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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
Study period:
2 November 1988 - 8 November 1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study was conducted under GLP conditions and according to a reliable method, but not an official guideline. The study report is well-documented.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report date:
1990

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
no functional observations
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
B100
IUPAC Name:
B100
Constituent 2
Reference substance name:
Peppermint oil
IUPAC Name:
Peppermint oil
Details on test material:
- Name of test material (as cited in study report): B100
- Physical state: Liquid
- Storage condition of test material: Protected from light in an amber jar under refrigeration
- Composition of test material, percentage of components: confidential information

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, North Carolina, USA
- Age at study initiation: 6 weeks
- Weight at study initiation:
Males: 198.4 - 226.1
Females: 139.3 - 170.5
- Housing: Individually
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.9 - 25.6
- Humidity (%): 50 +/- 20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Test material was weighed into a pre-calibrated beaker on an appropriate balance. Corn oil was added to achieve appropriate volume and stirred for 2-3 minutes. Test mixtures were prepared fresh weekly.

VEHICLE
- Justification for use and choice of vehicle (if other than water): No data
- Amount of vehicle (if gavage): 10 ml/kg
- Lot/batch no. (if required): BG 1314
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
No data
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 200, 400 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: No data
- Rationale for animal assignment: At random
- Section schedule rationale: Two-day period, fifty-fifty sacrificed and necropsied over the two days.
Positive control:
Not relevant

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS:
- Time schedule: Twice daily
- Cage side observations: mortality and moribundity

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Weekly

BODY WEIGHT:
- Time schedule for examinations: Weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined as g food/week.

HAEMATOLOGY:
- Time schedule for collection of blood: Prior to intitation of study and at termination
- Anaesthetic used for blood collection: Yes (ketamine)
- Animals fasted: Yes, overnight
- How many animals:
Prior to initiation: 10 healthy animals not selected for study
At termination: all surviving animals
- Parameters checked: corrected leukocyte count, leukocyte count, erythrocyte count, hemoglobin, hematocrit, platelet count, leukocyte differential count cell morphology, myeloid/erythroid ratio.

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: Prior to intitation of study and at termination
- Anaesthetic used for blood collection: Yes (ketamine)
- Animals fasted: Yes, overnight
- How many animals:
Prior to initiation: 10 healthy animals not selected for study
At termination: all surviving animals
- Parameters checked: sodium, potassium, chloride, total protein, albumin, calcium, total carbon dioxide, total bilirubin, blood urea nitrogen, creatinine, glucose, alanine aminotransferase, aspartate aminotransferase, gamma glutamyltransferase, alkaline phosphatase.
Sacrifice and pathology:
GROSS PATHOLOGY: Necropsy was performed on all surviving animals and the following was examined: external surfaces, all orifices, cranial cavity, external surface of the brain (external surface of the spinal cord and the cut surfaces of the brain and spinal cord were examined at the time of tissue trimming), nasal cavity and paranasal sinuses, thoracic, abdominal and pelvic cavities and their viscera, cervical tissues and organs.

ORGAN WEIGTHS: Organ weights of the following organs were measured for each sacrificed animal: brain (including brainstem), spleen, liver, heart, kidneys, testes with epididymides, thyroid with parathyroids, adrenals, ovaries, pituitary.

HISTOPATHOLOGY: The following tissues were examined from all control and high-dose animals: femoral bone marrow, lung (with mainstem bronchi), ovaries, gross lesions, kidneys, adrenals, testes with epididymides, duodenum and jejunum and ileum, brain with brainstem (medulla/pons, cerebellar cortex, cerebral cortex), pancreas, urinary bladder, pituitary, uterus, thyroid (parathyroids), heart, liver, spleen, colon and cecum and rectum, stomach, mesenteric lymph node. From the low and middose group only the heart, liver, kidneys and gross lesions were examined microscopically.
Other examinations:
None performed.
Statistics:
ANOVA was used to determine significant differences in parameters between the test groups.

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:
not examined
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:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
No mortality was observed. An increase in urine stains was observed for the high-dose males as compared to the other test groups.

