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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Justification for type of information:
Data is from secondary source NTRL

Data source

Reference
Reference Type:
other: NTRL
Title:
Repeated dose oral toxicity study of the test chemical
Author:
NTRL
Year:
1981
Bibliographic source:
NTRL

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: Refer below principle
Principles of method if other than guideline:
Repeated dose chronic/carcinogenicity study was conducted to determine the toxic nature of the test chemical
GLP compliance:
not specified
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material: Trichlorocarbanalide
- Molecular formula: C13H9Cl3N2O
- Molecular weight: 315.5861 g/mol
- Substance type: Organic
- Physical state: White powder
- Impurities (identity and concentrations): 100 % pure

Test animals

Species:
rat
Strain:
other: CD
Details on species / strain selection:
No data
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Wilmington, Massachusetts
- Age at study initiation: 46 days
- Weight at study initiation: No data
- Fasting period before study:
- Housing: The animals were housed individually in elevated stainless steel cages
- Diet (e.g. ad libitum): Standard laboratory diet (Purina Lab Chow R 5001) ad libitum
- Water (e.g. ad libitum): By automated water system ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Monitored twice daily
- Humidity (%): No data
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12 hrs light/dark cycle

IN-LIFE DATES: From: To: No data

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: Feed
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Appropriate amounts of test substance and standard laboratory diet were mixed weekly to give dose levels of 0, 25, 75 or 250 mg/Kg/day

DIET PREPARATION
- Rate of preparation of diet (frequency): Continuously
- Mixing appropriate amounts with (Type of food): No data
- Storage temperature of food: No data

VEHICLE
- Justification for use and choice of vehicle (if other than water): Feed
- Concentration in vehicle: 0, 25, 75 or 250 mg/Kg/day
- Amount of vehicle (if gavage): No data
- Lot/batch no. (if required): No data
- Purity: No data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
200g samples of control feed and each dietary level were taken weekly for analysis
Duration of treatment / exposure:
2 years
Frequency of treatment:
Daily
Doses / concentrations
Remarks:
0, 25, 75 or 250 mg/Kg/day
No. of animals per sex per dose:
0 mg/Kg/day: 80 males and 80 females
25 mg/Kg/day: 80 males and 80 females
75 mg/Kg/day: 80 males and 80 females
250 mg/Kg/day: 80 males and 80 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: No data
- Rationale for animal assignment (if not random): Random assignment
- Rationale for selecting satellite groups: No data
- Post-exposure recovery period in satellite groups: No data
- Section schedule rationale (if not random): No data
Positive control:
No data

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily for mortality
- Cage side observations checked in table [No.?] were included. Mortality and gross signs of toxicology or pharmacologic effects

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Twice pretest, weekly through 13 weeks, biweekly 14 thourgh 26 weeks and monthly thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, Twice pretest, weekly through 13 weeks, biweekly 14 through 26 weeks and monthly thereafter
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes, calculated from food consumption data

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: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations: No data

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pretest, 6, 12, 18 and 24 months
- Dose groups that were examined: All animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at 6 and 12 months for group receiving 0 and 250 mg/kg/day test chemical; 10/sex/group for all groups at 18 (females only) and 24 months
- Anaesthetic used for blood collection: Yes, light ether anaethesia
- Animals fasted: Yes, overnight fasting
- How many animals: Animals were selected randomly; 10/sex/group
- Parameters checked in table [No.?] were examined. Hemoglobin, hematocrit, eryhtocytes, reticulocytes, prothrombin time, total and differential leukocytes, erythrocyte morphology, Heinz bodies and methemoglobin

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at 6 and 12 months for group 0 and 250 mg/kg/day test chemical; 10/sex/group for all groups at 18 (females only) and 24 months
- Animals fasted: Yes, overnight fasting
- How many animals: Animals were selected randomly; 10/sex/group
- Parameters checked in table [No.?] were examined. Serum glutamic pyruvic transminase, alkaline phosphatase, blood urea nitrogen, fasting glucose, total bilirubin

URINALYSIS: Yes
- Time schedule for collection of urine: No data
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters checked in table [No.?] were examined. Gross appearance, specific gravity, pH, protein, glucose, ketones, bilirubin, occult blood, microscopic analysis

