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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 10, 1986 through September 10, 1986
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
This study was conducted in compliance with the Good Laboratory Practices Regulations as stated in the U.S. Environmental Protection Agency (EPA) Good Laboratory Practice Standards [Subpart I, Part 792, Chapter I of Title 40, Code of Federal Regulationsl, as well as the Organization for Economic Co-operation and Development (OECD Guidelines for Testing Chemicals, ISBN 92-64-12221-4, adopted by the council at its 535th meeting on May 12, 1981.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1987

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): diethyl toluene diamine (DETDA)
- Physical state: amber liquid
- Stability of test material: The stability under storage conditions (nitrogen blanketed at room temperature) was determined at days 0, 45, and at study termination.
-Stability under test conditions: The stability in the diet was determined to be 36 days. The stability under test conditions was determined and the diet was renewed twice a week.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Wilmington, Massachusetts, U.S.A.
- Age at study initiation: 41 days old
- Weight at study initiation:
- Fasting period before study:
- Housing: Rats were housed individually in stainless steel 1/2" wire mesh cages. Size in accordance with the "Guide for the Care and Use of Laboratory Animals" of the Institute of Laboratory Resources, U.S. National Research Council. Waste material was removed three times per week; cages and feeders were sanitized every two weeks.
- Diet (e.g. ad libitum): Purina Certified Rodent Meal, ad libitum. Feeders were designed to reduce soiling, bridging, and scattering. Certified food was used.
- Water (e.g. ad libitum): Availability - fresh tap water, ad libitum. Water was monitored for contaminants at periodic intervals.
- Acclimation period: The rats were acclimated twelve days. During this conditioning period, the rats were weighed and observed for any pharmacotoxic signs of disease or inadequate weight gain.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C +/- 3 °C
- Humidity (%): 30 to 70%
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12 hrs dark/12 hrs light


IN-LIFE DATES: From: June 10, 1986 To: September 9-10, 1986

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Preparation of the test article for administration to the rats consisted of incorporating the test article in the diet utilizing a liquid-solids blender, (Twin-Shell Intensifier Blender, Patterson-Kelly Company, East Stroudsburg, Pennsylvania). Homogeneity of the test article mixture in the feed was determined. Three different levels (top left, top right and bottom) from the blender were sampled. A total of three samples per mixing level, 9 samples for three dose levels, were collected each time the diet was prepared.

DIET PREPARATION
- Rate of preparation of diet (frequency): The diet was renewed twice a week.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
90 days
Frequency of treatment:
Daily for 90 days
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
50 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
125 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
320 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
20 animals per sex per dose
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Based upon a 28-day dose range finding study (Naismith 1986).
- Rationale for animal assignment (if not random): Treatment groups were housed by vertical cage positioning. Randomization was carried out by use of a random number table. One week before treatment commenced all rats were weighed, ranked according to body weight and assigned to treatment groups using a table of random numbers so that each treatment group would have a similar distribution according to body weight. An Analysis of variance Test was performed to insure that no statistical significance was present.
- Rationale for selecting satellite groups:
- Post-exposure recovery period in satellite groups:
- Section schedule rationale (if not random):
Positive control:
No

Examinations

Observations and examinations performed and frequency:
METHODS OF STUDY PERFORMANCE:
Four groups of rats (20 males and 20 females per group) were initially fed with the test article at dose levels of 0, 50, 125 and 320 ppm, seven days per week over a period of 90 days. Pharmacotoxic signs were recorded as they were observed, including the time of onset, degree and duration. Cageside observations included, but were not limited to changes in skin and fur, eyes and mucous membranes, and also respiratory, circulatory, autonomic and central nervous system, somatomotor activity and behavior patterns. Measurements were made of food consumption twice weekly, the diet was replenished with fresh freezer stored diet at the time of food consumption and the rats were weighed weekly.

