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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From August 03, 2005 to March 03, 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted in compliance with OECD Guideline No. 408.

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006
Reference Type:
publication
Title:
Ninety Day Repeated Dose Oral (Gavage) Toxicity Study with Alpha-Iso-Methylionone in Rats
Author:
Lapczynski AA, Politano VT and Api AM.
Year:
2007
Bibliographic source:
Society of Toxicology, 46th Annual Meeting and ToxExpo, The Toxicologist 96 (1): 193.
Reference Type:
publication
Title:
Ninety-Day Toxicity Study of Alpha-Iso-Methylionone in Rats
Author:
Politano VT, Lapczynski AA, Ritacco G and Api AM.
Year:
2012
Bibliographic source:
International Journal of Toxicology 31 (6): 595-601.

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Programme (inspected on August 30, 2005/signed on November 21, 2005 )
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical state: Extremely pale yellow liquid
- Storage condition of test material: Stored at room temperature in the dark.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent.
- Age at study initiation: Approximately 6-8 weeks
- Weight at study initiation: Males: 131-172 g; females: 122-155 g.
- Housing: Animals were housed in groups of three or four by sex in polypropylene grid-floor cages suspended over trays lined with absorbent paper.
- Diet: Pelleted diet (Rodent 5LF2 (Certified) Diet, BCM IPS Limited, London, UK), ad libitum
- Water: Mains drinking water, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 2 °C
- Humidity: 55 ± 15 %
- Air changes: At least 15 changes/h
- Photoperiod: 12 h dark/ 12 h light

IN-LIFE DATES: From: August 03, 2005 To: March 03, 2006

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Test material was prepared at the appropriate concentrations as a solution in Corn oil. Test material formulations were stable for at least 14 days and therefore prepared weekly and stored at 4 °C in the dark.

VEHICLE
- Concentration in vehicle: 1.25, 7.5 and 125 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- The concentration of test material in the formulations was determined by gas chromatography (GC) using an external standard technique. All the concentrations were analysed in weekly interval till the end of the study. Homogeneity and stability of test material formulations were determined. The test material formulations were sampled and analysed within 3 days of preparation for verification of concentrations.
- The results of analysis indicated that the prepared formulations were within acceptable limits.
Duration of treatment / exposure:
90 days
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
5 mg/kg bw/day (actual dose received)
Remarks:
actual ingested
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Remarks:
actual ingested
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected based on the results of range-finding study using 1000 mg/kg bw/day.
- Rationale for animal assignment (if not random): Animals were randomly allocated to treatment groups using a total randomisation procedure and the group mean bodyweights were then determined to ensure similarity between the treatment groups.
Positive control:
None

Examinations

Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing and 1 and 5 h after dosing during the working week. At weekends, animals were observed immediately before dosing and 1 h after dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual bodyweights were recorded on Day 1 and at weekly intervals thereafter. Bodyweights were also recorded at terminal kill.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Food consumption was recorded for each cage group at weekly intervals throughout the study.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: The eyes of animals were examined pre-treatment and before termination of treatment (during Week 12). Examinations included observation of the anterior structures of the eye, pupillary and corneal blink reflex. Detailed examination of the internal structure of the eye using a direct ophthalmoscope was performed after pupil dilation with 0.5 % Tropicamide solution (Alcon Laboratories (UK) Ltd., Imperial Way, Watford, Hertfordshire).
- Dose groups that were examined: Control and high dose group.

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the study (Day 90) from the lateral tail vein; where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 91.
- Animals fasted: No
- How many animals: All animal from each test and control group
- Parameters checked in table 7.5.1/1.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioural toxicity. During Week 12 functional performances tests were also performed on animals together with an assessment of sensory reactivity to different stimuli.
- Dose groups that were examined: 10 animals/sex from each dose group.
- Battery of functions tested:
(a) Behavioural assessments: Gait, tremors, twitches, convulsions, bizarre/ abnormal/stereotypic behaviour, salivation, pilo-erection, exophthalmia, lachrymation, hyper/hypothermia, skin colour, respiration, palpebral closure, urination, defecation, transfer arousal, tail elevation.
(b) Functional performance tests: motor activity, forelimb/hindlimb grip strength.
(c) Sensory reactivity to auditory, visual and proprioceptive stimuli: grasp response, vocalisation, toe pinch, tail pinch, finger approach, touch escape, pupil reflex, startle reflex, blink reflex.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- All animals were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on completion of the dosing period. All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

