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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
Study period:
2006
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP, guideline study, available as unpublished report, no restrictions, fully adequate for assessment.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2006

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
Description: extremely pale yellow liquid
Batch number: 929473
Composition: 61.7% alpha-isomethylionone (CASnr 127-51-5), 20.9% n-alphamethylionone (CASnr 7779-30-8), 7.9% n-betamethylionone (CASnr 127-43-5)
Date received: 21 July 2005
Storage conditions: room temperature in the dark
The integrity of supplied data relating to the identity, purity and stability of the test material is the responsibility of the Sponsor.

Test animals

Species:
rat
Strain:
other: Sprague-Dawley Crl:CD® (SD) BR strain
Sex:
male/female
Details on test animals and environmental conditions:
A sufficient number of male and female Sprague-Dawley Crl:CD® (SD) BR strain rats were obtained from Charles River (UK) Limited, Margate, Kent. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatised for 6 days during which time their health status was assessed. A total of eighty animals (forty males and forty females) were accepted into the study. At the start of treatment the males weighed 131 to 172g, the females weighed 122 to 155g, and were approximately 6 to 8 weeks old.
The animals were housed in groups of three or four by sex in polypropylene grid-floor cages suspended over trays lined with absorbent paper. The animals were allowed free access to food and water. A pelleted diet (Rodent 5LF2 (Certified) Diet, BCM IPS Limited, London, UK) was used. Certificates of analysis of the batches of diet used are given in Appendix 15. Mains drinking water was supplied from polycarbonate bottles attached to the cage. The diet and drinking water were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study. Environmental enrichment was provided in the form of wooden chew blocks (B & K Universal Ltd., Hull, UK) and cardboard fun tunnels (Datesand Ltd., Cheshire, UK).
The animals were housed in a single air-conditioned room within the Safepharm Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerised system, and print-outs of hourly mean temperatures and humidities were included in the study records. The temperature and relative humidity controls were set to achieve target values of 21 ± 2°C and 55 ± 15% respectively.
The 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. The cage distribution within the holding rack was also randomised. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
The test material was administered daily, for ninety consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 ml/kg/day of Corn oil.
The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at weekly intervals.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of alpha-iso-methylionone in the test material formulations was determined by gas chromatography (GC) using an external standard technique.
The test material formulations were extracted with methanol to give a final, theoretical test material concentration of approximately 0.1 mg/ml.
Standard solutions of test material were prepared in methanol at a nominal concentration of 0.1 mg/ml.
The standard and sample solutions were analysed by GC using the following conditions:
GC system: Agilent Technologies 5890, incorporating autosampler and workstation
Column: DB-5 (30 m x 0.25 mm id x 0.25 μm film)
Oven temperature program: initial 75°C for 0 mins rate 10°C/min final 225°C for 10 mins
Injection temperature: 250°C
Flame ionisation detector: 250°C temperature
Injection volume: 1 μl
Retention time From ~ 10.9 to 11.4mins
The test material formulations were mixed thoroughly and samples were taken from the top, middle and bottom of the container, shaking between sampling. Sampling was performed in triplicate. The test material formulations were sampled and analysed initially and then after storage at approximately +4°C in the dark for fourteen days. The test material formulations were sampled and analysed within three days of preparation.
Duration of treatment / exposure:
90 day
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
5 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
30 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
500 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
10 rats/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: based in the preliminary 14 day repeated dose oral (gavage) range-finder in the rat study.

Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
DETAILED CLINICAL OBSERVATIONS: Yes, immediately before dosing and one and five hours after dosing during the working week.
BODY WEIGHT: Yes, Individual bodyweights were recorded on Day 1 and at weekly intervals thereafter. Bodyweights were also recorded at terminal kill.
FOOD EFFICIENCY: Yes, Food consumption was recorded for each cage group at weekly intervals throughout the study.
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes, water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.
OPHTHALMOSCOPIC EXAMINATION: Yes, The eyes of all control and high dose 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. Following pupil dilation with 0.5% Tropicamide solution (Alcon Laboratories (UK) Ltd., Imperial Way, Watford, Hertfordshire), detailed examination of the internal structure of the eye using a direct ophthalmoscope was performed.
HAEMATOLOGY and CLINICAL CHEMISTRY: Yes, Haematological and blood chemical investigations were performed on all animals from each test and control group at the end of the study (Day 90). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 91. Animals were not fasted prior to sampling.
URINALYSIS: No data
NEUROBEHAVIOURAL EXAMINATION: Yes, 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 all animals together with an assessment of sensory reactivity to different stimuli.
Sacrifice and pathology:
On completion of the dosing period all animals were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination.
All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Organ Weights
The following organs, removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation: Adrenals, Ovaries, Brain, Spleen, Epididymides, Testes, Heart, Thymus, Kidneys, Uterus and Liver

Histopathology
Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin: Adrenals, Aorta (thoracic), Bone & bone marrow (femur including stifle joint), Brain (including cerebrum, cerebellum and pons), Caecum, Colon, Duodenum, Epididymides, Eyes, Heart, Ileum (including Peyer's patches), Jejunum, Kidneys, Liver, Lungs (with bronchi), Lymph nodes (cervical and mesenteric), Mammary glands, Muscle (skeletal), Oesophagus, Ovaries, Pancreas, Pituitary Bone & bone marrow (sternum), Prostate, Rectum, Salivary glands (submaxillary), Sciatic nerve, Seminal vesicles, Skin (hind limb), Spinal cord (cervical, mid-thoracic and Gross lesions lumbar), Spleen, Stomach, Testes, Thymus, Thyroid/parathyroid, Tongue, Trachea, Urinary bladder, Uterus
All tissues from control and 500 mg/kg/day dose group animals were prepared as paraffin blocks, sectioned at nominal thickness of 5 JlID 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:
no data
Statistics:
Data were processed to give group mean values and standard deviations where appropriate.
All data were summarised in tabular form. Where appropriate, quantitative data were analysed by the Provantis Tables and Statistics Module. 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 frnd 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. Finally, 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.
Chi squared analysis for differences in the incidence of lesions occurring with an overall frequency of 1 or greater.
Kruskal-Wallis one way non-parametric analysis of variance for the comparison of severity grades for the more frequently observed graded conditions.
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)