CLINICAL EXAMINATIONS:
Blood was drawn by percutaneous cardiocentesis. All animals were fasted overnight prior to the time of sacrifice. The following determinations were made on twenty rats (10/sex) prior to initiation. In addition, after 90 days of feeding, five (5) animals per sex per group were chosen by random number tables and the following were determined:
1) Hematology - Hemoglobin, Hematocrit, Erythrocyte count, Total and differential leucocyte counts, and Platelet count;
2) Clinical Biochemistry - Calcium, Phosphorus, Chloride, Sodium, Potassium, Fasting Glucose, Serum alanine aminotransferase, Serum aspartate aminotransferase, Gamma glutamyl transpeptidase, Urea, nitrogen, Albumen, Blood creatinine, Total bilirubin, and Total serum protein measurements.
3) Ophthalmologic - Ophthalmological examinations using an ophthalmoscope were made prior to the administration of the test substance on all animals. At termination of the study 5/sex/group chosen from a table of random numbers were evaluated. If changes in the eyes were detected all the animals would be examined.


Sacrifice and pathology:
SACRIFICE:
At the end of the 90 day period all surviving animals were sacrificed by carbon dioxide asphyxiation. Any weak or moribund animals were removed, sacrificed and necropsied during the study as deemed necessary by the study director and principle investigator.

GROSS NECROPSY:
All animals were subjected to necropsy which included examination of the external surface of the body, all orifices, and the cranial, thoracic, abdominal and pelvic cavities and their contents. The following organs were weighed: Iiver, gonads, adrenal glands, brain and kidneys.

The following organs and tissues from the animals at the 90 day sacrifice were preserved in 10% neutral buffered formalin for future histopathological examination: All gross lesions, Brain-including sections of medulla/pons, Cerebellar cortex and cerebral cortex, Pituitary, Thyroid/parathyroid, Thymus, Lungs, Trachea, Heart, Sternum with bone marrow, Salivary glands, Liver, Spleen, Kidneys/adrenals, Pancreas, Gonads, Uterus, Accessory genital organs (epididymides, prostate, and, if present, seminal vesicles), Aorta, Skin, Esophagus, Nasal turbinates, Stomach, Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum, Urinary bladder, Representative lymph node, Marmnary gland, Thigh musculature, Peripheral nerve, Eyes, Femur-including articular surface, Spinal cord at three levels - cervical, midthoracic and lumbar, and Exorbital lachrymal glands.

HISTOPATHOLOGY:
A full histopathologic evaluation of the above organs and tissues was evaluated in all control and high dose animals sacrificed at 90 days of treatment. All animals which died or were sacrificed in a moribund condition prior to completion of the study were also subjected to a full histopathologic evaluation of the above named tissues and organs. All gross lesions in all animals were examined histologically.


Statistics:
Evaluation of equality of means was made by the one way analysis of variance using the F distribution to assess significance. If significant differences among the means were indicated, Dunnett's test was used to determine significant differences from control means. Analysis of discrete data where appropriate was conducted using non-parametric procedures. All raw data were summarized and statistically evaluated using LABCAT modules designed by Innovative Programming Associates, Inc., One Airport Place, Princeton, NJ 08540.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
effects observed, treatment-related
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS:
Three control males exhibited lesions, alopecia and/or scab formations in the ventral cervical region. One control male, exhibited an opaque, buphthalmic left eye. Inferior, anterior synechia was subsequently diagnosed. This ocular abnormality resulted in chronic keratitis. There were no additional control male or female observations recorded. A similar lesion and alopecia formation were observed in one low dose male rat. Low dose females displayed no pharmacotoxic or abnormal signs. Alopecia and/or lesion formation with or without scab formation was observed from one mid dose male and two mid dose females. In addition, piloerection and chromodacryorrhea were exhibited from one mid dose male. These abnormalities were the result of a dislodged upper incisor. Transient piloerection (2 days) was observed from one mid dose male. Transient poor grooming was also observed from one mid dose female. With the exception of four rats (1 male and 3 females), all the high dose animals exhibited several combinations of pharmacotoxic observations. These included abnormal stance, abnormal gait, flaccid body tone, ptosis, decreased activity, chromodacryorrhea, elevated gait, loss of skin elasticity, poor grooming, dyspnea, tremors, ataxia, and morbidity. In general, these signs resulted in either moribund sacrifice or death in the majority of these high dose rats.

