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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Conducted per OECD guideline and GLP. The OECD guideline has been updated some years after the finalization of the present study. However, the updates made to the guideline are not considered to effect the integrity of the results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report Date:
1996

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
not specified
Principles of method if other than guideline:
The report authors cite OECD, Japanese MITI and US EPA guidelines as sources of design features for this study.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
Test animals of both sexes were purchased from Taconic Farms, New York at ~10 weeks of age. Rats were acclimated for 2 weeks prior to study initation, and housed 2 per cage. They were fed Purina 5018 NIH meal and presented drinking water ad libitum. The photoperiod was 12 hr light/12 hr dark in 72 +/- 8 degrees F, and 55 +/- 15% humidity. Air changes in the room ranged from 10 - 12/hr. Rats average weights on Day 1 were ~246 (males), and 162 (females) grams.

In-life dates: From: July 13, 1994 To: August 24, 1994

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
Test substance was prepared by grinding in a coffee mill, and sieved through 125 um mesh screen. This powder was mixed with rodent diet at 0, 120, 470 and 1900 mg/kg diet (delivers ~7.5, 30 & 120 mg W-100/kg bw/d to rats via the diet). Diets were prepared weekly with reserve diets stored in refrigerator until presented to test animals. Stability (for 46 days), homogeneity, and dose verification confirmed delivery and exposure to test compound as described.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Diet samples were shipped from AHF to Goodyear for analyses. Samples were extracted with toluene, and subjected to gas chromatography with N-P detection. Quantitation was achieved by comparing summed chromatographic peaks in test chemical standard and diet preparations. This procedure was carried out to confirm diet homogeneity of test cmpd, stability (periodic sampling over 46 days), and dietary contents. Results from assays confirmed all levels were within 16% variation from target levels in 46 day storage trials, homogeneities for top, middle and bottom samples of mixing vessel were within 13% variation, and diet samples were within 10% variation.
Duration of treatment / exposure:
Rats were exposed to dosed diets for a period of 4 weeks ad libitum.
Frequency of treatment:
Daily for 4 weeks
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
120, 470, 1900 mg/kg diet
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
7.5, 30, 120 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
8 males/8 females were present in all test groups for sacrifice after 28 days.
Additional rats (6M/6F) were added to control & high dose groups to assess recovery post-exposure.
Details on study design:
The dosed feed was presented to 8 male/8 female rats per test group at 12 wks of age for 4 wks. An additional six rats/sex/group were held post-exposure in 2-wk recovery groups in the high dose and control groups. Test rats were monitored for body wts (BW), feed consumption (FC), & clinical signs. Collections were performed on 6 or 3 rats/sex/groups at 28 and 42 sacrifice days for blood (hematologies and clin. chems) and urinalyses, respectively. Necropsies were performed on all rats, and organs were weighed (liver, kidneys, pituitary, uteri, heart, brain, spleen, thyroids, adrenals, testes, ovaries). These and other major organs were preserved in formalin, stained with H&E, and subjected to microscopic evaluations. Liver, kidney, and urinary bladder slices were subjected to immunohistochemical staining for proliferating cell nuclear antigen (PCNA) for assessment of cellular division
Positive control:
None

Examinations

Observations and examinations performed and frequency:
Animals were observed cage-side daily for evidence of mobidity or mortality.

Body weights were recorded at study initiation and weekly thereafter. Feed consumption was recorded for 5 day periods throughout the study. Dietary exposures to test compound and feed efficiencies were not calculated.

Blood samples were collected for hematology and clinical chemistry analyses from 6 rats per test group via cardiac puncture under CO2 anesthesia immediately prior to terminal sacrifice, and following overnight fasting. Parameters assayed were WBC, RBC, Hgb, Hct, MCV, MCH, MCHC, Retic, Plts, Segs, and Lymphs.

Clinical chemistry endpoints were:total bilirubin, total protein, alb, cholesterol, ALT/GPT, AST/GOT.

