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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:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Study period:
17 Feb - 30 Mar 2000
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions (no details on analytical purity of the test substance given).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
limited data on test material available
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
other: Cr:CD BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., New York, USA
- Age at study initiation: ca. 8 weeks
- Weight at study initiation: 246-287 g (males) and 178-206 g (females)
- Housing: animals were housed individually in suspended stainless steel and wire mesh cages with absorbent paper
- Diet: Certified Rodent Diet #5002 (PMI Feeds, Inc., Richmond, USA), ad libitum
- Water: water from an automatic watering system (ExxonMobil Research and Engineering Potable Water System), ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): ca. 19-22
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 01 Mar 2000 To: 30 Mar 2000

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
peanut oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: dosing solutions were prepared weekly by dissolving appropriate amounts of the test substance in peanut oil yielding a final concentration of 2, 6 and 20% (w/v) for dose levels of 100, 300 and 1000 mg/kg bw/day, respectively.

VEHICLE
- Justification for use and choice of vehicle (if other than water): the test substance was soluble in the carrier at the concentrations required for this study
- Concentration in vehicle: 2, 6 and 20% (w/v) corresponding to 100, 300 and 1000 mg/kg bw/day, respectively
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no.: 29H0162
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
ANALYSIS METHOD
Analysis for the stability, uniformity, and concentration of the test and control material solutions were performed by the testing laboratory. Analysis for stability and uniformity was initiated prior to or concomitant with the experimental start. Concentration analysis were performed for the Week 1 and 4 mixtures. Samples of dosing solutions ranging from 1 and 50% (w/v) in peanut oil were diluted in ethyl acetate and analysed for uniformity, stability and concentrations using Gas Chromatography with Flame Ionization Detection (GC/FID).

ANALYSIS RESULTS
Satisfactory uniformity was observed. All values were within ± 14% of the nominal value with a maximum relative standard deviation of 2.4%. Stability data indicated that the test substance in peanut oil was stable for at least 15 days at room temperature. Concentration verification analyses indicated that the test substance concentrations were within 9% of the nominal values.
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily, 7 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on a preliminary range-finding study, which revealed clinical signs of toxicity and mortality in treated animals, the high dose was set at 1000 mg/kg bw/day. The other dose levels (mid and low) were set at approximately half-log intervals from the high dose.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: the animals were examined for viability twice daily. Clinical signs of toxicity as well as the nature, onset, severity, and duration of these effects were observed daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once during Weeks 1, 2, and 3, all animals were observed in a standard arena for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, and unusual respiratory pattern). Changes in gait, posture, and response to handling as well as the presence of clonic or tonic movements, stereotypies or unusual behaviour also were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: body weights were determined prior to initiation of dosing, on the day of treatment (Day 0), on Days 7, 14, 21, and 27 as well as on the day of sacrifice.

FOOD CONSUMPTION:
- Food consumption for each animal determined: Yes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on Day 28
- Anaesthetic used for blood collection: Yes (halothane)
- Animals fasted: Yes, overnight
- How many animals: all
- Parameters checked: haematocrit, haemoglobin, erythrocyte count, leukocyte count (total and differential, platelet count, reticulocyte count, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, prothrombin time and thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on Day 28
- Animals fasted: Yes, overnight
- How many animals: all
- Parameters checked: albumin, urea nitrogen, calcium, creatinine, electrolytes (Na+, Cl-, K+), glucose, phosphorus, gamma glutamyl transpeptidase, serum alanine aminotransferase, serum aspartate aminotransferase, serum alkaline phosphatase, total protein, total bilirubin, cholesterol, triglycerides