MORTALITY:
Twenty-seven rats from the high dose group died during the study. Thirteen (9 females and 4 males) were found dead and fourteen (7 females and 7 males) were moribund sacrificed. At necropsy, there were no visible lesions recorded for seven females and four males. The majority of the observations recorded for both the males and females were non-specific. These observations recorded included stomach lesions, dark discoloration of the liver, kidneys and/or genital organs, discolored anal region, discoloration and/or distention of the intestines, emaciation and apparent dehydration of the animal, lung congestion, liver foci and lack of thymic tissue. The microscopic correlations of these necropsy observations with the histopathology are provided under the heading 'HISTOPATHOLOGICAL EVALUATION' below.

BODY WEIGHT:
The high dose (320 ppm) group mean male body weights were significantly reduced from study Day 7 through Day 90 when compared to the controls. The mid dose (125 ppm) group mean male body weights were significantly reduced from study Day 77 through Day 90.

The group mean high dose female body weights were significantly reduced from Day 14 through Day 90. In addition, the mid dose female weights were significantly reduced on Days 28 and 35, and from Day 49 through Day 90.

BODY WEIGHT GAINS:
The high dose male body weight gains were significantly reduced on Day 7 through Day 35. The high dose males also displayed a statistically significant body weight loss on Day 42 through Day 90. The total male high dose gain was also significantly decreased when compared to the controls. The mid dose male body weight gains were significantly reduced on Days 70 and 84. The total mid dose gain was also statistically decreased. A statistical increase in the low dose male body weight gains occurred on study Day 21.

In a similar manner, the high dose female body weight gains were significantly reduced on Day 7 through Day 35. A statistically significant weight loss was exhibited by the high dose females on Day 42 through Day 90. The total body weight gain was also significantly decreased. Mid dose females exhibited a statistically significant reduced gain on Days 21, 28, and 84. The total gain was also significantly decreased. No statistically significant changes were observed in the low female dosage group.

FOOD CONSUMPTION:
The amount of feed consumed by the high dose DETDA treated male rats was statistically reduced on Days 14, 21, 24, 28, 35, and 38. The mid dose male food consumption was significantly reduced on Days 45, 59, 63, 73, and 90. Statistically significant increases in the amount of feed consumed by the low dose male group were recorded on Day 3 through Day 28 and on Day 35.

Female high dose food consumption was significantly reduced on Day 17 through Day 52, Mid dose females exhibited a statistically significant reduced feed consumption on Days 45, 52, 59, 63, 70, 73 and Day 80 through 90. No differences were noted between control and low dose female feed consumption. Significant differences in mean food consumption among the four groups were also found on Days 31, 42, 56, 66, 70, and 77; however, no additional significance was noted between control and DETDA treated groups.

OPHTHALMOLOGIC EXAMINATION:
All rats in each dose group were examined prior to study initiation and at termination utilizing indirect ophthalmoscopic and biomicroscopic procedures. The examination evaluated adnexa, conjunctiva, sclera, cornea, anterior chamber, lens and posterior segment (vitreous, retina and optic disc). Eleven rats exhibited abnormal ocular defects prior to study initiation. These animals were subsequently replaced with normal animals. On Day 87 of the study, a pretermination ocular examination was performed on all the remaining rats. Twenty ocular lesions were noted. Two, one, four and thirteen from control to high dose, respectively. Striate retinopathy (right eye) and chronic keratitis were observed in two rats (males) from the control group. Striate retinopathy (right eye) was observed from one low dose female. Two males from the mid dose group also displayed striate retinopathy (right eye). In addition, hyaloid remnant (left eye) from one male and focal posterior cataract (left eye) from one female were also observed in the dosage group. With the exception of the focal posterior cataract, the abnormalities noted are inherent of this species' age group. Eleven high dose rats (males and females) exhibited nuclear sclerosis of both eyes. Two displayed pale fundus, one conjunctivitis, one multifocal cataract, one multifocal anterior cortical and equatorial cataract each from both eyes and three focal cataracts from the right eyes. These noted abnormalities may be considered to be a direct or indirect effect from test article administration.