HISTOPATHOLOGY: Yes
- Samples of the tissues (see table 7.5.1/2) were removed from all animals and preserved in buffered 10 % formalin.
- All tissues from control and 500 mg/kg bw/day dose group animals were prepared as paraffin blocks, sectioned at nominal thickness of 5 µm and stained with haematoxylin and eosin for subsequent microscopic examination.
- Since there were indications of treatment-related changes in the liver, kidneys, thyroid gland, and bone marrow, examination was subsequently extended to include sections of these tissues from all animals in the remaining groups.
Other examinations:
Organ weights:
- Adrenals, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, thymus and uterus were removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation.
Statistics:
- Data were processed to give group mean values and standard deviations where appropriate.
- All data were summarised in tabular form and quantitative data were analysed by the Provantis™ Tables and Statistics Module, where appropriate.
- For each variable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogeneity of means assessed using ANOVA or ANCOVA and Bartlett's test.
- The transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data.
- If no dose response was found, but the data showed non-homogeneity of means, the data were analysed by a stepwise Dunnett (parametric) or Steel (non-parametric) test to determine significant differences from the control group.
- If required, pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).
- Probability values (p) are presented as follows:
p < 0.01 **
p < 0.05 *
p ≥ 0.05 (not significant)
- Histopathology data were analysed using the following methods to determine significant differences between control and treatment groups for the individual sexes:
(a) Chi squared analysis for differences in the incidence of lesions occurring with an overall frequency of 1 or greater.
(b) Kruskal-Wallis one way non-parametric analysis of variance for the comparison of severity grades for the more frequently observed graded conditions.
(c) Probability values (p) are presented as follows:
p < 0.001 +++ --- ***
p < 0.01 ++ -- **
p < 0.05 + - *
p < 0.1 (+) (-) (*)
p ≥ 0.1 N.S. (not significant)
Plus signs indicate positive differences from the control group and minus signs indicate negative differences. Asterisks refer to overall between group variation which is non-directional.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
animals of either sex treated with 500 mg/kg bw/day showed a statistically significant increase in plasma creatinine, total protein and cholesterol as compared to control animals.
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):
animals treated with 500 mg/kg bw/day showed a statistically significant increase in liver and kidney weight (either sex); spleen weight (males).
Gross pathological findings:
no effects observed
Description (incidence and severity):
no macroscopic abnormalities were detected.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
(treatment related changes in liver, thyroid, bone marrow (at 500 mg/kg bw/day) and kidney (at 30 and 500 mg/kg bw/day))
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY:
- No mortality was observed. No clinically observable signs of toxicity were detected in test or control animals throughout the study period.
- Noisy respiration and increased salivation, together with associated findings of hunched posture and tiptoe gait were evident in 500 mg/kg bw/day treated animals throughout the treatment period. Incidents of increased salivation and noisy respiration were also evident in 5 and 30 mg/kg bw/day treated animals (respectively). These findings were not considered to represent systemic toxicity.
- At 30 and 500 mg/kg bw/day, females showed isolated episodes of tail elevation with the latter female also showing an episode of ataxia. The aetiology of these findings are unclear in the absence of any supporting behavioural assessments to suggest neurotoxicity and these changes were considered of no toxicological importance.
- Isolated instances of generalised fur loss, scabs formation or generalised red/brown stained fur were evident in a number of control and treated animals throughout the dosing period, however, these findings were considered to be entirely
incidental and unrelated to treatment.
- One control male showed episodes of noisy respiration whilst one control female developed hunched posture over a two day period. These findings were clearly unrelated to treatment.

BODY WEIGHT AND WEIGHT GAIN:
- No adverse effect on bodyweight development was detected. Bodyweight gain in test animals during the treatment period was similar to that of controls.
- Females treated with 500 mg/kg bw/day showed a statistically significant reduction in bodyweight gain during week 7 (p<0.05) together with an increase in bodyweight gain during weeks 9 and 13 (p<0.05 and p<0.001 respectively).
- Mean bodyweight at termination was at least 99 % of control and in the absence of any effect on overall bodyweight gain, differences in weekly bodyweight change were considered to reflect normal biological variation and to be of no toxicological significance.

FOOD CONSUMPTION AND FOOD EFFICIENCY:
- There was no adverse effect on food consumption during the entire study period. Food efficiency (the ratio of bodyweight gain to dietary intake) was similar to that of controls.