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: prior to dosing and during Week 4
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity, other: functional observational battery
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see Table 1 under “Any other information of materials and methods incl. tables")
HISTOPATHOLOGY: Yes (see Table 1 under “Any other information of materials and methods incl. tables")
Other examinations:
Organ weights of liver, kidneys, adrenals, testes, brain, heart, spleen, thymus and epididymides were determined.
Statistics:
Statistical evaluation of equality of means was done by an appropriate one-way analysis of variance and a test for ordered response in the dose groups. First, Bartlett's Test was performed to determine if the dose groups have equal variance. If the variances were equal, the testing was done using parametric methods, otherwise nonparametric techniques were used. For the parametric procedures, a standard one-way ANOVA using the F distribution to assess significance was used. If significant differences among the means were indicated, Dunnett's Test was used to determine which treatment groups differed significantly from control. In addition to the ANOVA, a standard regression analysis for linear response in the dose groups was performed. For the nonparametric procedures, the test of equality of means was performed using the Kruskal-Wallis Test. If significant differences among the means were indicated, Dunn's Summed Rank Test was used to determine which treatment groups differed significantly from the control. In addition to the Kruskal-Wallis Test, Jonckheere's Test for monotonic trend in the dose-response was performed. Bartlett's Test for equal variance was conducted at the 1% level of significance. All other tests were conducted at the 5% and 1% level of significance.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
300 and 1000 mg/kg bw/day (f): statistically significant decrease in mean body weight gain between Day 7 and Day 14 (non-adverse)
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:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: statistically significant decrease in mean corpuscular haemoglobin concentration (f, non-adverse); statistically significant decrease in neutrophils and statistically significant increase in lymphocytes (m, non-adverse)
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day (m): decrease in alanine aminotransferase; 300 and 1000 mg/kg bw/day (f): decreases in blood urea nitrogen and increases in sodium and calcium levels; 300 mg/kg bw/day (f): increase in cholesterol; non-adverse
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
300 and 1000 mg/kg bw /day (f): statistically significant increases in the mean relative liver weight (non-adverse)
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
300 and 1000 mg/kg bw/day (m): increased amount of hyaline droplets in the proximal cortical tubular epithelium (non-adverse)
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No mortalities and no treatment-related signs of toxicity occurred during the study period. There were single or very low incidences of dental abnormalities, dried red ocular discharge, and red material seen around the nose observed in one or more groups, including controls.

BODY WEIGHT AND WEIGHT GAIN
There were no biologically or statistically significant differences in mean body weight between treated and control animals. There was a statistically significant decrease in mean body weight gain between Day 7 and Day 14 in females treated with 300 and 1000 mg/kg bw/day. Since this change was not accompanied by any statistically significant difference in the mean body weights of females, it was not considered biologically significant.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
There were no statistically significant or biologically significant differences in mean food consumption during the study.

HAEMATOLOGY
A slight, statistically significant decrease in the mean corpuscular haemoglobin concentration was observed in females receiving 1000 mg/kg bw/day. However, this decrease was very small (-2%) and the parameters (haemoglobin and haematocrit) that are used to calculate this parameter were not statistically different from the control values. Thus, the change in mean corpuscular haemoglobin concentration was not considered biologically significant. In males, a statistically significant decrease in the neutrophils and a statistically significant increase in lymphocytes were noted at 1000 mg/kg bw/day. These changes appeared to be treatment-related, but due to the lack of any corresponding findings, these changes were not considered biologically significant.

CLINICAL CHEMISTRY
In males, a statistically significant decrease in alanine aminotransferase was observed at 1000 mg/kg bw/day compared to control. However, this alteration was not attributable to any toxic effects and thus considered to be of no biological relevance. In females, a statistically significant decrease in mean blood urea nitrogen and statistically significant increases in mean sodium and mean calcium were noted in the 300 and 1000 mg/kg bw/day groups. However, the biological significance of these differences was questionable. Additionally, there was a statistically significant increase in mean cholesterol in females receiving 300 mg/kg bw/day. This difference was not considered biologically significant due to the lack of a dose response relationship.

NEUROBEHAVIOUR
There were no statistically significant differences in the functional observational battery parameters between treated and control animals. There were no statistically significant differences between treated and control groups in motor activity.

ORGAN WEIGHTS
There were statistically significant increases in the mean relative liver weight in females treated with 300 and 1000 mg/kg bw/day (11 and 18%, respectively) when compared to the controls. Since the increase in mean relative liver weight was not correlated with any microscopic effects, it was not considered biologically significant.

GROSS PATHOLOGY
No treatment-related findings were observed at necropsy. Incidental findings included a single incidence of kinked tail in one male treated with 100 mg/kg bw/day and two instances of discoloured thymus in one male and one female of the control.

HISTOPATHOLOGY: NON-NEOPLASTIC
Microscopic examination did not reveal test substance-related microscopic changes in males treated at 100 mg/kg/day or in females treated with up to 1000 mg/kg bw/day. In contrast, an increased amount of hyaline droplets in the proximal cortical tubular epithelium were confirmed microscopically in the cytoplasm of the renal cortical tubular epithelial cells in male rats treated with 300 and 1000 mg/kg bw/day, respectively. this phenomenon is widely accepted to be specific to the male rat and as such is considered to have no relevance to manTherefore

Effect levels

Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion