXAMINATION: There were no test substance related differences in ophthalmic findings among the dose groups on Day 85 (males) or Day 86 (females).HAEMATOLOGY AND BLOOD COAUGULATION: No test substance related changes in hematology and coagulation parameters occurred during the study. Statistically significant differences were observed in some parameters (erythrocytes, hemoglobin, hematocrit, MCHC, mean corpuscular volume, prothrombin time, monocytes, segmented neutrophils) but these were not considered meaningful. Details are available in the study report.CLINICAL CHEMISTRY: The only test substance related change in clinical chemistry was an elevation in aspartate aminotransferase in the high dose males (group mean 134 ± 115.8 IU/L) and females (group mean 233 ± 155.6 IU/L) . The increase in males was not statistically significant and was observed in a minority of animals. This elevation in aspartate aminotransferase correlated with microscopic changes in the skeletal muscle of the thigh, diaphragm, tongue, and periocular muscle. At the end of the recovery period, aspartate aminotransferase levels in the high dose animals were comparable to those of controls. URINALYSIS: No test substance related differences in macroscopic or microscopic urinalysis parameters occurred during the study.NEUROBEHAVIOUR: There were no test substance related effects on functional observational battery findings during the study. Sporadic occurrences of head weaving/bobbing were noted in control and treated animals, and were therefore not considered to be test substance related. ORGAN WEIGHTS: No test substance related changes in organ weights occurred during the study. Statistically significant differences observed in both males and females were not regarded as meaningful.Details are available in the study report.GROSS PATHOLOGY: There were no test substance related gross findings during the study. The gross necropsy findings observed were not considered test substance related. HISTOPATHOLOGY: NON-NEOPLASTIC: Groups for assessment of toxicity (at 20 mg/kg bw/day)- Increase in mononuclear cell infiltrates in the skeletal muscle of the thigh, diaphragm, tongue, and periocular muscles but the tunica muscularis of the esophagus was not affected. . - In sections of thigh muscle, there was also an increased incidence of myofiber degeneration These changes in the muscle at these sites were considered related to test substance administration. Groups for assessment of recovery: No test substance related microscopic findings were noted. The gross findings observed were considered incidental, of the nature observed in this age of rat and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to treatment. Details of histopathological changes are available in study report.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Over all effects
Dose descriptor:
LOAEL
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Effects consisted of microscopic changes in the skeletal muscle of the thigh, diaphragm, tongue and periocular muscle.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Toluene-2,5-diamine sulphate (2 -methyl-p-phenylenediamine) when administered orally to Crl:CD (SD) rats at levels 2.5, 5, 10, 20 mg/kg bw/day revealed a NOAEL of 10 mg/kg bw/day based on microscopic changes in the skeletal muscle of the thigh, diaphragm, tongue, and periocular muscle at the highest dose of 20 mg/kg bw/day that correlated with elevations in AST.
Executive summary:

The purpose of this study was to evaluate the effects of Toluene-2,5-diamine sulphate (2 -methyl-p-phenylenediamine) in Crl:CD (SD)Sprague Dawley rats when administered once daily by oral gavage for 91 d following OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents).

A total of 170 Crl:CD (SD) rats (approx. 8 wk old) were used. The body weight of males was 243-275 g and that of females was 177 – 213 g. The feed used was PMI nutritional international certified Rodent Chow # 5002 (ad libitum).The animals were individually housed n suspended stainless steel cages. Standard laboratory conditions were maintained (temperature: 18 to 22°C; humidity: 42 -66%; artificial light: 12 h cycle).

The test material was tested at following concentrations:

0 (control), 2.5, 5, 10, 20 mg/kg bw/day

In this study, 15 animals per sex per dose were used for assessment of toxicity at the end of 91 d while additional 5 animals per sex in the control and the highest dose group were used for assessment of recovery

 

The following parameters and end points were evaluated in this study: clinical signs, functional observational battery (FOB), body weights, body weight changes, food consumption, ophthalmology, clinical pathology parameters (hematology, coagulation, clinical chemistry, and urinalysis), gross necropsy findings, organ weights, and histopathologic examinations.

No test substance related mortality occurred and there were no adverse test substance related effects on clinical signs, functional observational battery findings, body weights and body weight changes, food consumption, ophthalmic findings, hematology parameters, coagulation parameters, urinalysis parameters, gross necropsy findings, or organ weights. The only test substance related change in clinical chemistry was an elevation in aspartate aminotransferase (AST) in the high dose males (group mean 134 ± 115.8 IU/L) and females (group mean 233 ± 155.6 IU/L) on Day 92. The increase in males was not statistically significant and was observed in a minority of animals. This elevation in AST correlated with microscopic changes in the skeletal muscle of the thigh, diaphragm, tongue, and periocular muscle.. At the end of the recovery period, AST levels were comparable to those of controls.

Test substance related microscopic changes were only observed in animals at 20 mg/kg/day and were found in the skeletal muscle of the thigh, diaphragm and tongue and in the periocular muscle of the eye. These changes consisted of mononuclear cell infiltrates (eye, diaphragm, thigh and tongue), muscular degeneration (diaphragm, thigh and tongue), and muscular regeneration (diaphragm). After a 28 -day recovery period, the test substance findings noted at the terminal sacrifice were not present at the recovery sacrifice.

Gonadal tissues were examined for both gross pathology and histopathology and no treatment related effects were detected.

Under the test conditions, the NOAEL was established at 10 mg/kg bw/day based on microscopic changes in the skeletal muscle of the thigh, diaphragm, tongue, and periocular muscle at the highest dose of 20 mg/kg bw/day that correlated with elevations in AST.