Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was performed between 19 February 2008 and 16 Juune 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Sponsor's identification: C-Y9
Description: Orange Powder
Purity: 95.8%
Batch number: 071217
Date received: 24 January 2008
Storage conditions: Over silica gel 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: A sufficient number of male and female Sprague-Dawley Crl:CD (SD) lGS BR strain rats were obtained from Charles River (UK) Limited, Margate, Kent
- Age at study initiation: Approximately 5 to 6 weeks old.
- Weight at study initiation: Males weighed 137 yo 188 g, females weighed 129 to 169 g.
- Fasting period before study: No.
- Housing: The animals were housed in groups of five by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding.
- Diet (e.g. ad libitum): A pelleted diet was used. The animals were allowed free access to food.
- Water (e.g. ad libitum): Mains drinking water was supplied from polycarbonate bottles attached to the cage. The animals were allowed free access to water.
- Acclimation period: Five days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Set to achieve target values of 21 ± 2°C
- Humidity (%): Set to achieve target value of 55 ± 15% .
- Air changes (per hr): At least fifteen air changes per hour.
- Photoperiod (hrs dark / hrs light): Low intensity fluorescent lighting was controlled to give twelve hours continous light and tweleve hours darkness.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test material was prepared at the appropriate concentrations as solutions in Distilled water.

The test material was administered daily, for twenty-eight consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 5 ml/kg/day of Distilled water. Recovery group animals were maintained for a further fourteen days treatment-free period following termination of treatment.

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:
For the purpose of this study the test material was prepared at the appropriate concentrations as solutions in Distilled water. The stability and homogeneity of the test material formulations were determined. Results show the formulations to be stable for at least fourteen days. Formulations were therefore prepared weekly and stored at 4°C in the dark.

Samples of each test material formulation were taken and analysed for concentration of C-Y9 . The results indicate that the prepared formulations were within ± 8% of the nominal concentration.

The concentrations of C-Y9 in the test material formulations was determined spectrophotometrically.
Duration of treatment / exposure:
28 consecutive days.
Frequency of treatment:
The test material was administered daily.
Doses / concentrations
Remarks:
Doses / Concentrations:
0 (vehicle alone) 25, 150, 300 and 1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
The test material was administered to four groups, each of five males and five females at dose levels of 25, 150, 300 and 1000 mg/kg/day
A control group of five males and five females was dosed with vehicle alone.
Two recovery groups, each of five males and five females, were treated with the high dose (1000 mg/kg/day) or the vehicle alone for twenty-eight days and then maintained without treatment for a further fourteen days.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were chosen based on the results of a range-finder.

- Rationale for animal assignment (if not random):
The animals were randomly allocated to treatment groups using a total randomisation procedure and the group mean bodyweights were determined to ensure similarity between the treatment groups.
Positive control:
No positive control.

Examinations

Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS:
All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing, immediately post dosing and one and five hours after dosing during the working week. Animals were observed immediately before and after dosing and one hour after dosing at weekends and public holidays. During the treatment-free period, animals were observed twice daily, morning and afternoon (once daily at weekends). All observations were recorded.

FUNCTIONAL OBSERVATIONS:
Prior to the start of treatment and on Days 7, 14, 21 and 27, all animals were observed for signs of functional behavioural toxicity. Functional performance tests were also performed on all non-recovery animals during Week 4, together with an assessment of sensory reactivity to different stimuli. Observations were carried out from approximately two hours after dosing on each occasion.

BEHAVIOURAL ASSESSMENTS:
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:
Gait, Hyper/Hypothermia, Tremors, Skin colour, Twitches, Respiration, Convulsions, Palpebral closure, Bizarre/Abnormal/Stereotypic behaviour, Urination, Salivation, Defecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, Lachrymation

FUNCTIONAL PERFORMANCE TESTS:
Motor Activity:
Twenty purpose built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals of one sex were tested at each occasion and were randomly allocated to the activity monitors. The tests were performed at approximately the same time each day, under similar laboratory conditions. The evaluation period was one hour for each animal. The time in seconds each animal was active and mobile was recorded for the overall hour period and also during the final 20% of the period (considered to be the asymptotic period).

Forelimb/Hindlimb Grip Strength:
An automated grip strength meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal.

Sensory Reactivity:
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. The following parameters were observed:
Grasp response, touch escape, vocalisation, pupil reflex, toe pinch, startle reflex, tail pinch, blink reflex, finger approach.

BODYWEIGHT:
Individual bodyweights were recorded on Day 1 (prior to the start of treatment) and on Days 8, 15, 22 and 29 prior to terminal kill, and, in the case of recovery group animals, on Days 36 and 43 prior to terminal kill.