NEUROBEHAVIOURAL EXAMINATION: No data
- Time schedule for examinations: No data
- Dose groups that were examined: No data
- Battery of functions tested: sensory activity / grip strength / motor activity / other: No data

OTHER: No data
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, complete gross postmortem examination was performed

Organs weighed: Brains, pituitary, adrenals, testes, ovaries, heart, spleen, kidneys and liver

HISTOPATHOLOGY: Yes, adrenals, bone marrow, blood smear, brain, epididymis, eye, heart with coronary vessels, intestine, cecum, colon, duodenum, ileum, jejunum, kidneys, liver, lungs with mainstem bronchi, lymphnodes, ovary, pancreas, pituitary, prostate/urinary bladder/ seminal vesicles, salivary glands, skeletal muscle, sciatic nerves, spinal cord, spleen, stomach, testis, thymus, thyroid, parathyroid, trachea, uterus, urinary bladder, gross lesions, tissue masses or suspet tumors and regional lymph nodes were examined
Other examinations:
No data
Statistics:
Yes, statistical analysis was performed

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No physical observations were noted wich could be attributed week, to treatment. During 64-86 weeks, there was an overall increase in the number of control and treated males which exhibited varying degrees of emaciation , labored breathing and rales.
Mortality:
no mortality observed
Description (incidence):
No test substance related mortality was observed during the study. Almost twice as many animals were classified as moribund or died spontaneously in the control and treated males than in control and treated female groups throught the study.

During months 17, 18 and 19, 108 males and 24 female control and treated animals died or were sacrifised as moribund animals. This increase in mortality was attributed to respiratory infection which was present predominantly in the males during this time period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weights of 250 mg/Kg/day males and 75 and 250 mg/Kg/day females were lower than the controls during most of the study. However, differences from control never exceeded 9 and 12% in the females and 6% in the males
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The mean food consumption displayed normal variability, and was comparable to that of control
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No ocular abnormalities attributed to test chemical treatment were noted
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Mean hemoglobin and hematocrit values were significantly reduced for 250 mg/Kg/day dose males throughout the study and for 75 mg/Kg/day at 6 and 12 months. Mean erythrocyte counts were significanlt reduced in the 250 mg/Kg/day at 6 and 12 months and in 75 mg/Kg/day males at 12 months. Slight decrease were also observed in the 75 mg/Kg/day at months and in the 250 mg/Kg/day males at termination. The mean erythrocytes count of the 25 mg/Kg/day males was also significantly elevated in the 250 mg/Kg/day males at termination and were considered unremarkable in the 25 and 75 mg/Kg/day males at termination.

The mean leucocytes count was significantly elevates in the 25 and 75 mg/Kg/day males at 6 months and in the 25, 75 and 250 mg/Kg/day at 12 months. Mean total leukocyte counts were slightly elevated in the high dose males at termination.

In the females, mean erythrocyte counts were significanlt reduced in the 75 and 250 mg/Kg/day at 6 months ans in the 250 mg/Kg/day at 12 and 20 months. A slight increase was also observed at termination in the 250 mg/Kg/day group. A slight decrease was also observed at termination in the 250 mg/Kg/day group. Reductions in hemoglobin and hematocrit values for the 250 mg/Kg/day females were statistically significant at 6 and 20 months but not at 12 months and termination. In the 75 mg/Kg/day females, mean hemoglobin values were significantly reduced at 6 months and mean hematocrit values were slightly reduced at termination. Mean total and differential leucocytes at all study intervals and mean reticulocyte counts at termination were considered unremarkable.