WATER CONSUMPTION:
- No intergroup differences were detected.

OPHTHALMOSCOPIC EXAMINATION:
- There were no treatment-related ocular effects. The incidental findings recorded were those normally encountered in laboratory maintained rats of this age and strain.

HAEMATOLOGY:
- No treatment-related changes in the haematological parameters were measured.

CLINICAL CHEMISTRY:
- Animals of either sex treated with 500 mg/kg bw/day showed a statistically significant increase in plasma creatinine, total protein and cholesterol (p< 0.01) compared to control animals. Males from this treatment group also showed a statistically significant increase in plasma albumin (p< 0.01). No such effects were detected in animals of either sex treated with 5 or 30 mg/kg bw/day.
- Animals of either sex treated with 500 mg/kg bw/day showed a statistically significant reduction in aspartate aminotransferase (p<0.05 males and p<0.001 females) whilst females also showed a reduction in alkaline phosphatase (p<0.05). Males treated with 500 mg/kg bw/day also showed a statistically significant reduction in plasma chloride concentration (p<0.05). Females from all treatment groups showed a statistically significant reduction in plasma bilirubin (p<0.05). The majority of individual values were within the normal range for rats of the strain and age used and in the absence of a dose related response the intergroup differences were considered of no toxicological importance.

NEUROBEHAVIOUR:
- No treatment-related changes in the behavioural, functional performance parameters and sensory reactivity were measured.

ORGAN WEIGHTS:
- Animals of either sex treated with 500 mg/kg bw/day showed a statistically significant increase in liver weight (p<0.01) both absolute and relative to terminal bodyweight. Males treated with 500 mg/kg bw/day also showed statistically significant increases in spleen and kidney weight (p<0.01) both absolute and relative to terminal bodyweight. Females treated with 500 mg/kg bw/day also showed a statistically significant increase in absolute and relative kidney weight (p<0.01). No such toxicologically significant effects were detected in animals of either sex treated with 5 or 30 mg/kg bw/day.
- Animals of either sex treated with 30 mg/kg bw/day showed a statistically significant reduction in absolute and relative heart weight (p<0.05). Females treated with 30 mg/kg bw/day showed a statistically significant increase in absolute and relative liver weight (p<0.05). These effects were considered to be of no toxicological importance.
- Males treated with 5 mg/kg bw/day showed a statistically significant increase in absolute and relative adrenal weight (p<0.05). In the absence of a dose-related response or any histological correlates this intergroup differences was considered not to be toxicologically significant.

GROSS PATHOLOGY:
- No macroscopic abnormalities were detected.

HISTOPATHOLOGY: NON-NEOPLASTIC
- LIVER: Hepatocyte enlargement, centrilobular or generalised, was observed in relation to treatment for 4/10 (minimal) males and 9/10 (minimal) females with 500 mg/kg bw/day (p< 0.05 for males; p< 0.001 for females). Hepatocyte enlargement is commonly observed in the rodent liver following the administration of xenobiotics and in the absence of associated degenerative changes may be interpreted as adaptive in nature.
- KIDNEYS: A greater incidence of higher severity grades of globular accumulations of eosinophilic material were observed in the tubular epithelium of 3/10 (minimal), 5/10 (slight), 1/10 (moderate) males treated with 500 mg/kg bw/day (p< 0.01) or in 5/10 (minimal), 4/10 (slight) males treated with 30 mg/kg bw/day (p< 0.05) but not at 5 mg/kg bw/day. The kidney changes identified histopathologically were consistent with well documented changes that are peculiar to the male rat in response to treatment with some hydrocarbons. Two males treated with 500 mg/kg bw/day and one male treated with 30 mg/kg bw/day exhibited higher grades of tubular basophilia.
- THYROID: A higher incidence of follicular cell hypertrophy was seen in relation to treatment for 7/10 (minimal) males treated with 500 mg/kg bw/day (p< 0.01).
- BONE MARROW: A higher incidence of lower grades of severity of adipose infiltration of the bone marrow, indicative of marrow hyperplasia, was observed in relation to treatment for 7/10 (minimal) and 3/10 (slight) males treated with 500 mg/kg bw/day (p< 0.01).

All remaining morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed, and there were no differences in incidence or severity between control and treatment groups that were considered to be of toxicological significance.