HEMATOLOGY:
Statistically significant decreases were observed in the high dose male mean corpuscular volume, leucocyte and platelet counts. The high dose male erythrocyte and hemoglobin values were statistically larger when compared to controls. High dose females exhibited statistically significant increases in erythrocyte values. Hematocrit counts for each DETDA-treated female group were statistically larger than the control group. There were no additional statistically significant changes in any hematology parameters for either males or females when the test-article treatment groups were compared to the controls. However, a few instances of individual hematology values outside the normal range were found in each dosage group.

CLINICAL CHEMISTRY:
Males in the high dose group exhibited statistically significant decreases in albumin, globulin, calcium, phosphorus, creatinine, and total protein. Increases which were statistically significant by the high dose males were observed for the blood urea nitrogen, SGPT and SGOT values. The GGPT male values were markedly larger, although not statistically significant. High dosed females displayed statistically significantly larger blood urea nitrogen, GGTP, SGPT and SGOT values. The total protein values for the high dose females were also statistically reduced.

ABSOLUTE ORGAN WEIGHTS:
Statistically significant decreases in the high dose group male kidneys, liver, testes and brain weights were observed when compared to the controls. No significant differences were observed in the female organ weights among the four dose groups.

RELATIVE ORGAN TO BODY WEIGHTS:
Males and females from the high dose group exhibited statistically significant increases in adrenal, kidneys and brain weights to percent body weight. In addition, high dose male testes weights and female liver weights were significantly larger. Also, the male and female mid dose liver and kidney weights and female brain weights were statistically larger than control values.

RELATIVE ORGAN TO BRAIN WEIGHTS:
No statistically significant differences for the females were observed among the four groups. High dose males, however, exhibited a statistically significant increases in kidneys, liver and testes weights when compared to percent brain weight.

GROSS PATHOLOGY:
Similar observations such as kidneys, that appeared mottled, dilated pelvis, subcapsular cystic or presence of calculi, lobular or small livers, congested lungs, crystalline material in the gastrointestinal tract with or without gas distention were observed in each treatment group. There were, however, a few necropsy findings which occurred with greater frequency in the high dose group. The spleens of a few of the high dosed rats appeared small. There were a larger number of focal or linear stomach lesions. In general, the high dose animals appeared thin and dehydrated. With the exception of these observations, the majority of tissues examined appeared normal.