Mean methemoglobin levels and mean prothrombin times were unremarkable in the treated males and females throughout the study. Heinz body counts were considered comparable to control in the treated males and females at termination.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The mean alkanline phosphatase level in 75 mg/Kg/day males was significanlt increased at 12 months and slightly increased at termination. Mean values for the 250 mg/Kg/day males were significantly elevated at both intervals. Mean blood urea nitrogen levels were slightly increased in the 250 mg/Kg/day males at 6 and 12 month. Mean total bilirubin values were slightly but statistically significantly increased for high dose females at all study intervals.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No significant difference in treated and control animals was observed
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Mean absolute and relative testes weights ans testes/brain weight ratios were elevated in the high dose males at 6 and 12 months but not at termination. These increases were statistically significant with theexception of the relative testes weight at 6 months and the testes/brain weight rations at 12 months. Mean absolute spleen weights, spleen/body weight ratios and spleen/brain weight ratios were elevated in the 75 and 250 mg/Kg/day at 6 and 12 months and at termination. These increases were dose related and statistically significant. Increased spleen weights were observed in the 75 and 250 mf/Kg/day females at 6, 12 and 20 months and termination and in 25 mg/Kg/day females at termination. These increases were not as pronounced as those in the males and were not significant statistically.

Mean absolute liver weights, liver/body weight rations and liver/brain weight ratios were significantly increased in the 75 mg/Kg/day males at 6 months and in 250 mg/Kg/day at 6 and 12 months and at termination. Slight increase were observed in the 75 mg/Kg/day males at 12 months and in the 25 and 75 mg/Kg/day males at termination. Mean liver weight increases were also observed in 75 and 250 mg/Kg/day and in the 25, 75 and 250 mg/Kg/day females at 20 months and termination. These increase were statistically significant in the 25 mg/Kg/day at termination and in the 250 mg/Kg/day females at 12 and 20 months and termination. Statistically significance was also noted for liver/body weight rations in the 250 mg/Kg/day females at 6 months and in 75 mg/Kg/day females at 12 and 20 months and termination. Mean absolute and relative adrenal weight increases were also observed in the 75 and 250 mg/Kg/day at 6 and 12 months and in the 25, 75 and 250 mg/Kg/day females at 20 months and termination.

Mean absolute and relative adrenal weights were increased in the 250 mg/Kg/day females at 12 months, in the 25, 75 and 250 mg/Kg/day at 20 months and termination and in the 250 mg/Kg/day males at termination. These were not statistically significant.

Mean absolute heart weight, heart/body weight ratio and heart/brain weight ration were increase in the 250 mg/Kg/day males at 12 months and at termination. These increases were statistically significant with the exception of the heart/brain weight ratio at 12 months and the heart/body weight ratio at termination. These parameters were unremarkable in the treated females at all necropsy intervals.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The testes of the 250 mg/Kg/day males were frequently observed to be small and flaccid. These findings were considered to be treatment related.

No significant difference was seen in the incidence and number of rats with neoplasms in the control and 250 mg/Kg/day group.

A unusually large number of male rats died during months 17-19 of the study. The most common gross findings in these studies were consolidation and edema of the lungs with creamy pus-like material on the pleura. Proteus mirabilis was identified to be the causative agent behind the effects observed.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The 75 and 250 mg/Kg/day males and females exhibited microscopic changes considered to be compound related in the testes/epididymides, liver, spleen, kidneys, bone marrow and mesenteric lymph nodes. The tissue alterations were evident in rats in killed at scheduled necrospies, killed in extremis or dying spontaneously. No significant testicular or epididymal lesions were seen in treated male rats at 6 months. 250 mg/Kg/day male rats killed at the 12 month interim exhibited marked multifocal degeneration of the seminiferous tubules with mild to marked interstitial edema. Spermatocytic spermatidic giant cells were found only in 1 case of tubular degenaration. Similar gonadal lesions were seen in the 75 and 250 mg/Kg/day males killed at termination. The multi-focal tubular degeneration was judged mild to marked and the interstitial edema, moderate to marked. At terminal sacrifice, a fes 250 mg/Kg/day rats with an apparent intact tubular lining showed a mild to moderate decrease in the number of sperm. 250 mg/Kg/day rats which were killed moribund or found dead demonstrated focal to diffuse tubular degeneration with a high incidence of giant cell formation.

The changes in testes were reflected in epididymides. The sperm was either decreased in number mildly or moderately, or absent in the epididymal ducts of 250 mg/kg/day males that died spontanesouly or were sacrificed at 12 months and termination. On these occasions, the ductal lumens were occupied either by a mixture of sperm and cell debris or filled entirely with cell debris. Enlargement of secretory epithelium was first observed in 250 mg/Kg/day males at 12 months. This change persisted at termination but the degree of change was reduced from mild/moderate to minimal/mild.