Effect levels

open allclose all
Dose descriptor:
NOEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No treatment related effects were observed in females at 30 mg/kg bw/day.
Dose descriptor:
NOEL
Effect level:
5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No treatment related effects were observed in males at 5 mg/kg bw/day.
Key result
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
LOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry
organ weights and organ / body weight ratios
histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
Based on the results of the study, NOEL was considered to be 30 mg/kg bw/day for females and 5 mg/kg bw/day for males. Based on histopathological changes in the kidney in males, the NOAEL was 30 mg/kg bw/day.
Executive summary:

In a repeated dose toxicity study performed in accordance with OECD test Guideline No. 408 and in compliance with GLP, test material in corn oil was administered by oral route (gavage) to three groups of Sprague-Dawley Crl:CD® (SD) IGS BR strain rats (10/sex/dose) at dose levels of 5, 30 and 500 mg/kg bw/day for 90 consecutive days. Control rats were given the vehicle alone. Clinical signs, functional observations, bodyweight development and food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for all animals at the end of the study. Ophthalmoscopic examination was also performed on control group and high dose animals. All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed.

 

Following results were obtained:

 

- Mortality

No unscheduled deaths were observed.

 

- Clinical Observations

No clinically observable signs of toxicity were detected in test or control animals throughout the study period.

 

- Body weight

No adverse effect on bodyweight development was detected. Bodyweight gain in test animals during the treatment period was similar to that of controls.

 

- Food consumption

No adverse effect on dietary intake or food efficiency was detected.

 

- Water consumption

No intergroup differences were detected.

 

- Ophthalmoscopic examination

No treatment-related ocular effects were observed.

 

- Haematology

No treatment-related changes were detected.

 

- Clinical chemistry

Animals of either sex treated with 500 mg/kg bw/day showed a statistically significant increase in plasma creatinine, total protein and cholesterol (p< 0.01) compared to control animals. Males from this treatment group also showed a statistically significant increase in plasma albumin (p< 0.01). No such effects were detected in animals of either sex treated with 5 or 30 mg/kg bw/day.

 

- Neurobehavioral

No treatment-related changes in the behavioural, functional performance parameters and sensory reactivity were measured.

 

- Organ weights

Animals of either sex treated with 500 mg/kg bw/day showed a statistically significant increase in liver weight (p<0.01) both absolute and relative to terminal bodyweight. Males treated with 500 mg/kg bw/day also showed statistically significant increases in spleen weight (p<0.01) and in both sexes an increase in kidney weight (p<0.01) both absolute and relative to terminal bodyweight. No such toxicologically significant effects were detected in animals of either sex treated with 5 or 30 mg/kg bw/day.

 

- Gross pathology

No macroscopic abnormalities were detected.

 

- Histopathology

Liver: Microscopic examinations of liver sections revealed hepatocyte, centrilobular or generalised enlargement at 500 mg/kg bw/day. Hepatocyte enlargement is commonly observed in the rodent liver following the administration of xenobiotics and in the absence of associated degenerative changes may be interpreted as adaptive in nature.

Kidney: Microscopic examination of kidney sections revealed globular accumulations of eosinophilic material in the proximal tubular epithelium of males treated with 30 and 500 mg/kg bw/day. The kidney changes identified histopathologically were consistent with well documented changes that are peculiar to the male rat in response to treatment with some hydrocarbons. Two males treated with 500 mg/kg bw/day and one male treated with 30 mg/kg bw/day also exhibited higher grades of tubular basophilia.

Thyroid: A higher incidence of follicular cell hypertrophy was observed in males treated with 500 mg/kg bw/day. Thyroxine is ultimately excreted via the bile, having first been conjugated in the liver. A hypothesis is that conjugating hepatic enzymes may have been induced as a response to the test material therefore increasing thyroxine excretion and stimulating compensatory thyroxine stimulating hormone and thyroxine production possibly resulting in the microscopic changes identified.

Bone marrow: A higher incidence of lower grades of severity of adipose infiltration of the bone marrow, indicative of marrow hyperplasia, was observed in relation to treatment in males treated with 500 mg/kg bw/day. This observation may suggest a subtle effect on haematopoiesis, lessened by the absence of supporting changes in the haematological parameters.

 

Based on the results of the study, NOEL was considered to be 30 mg/kg bw/day for females and 5 mg/kg bw/day for males. Based on histopathological changes in the kidney in males, the NOAEL was 30 mg/kg bw/day.