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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: According to or similar to guideline study OECD method 408.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report Date:
1991

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): MCP 917
- Expiration date of the lot/batch: Aug 01, 1994
- Storage condition of test material: room temperature

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic, Germantown New York
- Age at study initiation: ca. 42 days
- Housing: individually
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum



ENVIRONMENTAL CONDITIONS
- Temperature (°F): 68-72
- Humidity (%): 40-60
- Photoperiod (hrs dark / hrs light): 12/12


Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test material was incorporated into the diet at levels of 0, 500, 5000, 20000 ppm. Trial diet was prepared and analyzed for homogeneity, stability and concentration. The diet had to be stable for a minimum of three weeks. The concentration of test material in the diet had to be at or no more than 10% the level specified in the protocol.

DIET PREPARATION
- Rate of preparation of diet (frequency): one week prior to study initiation and at approximately two week intervals thereafter.
- Mixing appropriate amounts with (Type of food): Purina Certified Lab Chow
- Storage temperature of food: room temperature

Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
13 weeks. Except for periods of deprivation required for hematology/serum chemistry studies and necropsy.
Frequency of treatment:
Daily for 13 weeks. Except for periods of deprivation required for hematology/serum chemistry studies and necropsy.
Doses / concentrations
Remarks:
Doses / Concentrations:
500, 5000, 20000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
20/sex/dose for a total of 40 animals/dose
Control animals:
yes, plain diet
Details on study design:
N/A
Positive control:
N/A

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily



DETAILED CLINICAL OBSERVATIONS: Yes / No / No data
- Time schedule:


BODY WEIGHT: Yes
- Time schedule for examinations: Day after receipt and the day of their release from quarantine. Animals allocated into experimental groups were weighed immediately before the initiation of treatment and approximately weekly 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: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No


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



OPHTHALMOSCOPIC EXAMINATION: Yes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to study initiation and during weeks 5 and 13
- Anaesthetic used for blood collection: Yes (diethyl ether)
- Animals fasted: Yes
- How many animals: 10/sex/dose for a total of 20 animals/dose



CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to study initiation and during weeks 5 and 13
- Animals fasted: Yes
- How many animals: 10/sex/dose for a total of 20 animals/dose



URINALYSIS: Yes
- Time schedule for collection of urine: weeks 5 and 13
- Metabolism cages used for collection of urine: No
- Animals fasted: No data


NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Adrenals, heart, ovaries, testes, brain, kidneys, prostate, thymus, epididymides, liver, spleen, uterus

HISTOPATHOLOGY: Yes
Adrenals, bone and marrow, brain, epididymides, esophagus, eye and optic nerve, gross lesions, heart and aorta, small and large intestine, kidneys, liver, lung, lymph nodes, mammary gland, ovaries, pancreas, peripheral nerve, pituitary, prostate and seminal vesicles, salivary gland, skeletal muscle, spleen, spinal cord, stomach, testes, thymus, thyroid/parathyroid, trachea, urinary bladder, uterus
Statistics:
Quantitative data were analyzed by parametric methods: analysis of variance and associated F-test followed by Tukey’s multiple comparison test, provided that there was statistical significance in ANOVA.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
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:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
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:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
Seven males (1 control, 6 high-dose) died or were sacrificed before the scheduled necropsy. No other clinical signs indicative of systemic toxicity were observed during the study.

HAEMATOLOGY
Statistically significant, dose-related alterations in hematology parameters reported for females included red blood cell count, hemoglobin, hematocrit, segmented neutrophils and lymphocytes. More specifically, red blood cell count, hemoglobin, and hematocrit were decreased, and segmented neutrophils were increased, at week 13 in females receiving 20,000 ppm of the test compound. Lymphocytes were reduced in 20,000 ppm females at week 1, and in females receiving 5000 and 20,000 ppm at week 13, with no significant differences observed at week 5. A linear dose-response relationship was found for all of these parameters, suggesting that the results were treatment-related. However, there were no statistically significant changes in hematology parameters among females receiving 500 ppm of the test compound, and the values in 5000 ppm females did not differ significantly from controls for any parameter other than lymphocytes. Comparison with historical reference values to further determine the significance of the results indicated that the dose-response for segmented neutrophils and lymphocytes at 20,000 ppm fell outside the normal range (10 - 90th percentile). The remaining hematology parameters were within normal ranges. No hematology effects were reported for male rats.