BODY WEIGHT AND WEIGHT GAIN
No significant differences in body weight (gain) were observed.

FOOD CONSUMPTION
No significant differences in food consumption were observed.

HAEMATOLOGY
A significant decrease in myeloid/erythroid ratio was observed for the high-dose males as compared to the control group.

CLINICAL CHEMISTRY
A significant decrease in glucose levels was observed for the high and mid-dose males as compared to the control group. A significant increase in alkaline phosphatase levels was observed for the high-dose group.

ORGAN WEIGHTS
A significant increase in absolute and relative liver weight was observed for females of the high-dose group as compared to the controls. Relative kidney weight was significantly higher for the high-dose males.

GROSS PATHOLOGY
An increased, non-significant, incidence in stomach dark area's was seen for the male dose groups as compared to control. This effect was also seen in the female groups, including the control group.

HISTOPATHOLOGY: NON-NEOPLASTIC
Histopathological changes (chronic inflammation, regeneration of tubules, droplets in tubule cells) in the kidney were observed for the male dose groups and not in the control group. This is supported by the increase in relative kidney weight. The observed effects consisted of renal tubular protein droplets, thought to be related to lysosomal handling of alpha-2-µ globulin. This effects is a known male-specific effect in rats caused by exposure to hydrocarbons.
For females, the same changes were found as compared to controls, but to a lesser extent and no droplets were observed in the tubule cells. The differences in relative and absolute liver weight in the female high-dose group were not supported by histopathology. No other remarkable histopathological differences between control and test groups were noted.

Effect levels

open allclose all
Dose descriptor:
LOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Histopathological changes (kidney) in all dose group: renal tubular protein resorption droplets (male-rat specific)
Dose descriptor:
NOAEL
Effect level:
400 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No observed adverse effects.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

No gross necrospy, organ weight and/or histopathological effects were observed for the brain stem (incl. medulla/pons, cerebellar cortex and cerebral cortex). No indication for neurotoxicity was found.

The individual result tables do not provide additional information on the effects that were observed (hematology, clinical chemistry, organ weights and histopathology).

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, no treatment related considered effects were noted in females. In all male dose groups treatment related kidney effects were observed, resembling the male rat-specific effect hyalin droplet nephropathy. Based on these findings, the NOAEL for females was established to be 400 mg/kg bw/day, while for males a LOAEL of 100 mg/kg bw/day was found based on male rat-specific effects.
Executive summary:

Rats were exposed to the substance B100 for 28 days by gavage. Animals were observed for mortality, moribundity, body weight and food consumption. Additionally, several hematology and clinical chemistry parameters were studied and gross necropsy, organ weight measurements and histopathology were performed.

No mortality was observed in the study. An increase in urine stains was observed for the high-dose males as compared to the other test groups. No significant differences in body weight (gain) and food consumption were observed. A significant decrease in myeloid/erythroid ratio and increase in alkaline phosphatase was observed for the high-dose males and a significant decrease in glucose levels was observed for the high and mid-dose males as compared to the control group.

A significant increase in absolute and relative liver weight was observed for females of the high-dose group as compared to the controls. Relative kidney weight was significantly higher for the high-dose males. An increased, non-significant, incidence in stomach dark area's was seen for the male dose groups as compared to control. This effect was also seen in the female groups, including the control group.

Histopathological changes (chronic inflammation, regeneration of tubules, droplets in tubule cells) in the kidney were observed for the male dose groups and not in the control group. This is supported by the increase in relative kidney weight. The observed effects consisted of renal tubular protein droplets, thought to be related to lysosomal handling of alpha-2-µ globulin. This effects is a known male-specific effect in rats caused by exposure to hydrocarbons. The differences in relative and absolute liver weight in the female high-dose group were not supported by histopathology. No other remarkable histopathological differences between control and test groups were noted.

Under the conditions of this study, no treatment related considered effects were noted in females. However, in all male dose groups treatment related kidney effects were observed. Based on these findings, the NOAEL for females was established to be 400 mg/kg bw/day, while for males a LOAEL of 100 mg/kg bw/day was found.

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