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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:
09 June 2008 to 18 December 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
other: US Food and Drug Administration, Redbook 2000, Toxicological Principles for the Safety Assessment of Food Ingredients (November 2003)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
(Date of inspection: 19 August 2008 Date of Signature: 29 January 2009)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd, Oxon, UK.
- Age at study initiation: Approximately six to eight weeks old
- Weight at study initiation: Males: 188 - 236 g, Females: 110 - 149 g
- Fasting period before study: not stated in report
- Housing: The animals were housed in groups of up to four by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Harlan Laboratories UK Ltd, Oxon, UK).
- Diet: ad libitum access to a ground diet (Rat and Mouse SQC Ground Diet No. 1 Diet, Special Diets Services Ltd., Witham, Essex, UK).
- Water: ad libitum access to mains drinking water supplied from polycarbonate bottles attached to the cages
- Acclimation period: Nine 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 15 changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours continuous light followed by 12 hours darkness

IN-LIFE DATES: From: Day 1 to Day 90 for test animals and control animals. Up to Day 119 for recovery animals

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: not applicable as dietary study

DIET PREPARATION
- Rate of preparation of diet (frequency): Prepared prior to treatment and then every two weeks during the treatment period i.e. at twice-monthly intervals

- Mixing appropriate amounts with (Type of food): A known amount of test material was mixed with a small amount of basal laboratory diet for nineteen minutes at a constant speed, setting 1 in a hobart QE200 mixer. an additional premix was prepared using the Blixer 4 mixture. These pre-mixes were then added to a larger amount of basal laboratory diet and mixed for a further 30 minutes at a constant speed, setting 1 in a Hobart H800 mixer.

- Storage temperature of food: ambient temperature.
(The diet was stored in labelled, double black plastic bags in labelled, covered plastic bins when not in use).

VEHICLE
- no vehicle used
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of DVS005u in the dietary admixtures was determined by HPLC using an external standard technique.

The dietary admixtures were extracted with acetonitrile to give a final, theoretical test material concentration of approximately 100 ppm. Standard solutions of test material were prepared in acetonitrile at a nominal concentration of 100 ppm.

The standard and sample solutions were analysed by HPLC using the following conditions:
HPKC: Agilent Technologies 1200, incorporating autosampler and workstation
Column: Gemini 5µ C18 (50 x 4.6 mm id)
Mobile phase: Acetonitrile
Flow rate: 1.0 ml/min
UV detector wavelength: 225 nm
Injection volume: 25 µl
Retention time; ~ 3, 7 and 17 minutes

The dietary admixtures were sampled and analysed initially and then after storage at ambient temperature in the dark for three weeks.

The dietary admixtures were sampled and analysed within four days of preparation.
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.
Duration of treatment / exposure:
90 days
Frequency of treatment:
Free access to food
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
1000 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
70 mg/kg bw/day
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
10000 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
759 mg/kg bw/day
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
20000 ppm (1531 mg/kg/ bw/day)
Basis:
nominal in diet
No. of animals per sex per dose:
20 males and 20 females per dose group.

10 males and 10 females in the recovery control
10 males and 10 females in the recovery high group
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The dietary concentrations were chosen based on the results of the range-finding / palatability investigation detailed as a suporting study for this endpoint in the IUCLID dossier.
- Rationale for animal assignment (if not random): not applicable, as animal selection was random
- Rationale for selecting satellite groups: not stated in report
- Post-exposure recovery period in satellite groups: not stated in report
- Section schedule rationale (if not random): not applicable
Positive control:
Not applicable, as no positive control used.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE AND NEUROBEHAVIOURAL OBSERVATIONS: Yes
- Time schedule for examinations: Prior to the start of treatment and at weekly intervals thereafter
- Dose groups that were examined: All non-recovery animals. Functional performance tests were also performed on all non-recovery animals during Week 12.
- Battery of functions tested: Behavioural assesment, sensory reactivity, forelimb/hindlimb grip strength, motor activity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health or behavioural changes once daily. All observations were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual bodyweights were recorded on Day 1 (prior to the start of treatment) and at weekly intervals thereafter. bodyweights were also recorded at terminal kill.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No
Food consumption was recoded for each group at weekly intervals throughout the study.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water consumption 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: Pre-treatment and during Week 12
- Dose groups that were examined: The eyes of ten males and females from each non-recovery group were examined pre-treatment. The eyes from ten males and females from the non-recovery control and high dose groups were examined before termination of treatment (during Week 12).

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during Week 2, Week 7 and 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 or Day 120 in the case of recovery animals.
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: Ten male and ten female non-recovery animals from each test and control group and all animals of the recovery group.
- Parameters examined: Haemoglobin (Hb) erythrocyte count (RBC), Haematocrit (Hct), erythrocyte indices, total leucocyte count (WBC), differebtial leucocyte count, platelet count (PLT), reticulocyte count (Retic), prothrombin time (CT) and activated partial thromboplastic time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during Week 2, Week 7 and 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 or Day 120 in the case of recovery animals.
- Animals fasted: No
- How many animals: Ten male and ten female non-recovery animals from each test and control group.
- Parameters examined: Urea, glucose, total protein, albumin, albumin/globulin (A/G) ratio, sodium (Na+), potassium (K+), chloride (Cl-), Calcium (Ca++), inorganic phosphorous (P), aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), alkaline phosphatase (AP), creatinine, total cholesterol, total bilirubin, triglycerides, gamma-glutamyltranspeptidase and sorbitol dehydrogenase

URINALYSIS: Yes
- Time schedule for collection of urine: Ten male and ten female non-recovery test and control group animals during the final week of the treatment period and on all recovery group animals during the final week of the treatment-free period.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: Volume, specific gravity, pH, protein, glucose, ketones, urobilinogen, bilirubin, blood - erythrocytes and haemoglobin, redcing substances and appearance

Sacrifice and pathology:
GROSS PATHOLOGY (Organ weights): Yes - The following organs were removed from test animals that were killed at the end of the study, were dissected free from fat and weighed before fixation: Adrenals, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, thymus, uterus and throid/parathyroid.

HISTOPATHOLOGY: Yes - samples of the following tissues were removed from all animals and preserved in buffered 10% formalin, except where stated: Adrenals, thoracic aorta, bone and bone maarow (for the sternum, femur including stifle joint), brain (including cerebrum, cerebellum and pons), caecum, colon, duodenum, epididymides (preserved in Bouin's fluid then transferred to IMS up to 48 hours later), eyes, gross lesions, harderian gland, heart, ileum (including Payer's patches) jejunum, kidneys, liver, lungs (with bronchi), lymph nodes (cervical and mesenteric), ammary glands, skeletal muscles, nasal turbinates, oesophagus, ovaries (with fallopian tubes, pancreas, pituitary, prostate, rectum, salivary glands (submaxillary), sciatic nerve, seminal vesicles, skin (hind limb), spinal cord (cervical, mid-thoracic and lumbar), spleen, stomach, testes, thymus, throid/parathyroid, tongue, trachea, urinary bladder, uterus (corpus and cervuix, vagina.

Since there were indications of treatment-related bone marrow changes, examination was subsequently extended to include similarly prepared sections of bone marrow from all animals in the other treatment groups.

Additional information regarding histopathology
All tissues from non-recovery control and 20000 ppm dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 µm and stained with haematoxylin and eosin for subsequent microscopic examination. All macroscopically observed lesions were also processsed.
Statistics:
Data were processed to give group mean values and standard deviations where appropriate.

Where appropriate, quantitative data were analysed by the Provantis Tables and Statistics Module. For each variable, the most suitable transformation of the data were founf, 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 Willians 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).

Sorbitol dehydrogenase values were not captured directly onto the Provantis data capture system as this facility was not available. The statistical analyses were undertaken using the SPSS statisticak program (version 10). ANOVA, incorporating a test for homogeneity of variance was used. Dose response relationshps were investigated by linear regression. Where variances were shown to be homogenous, pair-wise comparisons were conducted using dunnetts test. Where appropriate, data was analysed using non-parametric methods; Kruskal-Wallis, ANOVA and Mann-witney U test.

Probability values are presented as follows: p <0.01, p < 0.05, p = 0.05 (not significant).

PLEASE SEE FOLLOWING SECTION FOR ADDITIONAL INFORMATION REGARDING STATISTICS

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):
Findings not considered to represent an adverse health effect. (Please see following section for details on observations)
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Findings not considered to represent an adverse health effect. (Please see following section for details on observations)
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no unscheduled deaths.
No clinically observable signs of toxicity were detected.

BODY WEIGHT AND WEIGHT GAIN
No adverse effect on bodyweight change was detected.
Males treated with 20000 and 10000 ppm displayed statistically significant reductions in bodyweight gain during Week 4 (p<0.01). Statistically significant reductions in bodyweight gains were also detected for males from all treatment groups when compared to controls during Week 8 (p<0.05 to p<0.01). Recovery 20000 oom males showed a statistically significant increase in bodyweight gain during Week 17 (p<0.01) and a statistically significant reduction during Week 18 (p<0.05). These significant differences detected during the study were confinded to the male dose groups, and in the absence of convincing reductions throughout the treatment period, these findings were not considered to represent an adverse effect of treatment.

FOOD CONSUMPTION, FOOD EFFICIENCY, AND COMPOUND INTAKE (if feeding study)
No adverse effect on dietary intake or food efficiency was detected.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
No intergroup differences were detected.

OPHTHALMOSCOPIC EXAMINATION
No ocular effect were detected.

HAEMATOLOGY
No treatment-related effects were detected.

CLINICAL CHEMISTRY
Aspartate aminotransferase levels for animals treated with 20000 and 10000 ppm were higher than controls during the treatment period and the increase was still evident for recovery 20000 ppm males following the treatment-free period. No such effects were detected for animals of either sex treated with 1000 ppm.
Findings not considered to represent an adverse health effect.

URINALYSIS
No treatment-related effects were detected.