CLINICAL CHEMISTRY
Parameters affected at week 13 included serum urea nitrogen, uric acid, alanine aminotransferase (ALT), albumin, potassium, phosphorus, calcium, and chloride in males; and ALT, alkaline phosphatase (ALP), cholesterol, total bilirubin, total protein, albumin, globulin, sorbitol dehydrogenase (SDH), chloride, and cholinesterase in females.

A linear dose-response relationship was found with regard to ALT, uric acid, albumin, potassium, and phosphorus in males; and with regard to ALT, ALP, cholesterol, total protein, albumin, SDH, and cholinesterase in females, suggesting that these results were treatment related. However, changes observed in males at week 13 were statistically significant only at 20,000 ppm, except for a small but significant (p<0.05) decrease in chloride at 5000 and 20,000 ppm. Calcium was significantly increased in males at 500 ppm but not at higher doses; the lack of a dose-response suggests the change was not substance-related.

In females, ALT, ALP, and SDH levels were significantly increased only at 20,000 ppm, while cholesterol, protein, albumin, and cholinesterase were significantly decreased at both 5000 and 20,000 ppm. Bilirubin was decreased at 500 ppm at week 13, but was not affected at higher doses, suggesting a non-treatment related effect. A small, but statistically significant reduction in chloride levels was found at all doses at week 13, though chloride levels were unaffected at weeks 1 and 5. Chloride levels did not appear to be dose-related, and amounted to only a 2-3% reduction from controls. The A/G ratio was significantly increased at all doses at week 5, reflecting increased albumin and decreased globulin levels at this sampling interval. However, at week 13, globulin was significantly decreased only at 20,000 ppm and the A/G ratio was not affected.

When compared with historical serum reference values, the dose-response curves for male ALT, and phosphorus at 20,000 ppm, and female ALP and cholesterol at 20,000 ppm were outside normal ranges (10 - 90th percentile). The remaining serum chemistry values were within normal ranges.


ORGAN WEIGHTS
Statistically significant increases in the absolute and/or relative weight of the kidney (both sexes) and of the adrenals and liver (females) were seen at 5000ppm and higher. At 20000ppm, significant increases in absolute and/or relative weight of the liver (males) and of the spleen (females) were observed.

GROSS PATHOLOGY
The observations recorded most frequently in the animals sacrificed on schedule included enlarged lymph nodes and abnormal areas of the liver. In general, lymph node enlargement was seen in all groups with equal frequency. Abnormal areas of the liver were observed in approximately half of the females dosed at 20,000ppm. All other observations were seen sporadically and regarded as non-treatment related, spontaneous, unremarkable and/or self-explanatory.

HISTOPATHOLOGY: NON-NEOPLASTIC
At 5000ppm at higher, macrophages were observed in the lymph nodes and liver. There was no substantial difference between the lesions induced at 5000ppm and 20000ppm. The liver lesions indicated that females were more susceptible to the test material than males. The mesenteric lymph node lesions showed no sex predilection. The microscopic changes seen in the mesenteric lymph nodes and liver of rats sacrificed in extremis suggest that the onset of lesions was earlier in the mesenteric lymph nodes than in the liver. No changes were detectable at 500ppm.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
>= 500 ppm
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
LOAEL
Effect level:
<= 5 000 ppm
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The NOAEL for rats orally exposed to the test material is 500ppm (34.6-38.4 mg/kg bw/day). Effects on organ weights were observed at the next highest dose of 5000ppm (340-380 mg/kg/day) and were limited to increases in the absolute and/or relative weight of the kidney (both sexes) and of the adrenals and liver (females). However, there was no evidence of organ dysfunction at this dose. These findings do not warrant classification of the submission substancel for target organ toxicity (repeated exposure) under the new Regulation (EC) 1272/2008 on classification, labeling, and packaging of substances and mixtures (CLP), under the Directive 67/518/EEC for dangerous substances and Directive 1999/45/EC for preparations, or under the Globally Harmonzied System of Classification and Labelling of Chemicals (GHS).
Executive summary:

A 90-day oral toxicity study of the test material was conducted in rats (20/sex/dose) at dietary concentrations of 0, 500, 5,000 and 20,000 ppm. Seven animals died or were sacrificed during the study (1 control, 6 high dose). No other clinical signs indicative

of systemic toxicity were observed during the study. Most of the clinical observations were localized effects (e.g., neck irritation and/or alopecia on neck and forepaws from rubbing or grooming, ear irritation tags, etc). Chromodacryorrhea (red tears) and reddish nasal discharge were likely the result of transitory stress from procedures such as bleeding, handling, etc.