FOOD CONSUMPTION:
Food consumption was recorded for each cage group at weekly intervals throughout the study.

WATER CONSUMPTION:
Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes. Water intake was measured and recorded daily during Week 3 for each cage group and revealed possible intergroup differences. Water consumption was, therefore, continued to be measured and recorded for each cage group until study termination.

LABORATORY INVESTIGATIONS:
Haematological and blood chemical investigations were performed on all non-recovery test and control group animals at the end of the treatment period (Day 28) and on all recovery group animals at the end of the treatment-free period (Day 42). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Days 29 and 43. Animals were not fasted prior to sampling.
Urinalytical investigations were performed on all non-recovery test and control group animals during Week 4 and on all recovery group animals during Week 6. Urine samples were collected overnight by housing the rats in metabolism cages. Animals were maintained under conditions of normal hydration during collection but without access to food.

HAEMATOLOGY:
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:
Haemoglobin (Hb)
Erythrocyte count (RBC)
Haematocrit (Hct)
Erythrocyte indices - mean corpuscular haemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular haemoglobin concentration (MCHC)
Total leucocyte count (WBC)
Differential leucocyte count - neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Methaemoglobin (Meth)
Reticulocyte count (Retic) - Cresyl blue stained slides were prepared but reticulocytes were not assessed

Prothrombin time (CT) was assessed by 'Thrombomax HS with calcium' and Activated partial thromboplastin time (APTT) was assessed by 'Actin FS' using samples collected into sodium citrate solution (0.11 mol/l).

BLOOD CHEMISTRY:
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea, Calcium (Ca++), Glucose, Inorganic phosphorus (P), Total protein (Tot.Prot.), Aspartate aminotransferase (ASAT), Albumin, Alanine aminotransferase (ALAT), Albumin/Globulin (A/G) ratio (by calculation), Alkaline phosphate (AP), Sodium (Na+), Creatinine (Creat), Potassium (K+), Total cholesterol (Chol), Chloride (Cl¯), Total bilirubin (Bili), Gamma glutamyltranspeptidase (GT), Triglycerides (Tri).

URINALYSIS:
The following parameters were measured on collected urine:
volume, specific gravity, pH, protetin, glucose, ketones, bilirubin, urobilinogen, reducing substances, blood.









































Sacrifice and pathology:
PATHOLOGY:
On completion of the dosing period, or in the case of recovery group animals, at the end of the treatment-free 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 either at the end of the dosing period or at the end of the treatment-free period, were dissected free from fat and weighed before fixation:
- Adrenals, Ovaries, Brain, Spleen, Epididymides, Testes, Heart, Thymus, Kidneys, Liver.

HISTOPATHOLOGY:
Samples of tissues were removed from all animals and preserved in buffered 10% formalin
(Please see Any other information on materials and methods section for list of tissues examined).








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 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. 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 <0.001**
p <0.05*
p ≥0.05 (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):
not examined
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
MORTALITY:
There were no unscheduled deaths during the study.

CLINICAL OBSERVATIONS:
No toxicologically significant signs of toxicity were detected throughout the treatment period or during the treatment free period.

The isolated findings of yellow fur staining by the test material were considered to be of no toxicological importance.

FUNCTIONAL OBSERVATIONS:
Behavioural Assessments:
All inter and intra group differences in behavioural scores were considered to be a result of normal variation for rats of the strain and age used, and were of no toxicological importance.

Functional Performance Tests:
There were no toxicologically significant changes in the functional performance parameters measured.

The statistically significant intergroup difference detected in hind limb grip strength was considered to be of no toxicological importance.

Sensory Reactivity Assessments:
There were no treatment-related changes in sensory reactivity.

All inter and intra group differences in sensory reactivity scores were considered to be a result of normal variation for rats of the strain and age used and were of no toxicological importance.

BODYWEIGHT:
No toxicologically significant effects on bodyweight development were detected.

The statistically significant intergroup differences detected during Weeks 1, 4 and 5 were considered to be of no toxicological importance.

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

WATER CONSUMPTION:
Gravimetric measurement of water consumption during Week 3 revealed an increase for males treated with 1000 mg/kg/day. An increase in water consumption remained evident in 1000 mg/kg/day males during the final week of treatment and continued in recovery 1000 mg/kg/day males during the treatment free period.

No such effects were detected in 1000 mg/kg/day females, animals of either sex treated with 300, 150 or 25 mg/kg/day or in recovery 1000 mg/kg/day females following fourteen days without treatment.

LABORATORY INVESTIGATIONS:-

HAEMATOLOGY:
There were no toxicologically significant changes in the haematological parameters measured.

The intergroup differences detected in total leucocyte count, lymphocyte count and mean cell haemoglobin concentration were considered to be of no toxicological importance.