Mild hepatocellular hypertrophy, centrilobular to midzonal was observed in 25, 75 and 250 mg/Kg/day males and females at 6 and 12 months. An increase incidence of mild hepatocellular hypertrophy was observed in 75 and 250 mg/Kg/day males and females during last 12 months of the study. Brown pigment in kupffer cells was noted as early as 6 months in the 75 and 250 mg/Kg/day in males and females was observed continuously throughout the study. The amount of pigment increased slightly with age as more animals manifested moderate to marked pigmentation. The overall incidence of bile duct hyperplasia and cholangiofibrosis was increased significantly for the 75 and 250 mg/Kg/day males.

No significant difference was noted in the intensity of splenic hemosiderosis among females at 6 and 12 months. Among 250 mg/Kg/day males, the frequency of mild hemosiderosis was increased when compared to control males. Hemosiderosis ceased to be significant at 20 months and termination. Congestion of the spleen was significant for the 25 mg/Kg/day females and 75 and 250 mg/Kg/day males and females at 6 and 12 months. During the last half of the study, an increased incidence of congestion was observed for males and females at all dose levels. The degree of congestion changed from minimal to moderate at 6 months, to mild to moderate at 12 months. Extramedullary hematopoiesis was increased in the high dose males and females at 6 and 12 months, but not at 20 months and termination for the females. The overall incidence of extramedullary hematopoiesis was significantly increased in the 250 mg/Kg/day males and females.

Brown pigment in the cytoplasm of the proximal convulted tubules was present in the 75 and 250 mg/Kg/day males at 6, but not at 12 months. The dgree of pigmentation was slightly increased at 12 months when a few rats exhibited a moderate amount of pigment. Similar pigment was noted in 75 and 250 mg/Kg/day at 20 months and on 75 and 250 mg/Kg/day and 250 mg/Kg/day females at termination.

Hypercellularity of the bone marrow, judged minimal to mild, was seen in males and females at 75 and 250 mg/Kg/day at 6 and 12 months. 250 mg/Kg/day males and females at 12 months also exhibited minimal to mild congestion of the bob=ne marrow. No difference in the cellularity of the female bone marrow was discernible at 20 months. Increase in cellularity and marrow congestion became apparent in the 75 and 250 mg/Kg/day males and females and 250 mg/Kg/day males and females respectively at termination.

Increased brown pigment-laden macrophages in the mesentic lymph nodes was demonstrated by mild and high dose males at 6 and 12 months and high dose females at 12 months, a few affected males and females exhibited a moderate increase in pigment cells at 12 months. This change was not apparent at the 20 month and terminal sacrifice. When a total incidence for each group was examined, the incidence in 75 and 250 mg/Kg/day males was significant.

No other microscopic findings are considered treatment related. The other changes noted represent spontaneous or common incidental histological findings. No morphologic evidence of the carcinogenic potential of the test chemical was observed.
Histopathological findings: neoplastic:
not specified
Description (incidence and severity):
No significant difference was seen in the incidence and number of rats with neoplasm, both benign and malignant, in the control and high dose groups.

Rats with malignant neoplasms wer enot diagnosed in the 250 mg/Kg/day males and females. The incidence of neoplasm at the 25 and 75 mg/Kg/day levels were comparable to or lower than control and 250 mg/Kg/day dosed animals.
Other effects:
not specified

Effect levels

Dose descriptor:
NOAEL
Effect level:
25 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Significant effects were observed at the dose levels of 75 or 250 mg/Kg/day

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The no observed adverse effect level (NOAEL) is considered to be 25 mg/Kg/day when male and female rats were exposed to the test chemical for 2 years.
Executive summary:

Repeated dose chronic/carcinogenicity study was conducted to determine the toxic nature of the test chemical. The study was performed using male and female CD rats for 2 years. The test chemical was mixed with feed and used at dose level of 0, 25, 75 or 250 mg/Kg/day. During the study period, the animals were observed for mortality, clinical signs, body weight and food consumption changes, ophthalmology, hematology, clinical chemistry, urinalysis, gross pathology and histopathology. During the study, no treatment related mortality was noted. No physical observations were noted which could be attributed were to treatment. During 64-86 weeks, there was an overall increase in the number of control and treated males which exhibited varying degrees of emaciation, labored breathing and rales. The mean body weights of 250 mg/Kg/day males and 75 and 250 mg/Kg/day females were lower than the controls during most of the study. However, differences from control never exceeded 9 and 12% in the females and 6% in the males. The mean food consumption displayed normal variability, and was comparable to that of control. No ocular abnormalities attributed to test chemical treatment were noted. Slight decreases were observed in mean hemoglobin, hematocrit and erythrocyte counts in the high-dose males throughout the study. These decreases were associated with an increased mean reticulocyte count at termination. Less marked decreases in mean hemoglobin, hematocrit and erythrocyte counts were noted in the mid-dose males at 6 and l2 months. The mean erythrocyte counts of the low-dose males was also slightly reduced at 12 months. Mean reticulocyte counts were considered unremarkable in the low- and mid-dose males at termination. Mean total leukocyte counts were slightly elevated in the 25 and 75 mg/Kg/day dose males at 6 months, in the 25, 75 and 250 mg/Kg/day dose males at 12 months and in the high-dose males at termination. In the females, mean hemoglobin values and erythrocyte counts were reduced for the 75 and 250 mg/Kg/day dose groups at 6 months and for the 250 mg/Kg/day group at 12 and 20 months and termination. Mean hematocrit values were slightly reduced in the high-dose females at 6 and 20 months and termination. Mean alkaline phosphatase levels were slightly elevated in the 75 and 250 mg/Kg/day dose males at 12 months and at termination. Mean blood urea nitrogen levels were slightly increased in the high-dose males at all study intervals. Mean total bilirubin values were slightly increased in the high-dose females at termination. Mean absolute testes weights, testes/body weight ratios and testes/brain weight ratios were elevated in the high-dose males at 6 and 12 months, but not at termination. Mean absolute and relative (to body and brain weights) spleen weights were elevated in the 75 and 250 mg/Kg/day dose males at all necropsy intervals. Increased Spleen weights were observed in the 75 and 250 mg/Kg/day dose females at 6, 12 and 20 months and at termination and in the 25 mg/Kg/day dose females at termination. Mean liver weights (absolute, liver/body weight and liver/brain weight ratios) were increased in the mid- and high-dose males at 6 and 12 months and in the 25, 75 and 250 mg/Kg/day dose males at termination. Mean liver weight increases were also observed in the 75 and 250 mg/Kg/day dose females at 6 and 12 months and in all treated females at 20 months and termination. Mean absolute and relative adrenal weights were increased in the high-dose females at 12 months, in the 25, 75 and 250 mg/Kg/day dose females at 20 months and termination and in the high-dose males at termination. Mean absolute and relative heart weights were increased in the 250 mg/Kg/day males at 12 months and at termination. Compound-related pathological changes were seen grossly in the 250 mg/Kg/day males only, and microscopically in the 75 and 250 mg/Kg/day dose males and females. Grossly, flaccidity and decrease in size of the testes were shown by a large number of 250 mg/Kg/day dose males. Microscopically significant findings were seen in the testes/epididymides, liver, kidneys, spleen, bone marrow and mesenteric lymph nodes. The microscopic pathological changes consisted of degeneration of seminiferous tubules; enlargement of epididymal secretory epithelium; decrease or absence of sperm in epididymal ducts; hepatocellular hypertrophy; brown pigment in Kupffer‘s cells; cholangiofibrosis; brown pigment in cytoplasm of proximal convoluted tubules; splenic congestion; hypercellularity and congestion of bone marrow. The most common findings in male rats dying at 17-19 months of study were pulmonary consolidation and edema with pus-like material on the pleural surface grossly, and acute multifocal suppurative bronchopneumonia microscopically. No morphologic evidence of the carcinogenic potential of the test chemical was observed. Based on findings of the study, the no observed adverse effect level (NOAEL) is considered to be 25 mg/Kg/day when male and female rats were exposed to the test chemical for 2 years.