HISTOPATHOLOGY (non-neoplastic):
Treatment-related microscopic changes were present in all of the male and female rats receiving 320 ppm. In these high dose rats, there was a high incidence of bilateral cataractous change in the eyes, diffuse atrophy of the acinar cells of the pancreas, bone marrow depletion, tubular vacuolation ( hydropic change) of the kidneys and vacuolation of the islet cells of the pancreas, atrophy of many organs, lymphoid depletion of the spleen, thymus and mesenteric lymph node, and increased pigmentation of the liver and spleen. A minimal to moderate multifocal degeneration of the acinar cells of the pancreas and increased splenic pigmentation in the females were present in tissues examined from the rats receiving 50 and 125 ppm of DETDA in the diet.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
>= 125 ppm
Sex:
male/female
Basis for effect level:
other: clinical signs (male = 21 mg DETDA/kg bw; female = 27 mg DETDA/kg bw)
Dose descriptor:
LOAEL
Effect level:
>= 320 ppm
Sex:
male/female
Basis for effect level:
other: clinical signs (male = 122 mg DETDA/kg bw; female = 125 mg DETDA/kg bw)
Dose descriptor:
LOAEL
Effect level:
>= 320 ppm
Sex:
male/female
Basis for effect level:
other: mortality/moribundity (male = 122 mg DETDA/kg bw; female = 125 mg DETDA/kg bw)
Dose descriptor:
NOEL
Effect level:
>= 50 ppm
Sex:
male/female
Basis for effect level:
other: body weight (male = 8 mg DETDA/kg bw; female = 10 mg DETDA/kg bw)
Dose descriptor:
LOAEL
Effect level:
>= 125 ppm
Sex:
male/female
Basis for effect level:
other: body weight (male = 21 mg DETDA/kg bw; female = 27 mg DETDA/kg bw)
Dose descriptor:
NOEL
Effect level:
>= 50 ppm
Sex:
female
Basis for effect level:
other: food consumption (female = 10 mg DETDA/kg bw)
Dose descriptor:
NOAEL
Effect level:
>= 50 ppm
Sex:
male
Basis for effect level:
other: increased food consumption (male = 8 mg DETDA/kg bw)
Dose descriptor:
LOAEL
Effect level:
>= 125 ppm
Sex:
male/female
Basis for effect level:
other: decreased food consumption (male = 21 mg DETDA/kg bw; female = 27 mg DETDA/kg bw)
Dose descriptor:
NOAEL
Effect level:
>= 125 ppm
Sex:
male/female
Basis for effect level:
other: ophthalmoscopic examination (male = 21 mg DETDA/kg bw; female = 27 mg DETDA/kg bw)
Dose descriptor:
LOAEL
Effect level:
>= 320 ppm
Sex:
male/female
Basis for effect level:
other: ophthalmoscopic examination (male = 122 mg DETDA/kg bw; female = 125 mg DETDA/kg bw)
Dose descriptor:
LOAEL
Effect level:
>= 320 ppm
Sex:
male/female
Basis for effect level:
other: haematology (male = 122 mg DETDA/kg bw; female = 125 mg DETDA/kg bw)
Dose descriptor:
LOAEL
Effect level:
>= 320 ppm
Sex:
male/female
Basis for effect level:
other: clinical chemistry (male = 122 mg DETDA/kg bw; female = 125 mg DETDA/kg bw)
Dose descriptor:
NOEL
Effect level:
>= 125 ppm
Sex:
male
Basis for effect level:
other: absolute organ weights (male = 21 mg DETDA/kg bw)
Dose descriptor:
NOEL
Effect level:
>= 320 ppm
Sex:
female
Basis for effect level:
other: absolute organ weights (female = 125 mg DETDA/kg bw)
Dose descriptor:
LOAEL
Effect level:
>= 320 ppm
Sex:
male
Basis for effect level:
other: absolute organ weights (male = 122 mg DETDA/kg bw)
Dose descriptor:
NOEL
Effect level:
>= 50 ppm
Sex:
male/female
Basis for effect level:
other: relative organ-to-body weights (male = 8 mg DETDA/kg bw; female = 10 mg DETDA/kg bw)
Dose descriptor:
LOAEL
Effect level:
>= 125 ppm
Sex:
male/female
Basis for effect level:
other: relative organ-to-body weights (male = 21 mg DETDA/kg bw; female = 27 mg DETDA/kg bw)
Dose descriptor:
NOEL
Effect level:
>= 125 ppm
Sex:
male
Basis for effect level:
other: relative organ-to-brain weights (male = 21 mg DETDA/kg bw)
Dose descriptor:
NOEL
Effect level:
>= 320 ppm
Sex:
female
Basis for effect level:
other: relative organ-to-brain weights (female = 125 mg DETDA/kg bw)
Dose descriptor:
LOAEL
Effect level:
>= 320 ppm
Sex:
male
Basis for effect level:
other: relative organ-to-brain weights (male = 122 mg DETDA/kg bw)

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Dietary administration of DETDA was well-tolerated by animals receiving DETDA in chow at 50 ppm (male = 8 mg DETDA/kg bw; female = 10 mg DETDA/kg bw). This level represents the no-observed-adverse-effect level (NOAEL) for male and female rats. Signs of toxicity were evident in animals receiving DETDA in chow at 125 ppm or higher (males = 21 mg DETDA/kg bw; females = 27 mg DETDA/kg bw). The concentration of 125 ppm was designated as the lowest-observed-adverse-effect level (LOAEL) based on decreased body weight and food consumption. Concentration-dependent changes in the pancreas were observed with increasing severity in animals receiving 125 or 320 ppm DETDA in diet. The liver, kidney, and pancreas were target organs of toxicity in animals receiving DETDA in diet at 320 ppm (males = 122 mg DETDA/kg bw; females = 125 mg DETDA/kg bw).