BLOOD CEHMISTRY:
There were no toxicologically significant changes in the blood chemical parameters measured.

The intergroup differences detected were considered to be of no toxicological importance.

URINALYSIS:
There were no toxicologically significant changes in the urinalytical parameters measured.

Animals of either sex treated with 1000 mg/kg/day and one male treated with 300 mg/kg/day showed yellow or yellow/orange coloured urine. This observation is often reported following oral administration of a coloured test material or excretion of a coloured metabolite and is considered not to be indicative of toxicity.

PATHOLOGY:-

ORGAN WEIGHTS:
There were no toxicologically significant changes in the organ weights measured.

The intergroup differences detected in liver and spleen was considered to be of no toxicological importance.

NECROPSY:
No toxicologically significant macroscopic abnormalities were detected.

Two males treated with 1000 mg/kg/day had a yellow stained stomach at necropsy. This observation is often reported following oral administration of a coloured test material and is considered not to be indicative of toxicity.

The remaining findings recorded at necropsy were considered to be of no toxicological importance.

HISTOPATHOLOGY:
The following treatment-related stomach changes were detected:

Agglomeration of secretion, superficial mucosal basophilia, and hyperplasia of mucous cells were observed in the gastric mucosa immediately adjacent to the limiting ridge among animals of either sex treated with 1000 mg/kg/day. Acanthosis and hyperkeratosis of the limiting ridge were also seen among both sexes at this dose level. There was probably also an effect of treatment on animals at the 300 mg/kg/day dose level with two males and one female being similarly affected to a greater or lesser extent. There was, however, no evidence of an effect at either of the remaining treatment levels. Although agglomeration of secretion was seen for one female rat treated with 25 mg/kg/day, this condition is seen occasionally among control animals as a spontaneous change.

There were regressions of the conditions among Recovery 1000 mg/kg/day animals following an additional fourteen days without treatment.

Treatment-related gastric changes of this type are relatively commonly seen as a response to orally administered xenobiotics and at the levels of severity seen in this investigation they are regarded as not adverse.

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 treated groups that were considered to be of toxicological significance.































































Effect levels

open allclose all
Dose descriptor:
NOEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects were detected in animals of either sex treated with 150 or 25 mg/kg/day and the "No observed Effect Level" (NOEL) was, therefore, considered to be 150 mg/kg/day.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Discussion:

The administration of C-Y9 by oral gavage at dose levels of 1000, 300, 150 and 25 mg/kg/day for a period of twenty-eight consecutive days resulted in treatment related effects in animals of either sex treated with 1000 and 300 mg/kg/day.

The visible signs of yellow fur staining were detected in a number of females treated with 1000 mg/kg/day and males treated with 300 mg/kg/day during the study (confirmed as coloured urine following urinalytical investigations). Such observations are often reported following administration of a coloured test material or excretion of coloured metabolites. This simply represents normal excretion of the coloured compound followed by normal grooming behaviour and subsequent dispersal of the material onto the external body surface. Such observations do not, therefore, represent systemic toxicity.

Histopathological examination revealed changes in the stomach. These were identified as agglomeration of secretion, mucosal basophilia/atrophy, and hyperplasia of mucous cells observed in the gastric mucosa immediately adjacent to the limiting ridge among animals of either sex treated with 1000 mg/kg/day. Acanthosis and hyperkeratosis of the limiting ridge were also seen among either sex at this dose level. There was also an effect of treatment on animals at the 300 mg/kg/day dose level with two males and one female being similarly affected to a greater or lesser extent. Such changes may be associated with gastric irritancy however, there were no clinical observations to support this and the changes observed are also commonly seen as a response to orally administered xenobiotics and at the levels of severity seen in this investigation they are regarded as not adverse.

No such toxicologically significant effects were detected in animals of either sex treated with 150 or 25 mg/kg/day.

The microscopic effects seen in the stomach had appreciably regressed following fourteen days without treatment.

Applicant's summary and conclusion

Conclusions:
The oral administration of C-Y9 at dose levels of 25, 150, 300 and 1000 mg/kg/day for a period of twenty-eight consecutive days resulted in treatment related effects in animals of either sex treated with 1000 and 300 mg/kg/day. No such effects were detected in animals of either sex treated with 150 or 25 mg/kg/day and the "No observed Effect Level" (NOEL) was, therefore, considered to be 150 mg/kg/day.

The gastric changes observed in this study showed regression following the fourteen day recovery period and therefore the effects were generally regarded as not being adverse. For these reasons the histopathological changes were considered not to represent an adverse health effect and the "No Observed Adverse Effect Level" (NOAEL) was, therefore, considered to be 1000 mg/kg/day.
Executive summary:

Introduction.