Urine samples were collected from 3 rats/test group/sex overnight period during fasting and immediately prior to sacrifice. Collections were kept cold by using dry ice cooling. Samples were analyzed for: appearance, volumes, protein, glucose, sp. gravity, occult blood, bilirubin, urobilinogen, pH and ketone bodies.

Sacrifice and pathology:
Necropsies were performed on Days 28-30 and 42 (recovery) following euthanasia by CO2 anesthesia. Organ weights were recorded for liver, kidneys, pituitary, uterus, heart, brain, spleen, thyroids, adrenals, testes, and ovaries. Microscopic pathology was conducted on control & high dose rats of both sexes for liver, kidneys, urinary bladder, brain, pituitary, thyroid/parathyroid, testes/epididymides, spleen, pancreas, lung, and gross lesions.
Other examinations:
Immunohistochemical staining was conducted on liver, kidney, urinary bladder to assess cellular proliferation in all test animals. Results were expressed as the number of cells displaying proliferation in treated groups relative to those values for control animals.
Statistics:
Conducted for all major endpoints.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
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:
not examined
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:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
Body weights were slightly depressed (~5%) in high dose female rats following study initiation. This difference disappeared during 2-week recovery phase. No effect on BWs were seen in males.

Feed consumption was depressed up to ~20% in high dose animals of both sexes during the test chemical exposure period.

Hematological results indicated MCVs (mean corpuscular volumes) were significantly increased in high dose groups of both sexes while mean corpuscular hemoglobin levels were decreased. This latter change was also observed in females at the mid dose. These changes diminished significantly during recovery phase. Macrocytic anemia was the primary change in rats related to test chemical exposure although these changes were reversible within 2 weeks following dietary exposure cessation. Reticulocyte counts increased substantially during the recovery phase.

Findings for clinical chemistry indicated cholesterol increased in high dose rats of both sexes with total recovery in males, females less recovery. Total bilirubin was elevated in males for mid and high dose groups with total recoveries. Increased total protein & alobumin were noted in high dose male recovery groups.

Liver weights were increased in both sexes in the high dose groups. This finding reversed during recovery period. Kidney weights were slightly increased. Heart & spleen wts were increased in females at high dose.

The PCNA exams showed significant increases relative to controls for liver tissue in the low dose groups of both sexes, and the mid-dose males. In kidneys, there was decrease in labeling in the high dose at 28 days, but an increase after 2-weeks recovery. There was increase in labeling in the low and mid doses for females but not males.

Effect levels

open allclose all
Dose descriptor:
LOAEL
Effect level:
ca. 1 900 mg/kg diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
haematology
clinical biochemistry
urinalysis
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
histopathology: neoplastic
Dose descriptor:
NOAEL
Effect level:
ca. 470 mg/kg diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
haematology
clinical biochemistry
urinalysis
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
histopathology: neoplastic
Dose descriptor:
NOEL
Effect level:
ca. 120 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
haematology
clinical biochemistry
urinalysis
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
histopathology: neoplastic

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Various effects were observed in dietary exposures of 470 & 1900 ppm including reversible macrocytic anemia, liver weights, and possibly enhanced cellular division in the liver & bladder. 120 ppm was a NOEL for all effects.
Executive summary:

A 4 -week toxicity study was conducted in rats at levels of 120, 470 & 1900 ppm in the diet. The study was similar to various guidelines and was conducted per GLPs. Macrocytic anemia was the primary change in rats related to chemical exposure. These changes were reversible within 2 weeks following dietary exposure cessation, as were liver weight and serum cholesterol elevations. These changes were minor, and were associated with no apparent prolonged adverse effects. Based upon these findings, the NOEL for test chemical in rats was ~7.5 mg/kg bw/day (120 ppm in the diet). The PCNA data for liver and bladder indicated increased cellular division at the higher doses, but the lack of dose-responsiveness provides results of uncertain importance to the assessment of the toxicity of this chemical