 

Additional parameters evaluated included physical examinations, hematology, serum chemistry, ophthalmology, body weight, gross pathology, histopathology, and urinalysis. No statistically significant differences between dose groups were observed for body weights, food consumption, ophthalmology, or urinalysis.

 

Statistically significant, dose-related alterations in hematology parameters reported for females included red blood cell count, hemoglobin, hematocrit, segmented neutrophils and lymphocytes. More specifically, red blood cell count, hemoglobin, and hematocrit were decreased, and segmented neutrophils were increased, at week 13 in females receiving 20,000 ppm of the test compound. Lymphocytes were reduced in 20,000 ppm females at week 1, and in females receiving 5000 and 20,000 ppm at week 13, with no significant differences observed at week 5. A linear dose-response relationship was found for all of these parameters, suggesting that the results were treatment-related. However, there were no statistically significant changes in hematology parameters among females receiving 500 ppm of the test compound, and the values in 5000 ppm females did not differ significantly from controls for any parameter other than lymphocytes. Comparison with historical reference values to further determine the significance of the results indicated that the dose-response for segmented neutrophils and lymphocytes at 20,000 ppm fell outside the normal range (10 - 90th percentile). The remaining hematology parameters were within normal ranges. No hematology effects were reported for male rats.

 

Serum chemistry for males and females showed some statistically significant differences between untreated and treated animals. Parameters affected at week 13 included serum urea nitrogen, uric acid, alanine aminotransferase (ALT), albumin, potassium, phosphorus, calcium, and chloride in males; and ALT, alkaline phosphatase (ALP), cholesterol, total bilirubin, total protein, albumin, globulin, sorbitol dehydrogenase (SDH), chloride, and cholinesterase in females.

 

A linear dose-response relationship was found with regard to ALT, uric acid, albumin, potassium, and phosphorus in males; and with regard to ALT, ALP, cholesterol, total protein, albumin, SDH, and cholinesterase in females, suggesting that these results were treatment related. However, changes observed in males at week 13 were statistically significant only at 20,000 ppm, except for a small but significant (p<0.05) decrease in chloride at 5000 and 20,000 ppm. Calcium was significantly increased in males at 500 ppm but not at higher doses; the lack of a dose-response suggests the change was not substance-related.

 

In females, ALT, ALP, and SDH levels were significantly increased only at 20,000 ppm, while cholesterol, protein, albumin, and cholinesterase were significantly decreased at both 5000 and 20,000 ppm. Bilirubin was decreased at 500 ppm at week 13, but was not affected at higher doses, suggesting a non-treatment related effect. A small, but statistically significant reduction in chloride levels was found at all doses at week 13, though chloride levels were unaffected at weeks 1 and 5. Chloride levels did not appear to be dose-related, and amounted to only a 2-3% reduction from controls. The A/G ratio was significantly increased at all doses at week 5, reflecting increased albumin and decreased globulin levels at this sampling interval. However, at week 13, globulin was significantly decreased only at 20,000 ppm and the A/G ratio was not affected.

 

When compared with historical serum reference values, the dose-response curves for male ALT, and phosphorus at 20,000 ppm, and female ALP and cholesterol at 20,000 ppm were outside normal ranges (10 – 90th percentile). The remaining serum chemistry values were within normal ranges.

 

Statistically significant increases in the absolute and/or relative weight of the kidney (both sexes) and of the adrenals and liver (females) were observed at ≥ 5,000 ppm. At 20,000 ppm, significant increases in the absolute and/or relative weight of the liver (males) and spleen (females) were observed.

 

During necropsy of the animals sacrificed on schedule, the most frequent observation was enlarged lymph nodes and “abnormal areas” of the liver. In general, lymph node enlargement was seen in all groups with equal frequency. “Abnormal areas” of the liver were observed in approximately half of the females dosed at 20,000 ppm. Other gross observations were sporadic and non-treatment related.

 

No statistically significant changes were detectable in any of the endpoints examined at the 500ppm dose level for either sex. Based on these data, the NOAEL for this study is considered to be 500 ppm. 

 

The conversion of NOAEL from ppm in the diet to mg/kg bw/day was calculated using the concentration of the test substance in the diet, the food consumption factor and the body weight factor. On this basis, the NOAEL for the study is calculated to be 34.6 to 38.4 mg/kg/day.

Classification for target organ toxicity (repeated exposure) under the new Regulation (EC) 1272/2008 on classification, labeling, and packaging of substances and mixtures (CLP), under the Directive 67/518/EEC for dangerous substances and Directive 1999/45/EC for preparations, or under the Globally Harmonzied System of Classification and Labelling of Chemicals (GHS).