The study was designed to investigate the systemic toxicity of the test material and complies with the following regulatory guidelines:

i) Comission Dirctive 96/54/EC (Method B7).

ii) The Japanese Ministry of Economy Trade and Industry (METI), Ministry of Health, Labour and Welfare (MHLW) and Ministry of the Environment (MOE) Guidelines of 21 November 2003 for a twenty-eight day repeat dose oral toxicity study as required by the

Law Concerning the Evaluation of Chemical Substances and Regulation of their Manufacture, etc (Chemical Substance Control Law) 1973 of Ministry of International Trade and Industry (MITI) amended 2004.

iii) The OECD Guidelines for Testing of Chemicals No. 407 "Repeated Dose 28 Day Oral Toxicity Study in Rodents" (adopted 27 July 1995).

iv) USA Environmental Protection Agency (EPA) Health Effects Test Guidelines, OPPTS 870.3050 Repeated Dose 28 -Day Oral Toxicity Study in Rodents, July 2000.

Methods.

The test material was administered by gavage to four groups, each of five male and five female Sprague-Dawley Crl:CD (SD) IGS BR strain rats, for twenty-eight consecutive days, at dose levels of 25, 150, 300 and 1000 mg/kg/day (incorporating a correction factor for 95.8% purity). A control group of five males and five females was dosed with vehicle alone (Distilled water). Two recovery groups, each of five males and five females, were treated with the high dose (1000 mg/kg/day) or the vehicle alone for twenty-eight consecutive days and then maintained without treatment for a further fourteen days.

Clinical signs, bodyweight development and food and water consumption were monitored during the study. Haematology, blood chemistry and urinalysis were evaluated for all non-recovery group animals at the end of the treatment period and for all recovery group animals at the end of the treatment-free period.

Results.

Mortality.

There were no unscheduled deaths during the study.

Clinical Observations.

No toxicologically significant signs of toxicity were detected throughout the treatment period or during the treatment free period.

Behavioural Assessment.

There were no toxicologically significant changes in the behavioural assessments observed.

Functional Performance Test.

There were no toxicologically significant changes in the functional performance parameters observed.

Sensory Reactivity Assessments.

There were no treatment-related changes in sensory reactivity.

Bodyweight.

No toxicologically significant effects on bodyweight development were detected.

Food Consumption.

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

Water Consumption.

Males treated with 1000 mg/kg/day showed an increase in water consumption during the final two weeks of treatment. Recovery 1000 mg/kg/day males continued to show an increase in water consumption during the fourteen day treatment free period. No such

effects were detected in 1000 ml/kg/day females or animals of either sex treated with 300, 150 or 25 mg/kg/day.

Haematology.

No toxicologically significant effects were detected.

Blood Chemistry.

No toxicologically significant effects were detected.

Urinalysis.

No toxicologically significant effects were detected.

Organ Weights.

No toxicologically significant effects were detected.

Necropsy.

No toxicologically significant effects were detected.

Histopathology.

The following treatment-related stomach changes were detected:

 

Agglomeration of secretion, superficial mucosal basophilia, and hyperplasia of mucous cells were observed in the gastric mucosa immediately adjacent to the limiting ridge among animals of either sex treated with 1000 mg/kg/day. Acanthosis and hyperkeratosis of the limiting ridge were also seen among both sexes at this dose level. There was probably also an effect of treatment on animals at the 300 mg/kg/day dose level with two males and one female being similarly affected to a greater or lesser extent. There was, however, no evidence of an effect at either of the remaining treatment levels. Although agglomeration of secretion was seen for one female rat treated with 25 mg/kg/day, this condition is seen occasionally among control animals as a spontaneous change.

 

There were regressions of the conditions among Recovery 1000 mg/kg/day animals following an additional fourteen days without treatment.

 

Treatment-related gastric changes of this type are relatively commonly seen as a response to orally administered xenobiotics and at the levels of severity seen in this investigation they are regarded as not adverse.

Conclusion.

The oral administration of C-Y9 at dose levels of 25, 150, 300 and 1000 mg/kg/day for a period of twenty-eight consecutive days resulted in treatment related effects in animals of either sex treated with 1000 and 300 mg/kg/day. No such effects were detected in animals of either sex treated with 150 or 25 mg/kg/day and the "No observed Effect Level" (NOEL) was, therefore, considered to be 150 mg/kg/day.

The gastric changes observed in this study showed regression following the fourteen day recovery period and therefore the effects were generally regarded as not being adverse. For these reasons the histopathological changes were considered not to represent an adverse health effect and the "No Observed Adverse Effect Level" (NOAEL) was, therefore, considered to be 1000 mg/kg/day.