NEUROBEHAVIOUR
No treatment-related effects were detected in the functional peformance tests, and the sensory reactivity and beavioural assesments.

ORGAN WEIGHTS
Females treated with 20000 ppm showed an increase in liver and spleen weights, both absolute and relative to terminal weights, when compared to controls. no such effects were evident following the treatment free period.
No such effects were detected for males treated with 20000 ppm or for animals of either sex treated with 10000 or 1000 ppm.
Findings not considered to represent an adverse health effect.

GROSS PATHOLOGY
No treatment-related macroscopic abnormalities were detected.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 531 mg/kg bw/day (actual dose received)
Based on:
other: dietary concentration
Sex:
male/female
Basis for effect level:
other: overall effects - minor treatment related changes at 20000 and 10000 which were not considered to represent an adverse health effect.
Dose descriptor:
NOEL
Effect level:
70 mg/kg bw (total dose)
Based on:
other: dietary concentration
Sex:
male/female
Basis for effect level:
other: No treatment related effects were detected at the lowest dietary concentration.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Oral administration of the test material DVS005u, to rats for a period of ninety consecutive days at dietary concentrations of 20000, 10000 and 1000 ppm resulted in minor treatment-related changes at 20000 and 10000 ppm. these findings were not considered to represent an adverse health effect, therefore the No Observed Adverse Effect Level (NOAEL) was considered to be 20000 ppm.

No treatment-related effects were detected at the lowest dietary concentration, therefore the 'No Observed Effect Level' (NOEL) was considered to be 1000 ppm.
Executive summary:

Introduction: The study was designed to investigate the systemic toxicity of the test material and complies with the recommendations of the OECD Guidelines for Testing of Chemicals No. 408 "Subchronic Oral Toxicity - Rodent: 90 Day study" (adopted 21 September 1998) and reflects the requirements of the US Food and Drug Administration, Redbook 2000, Toxicological Principles for the Safety Assessment of Food Ingredients (November 2003).

Methods: The test material was administered by dietary admixture to three groups, each of twenty male and twenty female Wistar HAN(TM): HsdRccHan (TM):WIST strain rats, for ninety consecutive days, at dietary concentrations of 1000, 10000 and 20000 ppm (equivalent to a mean achieved dosage of 70, 759 and 1531 mg/kg/day respectively). A further group of twenty males and twenty females was exposed to basal laboratory diet to serve as a control. Two recovery groups, each of ten males and ten females, were treated with the high dose (20000 ppm0 or basal laboratory diet for ninety consecutive days and then maintained without treatment for a further twenty-nine days on basal laboratory diet.

clinical signs, functional observations, bodyweiight development and food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for ten males and ten females from each non-recovery dose group during Week 2, Week 7 and at the end of the treatment phase. These investigations were also performed on recovery control and high dose group animals at the end of the treatment-free period. Opthalmoscopic examination was also peformed on ten males and ten females from each non-recovery dose group, prior to the start of treatment and for ten male and ten female non-recovery control and high dose animals during Week 12 of the study. Urianalytical investigations were undertaken for ten males and ten females from each non-recovery dose group during the final week of treatment and for all recovery group animals during the final week of the treatment-free period.

All animals were subjected to gross necropsy examination and a comprehensive histropathological evaluation of tissues was peformed.

Results

Mortality: There were no unscheduled deaths.

Clinical observations: No clinically observable signs of toxicity were detected.

Behavioural assessment: No treatment-related effects were detected.

Functional performance tests: No treatment-related effects were detected.

Sensory reactivity assessments: No treatment-related effects were detected.

Bodyweight: No adverse effect on bodyweight changes was detected.

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

Water consumption: No intergroup differences were detected.

Opthalmoscopy: No intergroup differences were detected.

Urinalysis: No treatment-related effects were detected.

Haematology: No treatment-related effects were detected.

Blood chemistry: Aspartate aminotransferase levels for animals treated with 20000 and 10000 ppm were higher than controls during the treatment period and the increase was still evident for recovery 20000 ppm males following the treatment-free period. No such effects were detected for animals of either sex treated with 1000 ppm.

Organ weights: Females treated with 20000 ppm showed an increase in liver and spleen weights, both absolute and relative to terminal weights, when compared to controls. no such effects were evident following the treatment free period. No such effects were detected for males treated with 20000 ppm or for animals of either sex treated with 10000 or 1000 ppm.

Necropsy: No treatment-related macroscopic abnormalities were detected.

Histopathology: No treatment-related changes were detected.

Conclusion: Oral administration of the test material DVS005u, to rats for a period of ninety consecutive days at dietary concentrations of 20000, 10000 and 1000 ppm resulted in minor treatment-related changes at 20000 and 10000 ppm. these findings were not considered to represent an adverse health effect, therefore the No Observed Adverse Effect Level (NOAEL) was considered to be 20000 ppm.

No treatment-related effects were detected at the lowest dietary concentration, therefore the 'No Observed Effect Level' (NOEL) was considered to be 1000 ppm.