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
Urinalysis findings:
not specified
Behaviour (functional findings):
no effects observed
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:
Blood chemistry: Animals of either sex treated with 500 mg/kg/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 treatment-related effects were detected in animals of either sex treated with 30 or 5 mg/kg/day. Animals of either sex treated with 500 mg/kg/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). In the absence of any supporting data to suggest an effect of treatment these intergroup differences were considered of no toxicological importance. Males treated with 500 mg/kg/day also showed a statistically significant reduction in plasma chloride concentration (p<0.05). All individual values were within the normal range for rats of the strain and age used and the intergroup difference was considered not to be toxicologically significant. 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 usedand in the absence of a dose related response the intergroup differences were considered of no toxicological importance.

Organ Weights: Animals of either sex treated with 500 mg/kg/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/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/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 30 or 5 mg/kg/day.
Animals of either sex treated with 30 mg/kg/day showed a statistically significant reduction in absolute and relative heart weight (p<0.05). All 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 to be of no toxicological importance. Females treated with 30 mg/kg/day showed a statistically significant increase in absolute and relative liver weight (p<0.05). In the absence of any histological correlates at this level the intergroup difference was considered not to be toxicologically significant. Males treated with 5 mg/kg/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.

Histopathology
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/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/day (p< 0.01) or in 5/10 (minimal), 4/10 (slight) males treated with 30 mg/kg/day (p< 0.05) but not at 5 mg/kg/day. This finding may indicate the presence of hydrocarbon nephropathy, which results from the excessive accumulation of alpha-2-microglobulin in renal proximal tubular epithelial cells. Aplha-2-microglobulin is found only in the proximal tubular epithelium of adult male rats. Two males treated with 500 mg/kg/day and one male treated with 30 mg/kg/day exhibited associated 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/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/day (p< 0.01).

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: Based on several adverse effects observed at the level of 500 mg/kg bw/day, including an increase in creatinine, absolute and relative increase in kidney and spleen weight and follicular cell hypertrophy in the thyroid.
Dose descriptor:
NOEL
Effect level:
5 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

The administration of Alpha-iso-methylionone by oral gavage, at dose levels of 500, 30 and 5 mglkg/day for a period of ninety consecutive days resulted in treatment related effects at 500 mglkg/day for females and at 500 and 30 mg/kg/day for males.

There was no apparent adverse effect on the physical condition of the animals. Animals of either sex treated with 500 mg/kg/day did showed instances of noisy respiration and transient increased salivation together with hunched posture and tiptoe gait. Such changes are commonly observed following oral administration of an unpalatable or slightly irritant test material formulation and are therefore considered not to be indicative of systemic toxicity. Blood chemical investigations at 500 mg/kg/day revealed an increase in total protein, cholesterol, creatinine and in males only an increase in albumin. Whilst some of these parameters are an indication of liver function, a true elevation in albumin is unlikely, in the absence of any evidence of dehydration because albumin synthesis normally occurs at close to the maximum possible rate. Furthermore, the elevation in cholesterol was not supported by any other evidence of obstructive liver damage. An increase in creatinine may suggest an effect on kidney function. However, there was no concomitant change in electrolyte balance but, in view of the significant changes seen in 500 mg/kg/day kidney weights, a toxicologically significant effect cannot be entirely ruled out. Microscopic examinations of liver sections revealed hepatocyte, centrilobular or generalised enlargement at 500 mg/kg/day. Increased hepatic activity of this type and in the absence of associated inflammatory or degenerative changes is considered a normal adaptive biological response to xenobiotics and is usually the result of detoxification mechanisms involving hepatic enzyme induction. Organ weight data supported this finding with increased absolute and relative liver weights detected in this treatment group. Follicular cell hypertrophy in the thyroids was observed in males treated with 500 mglkg/day. Thyroxine is ultimately excreted via the bile, having first been conjugated in the liver. It is conceivable 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.

Microscopic examination of kidney sections revealed globular accumulations of eosinophilic material in the proximal tubular epithelium of males treated with 500 and 30 mg/kg/day. This is a well documented effect, peculiar to the male rat, which occurs in response to treatment with certain hydrocarbons. Female rats and other species do not develop "hydrocarbon nephropathy" and for this reason., the effect may not be indicative of hazard to human health. Two males treated with 500 mg/kg/day and one male treated with 30 mg/kg/day also exhibited associated higher grades of tubular basophilia.

The remaining organ weight change seen at 500 mg/kg/day included an increase in absolute and relative spleen weight for males. The absence of pathological changes identified in the spleen minimises the toxicological significance of this finding, however, microscopic examinations of bone marrow did reveal a higher incidence of lower grades of severity of adipose infiltration in these animals which may suggest a subtle effect on haemopoeisis. The importance of these findings however, is lessened by the absence of supporting changes in the haematological parameters.

No such effects were detected in females treated with 30 mg/kg/day or animals of either sex treated with 5 mg/kg/day.

Applicant's summary and conclusion