Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 January 2009 to 29 June 2009
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 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 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
Qualifier:
according to
Guideline:
other: EPA: OPPTS 870.3050 Repeated Dose 28-Day Oral Toxicity Study in Rodents
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Date of inspection: 19 August 2008; Date of signature: 04 March 2009
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Sponsor's identification : H-CB sodium salt
Description : blue solid
Purity : 94.8%
Lot number : MB-2
Date received : 04 December 2008
Storage conditions : room temperature in the dark

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd, Blackthorn, Bicestoer, Oxon.
- Age at study initiation: males and females: 6 to 8 weeks
- Weight at study initiation: males: 214 to 247 g; females: 134 to 180 g
- Fasting period before study: Not stated
- Housing: The animals were housed in groups of five by sex in solid floor poluproylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd, Cheshire, UK).
- Diet (e.g. ad libitum): Free access to food (Rodent 2014C Teklad Global Diet Harlan Laboratories Ltd, UK, Oxon, Uk) throughout the study.
- Water (e.g. ad libitum): Free access to mains drinking water via polycarbonate drinking bottles sttached to the cages throughout the study.
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.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 2°C
- Humidity (%): 55 +/- 15%
- Air changes (per hr): At least fifteen per hour
- Photoperiod (hrs dark / hrs light): Twelve hours continous light and twelve hours darkness.

IN-LIFE DATES: From: Day 0 To: Termination 28 days later for both male and female groups in all groups bar recovery group. 42 days later for males and females in the recovery groups.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Concentration in vehicle: 0, 6, 60 and 200 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of H-CB Sodium Salt in the test material formulations was determined spectrophotometrically.

Samples
Standard solutions of test material were diluted with water to gove a final, theoretical test material concentration of approximately 0.01 mg/ml

Procedure
The standard and sample solutions were analysed spectrophotometrically using the following conditions:

Spectrophotometer : Camspec M550
Wavelength : λ max at ~ 608 nm
Cell path length : 1 cm
Reference medium : Water

Homogeneity Determinations
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 perfomred in triplicate.

Stability Determinations
The test material formulations were sampled and analysed initially and then after storage at approximately + 4°C in the dark for thirteen days.

Verification of Test Material Formulation Concentrations
The test material formulations were sampled and analysed wihtin two days of preparation.
Duration of treatment / exposure:
Test duration: 28 days
Frequency of treatment:
Dosing regime: once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
Dose levels of 30, 300 and 1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
Male and Female: 5 animals per sex at 0 mg/kg/day
Male and Female: 5 animals per sex at 30 mg/kg/day
Male and Female: 5 animals per sex at 300 mg/kg/day
Male and Female: 5 animals per sex at 1000 mg/kg/day
Male and Female: 5 animals per sex at 1000 mg/kg/day (Recovery)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Not stated in report.
- Rationale for animal assignment (if not random): Random
- Rationale for selecting satellite groups: Not stated
- Post-exposure recovery period in satellite groups: 14 days.
- Section schedule rationale (if not random): Not stated

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Pre, 1 hour, 5 hours and 28 days after dosing for all groups other than the recovery group.
Recovery group: Pre, 1 hour, 5 hours, 28 days and then daily (am and pm) until day 42.
- Cage side observations checked in table were included: yes

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Days 7, 13, 20 nd 26

BODY WEIGHT: Yes
- Time schedule for examinations: Day 1 and weekly thereafter.

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: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: End of treatment period
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: 30 (15 male, 15 female) Non-recovery and 10 (5 male and 5 female) control animals (10 (5 male and 5 female) Recovery obtained at the end of the treatment free period).
- The following parameters were examined: Haemaglobin, Erythrocyte count, Haematocrit, Erythrocyte indices, Total leucocyte count (WB), Differential leucocyte count, Platelet count (PLT), Reticulocyte count, Prothrombin count and Prothrombin time

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

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: End of treatment period
- Animals fasted: No
- How many animals: Non-recovery and control animals
- The following parameters were examined: Urea, Glucose, Total protein, Albumin, Albumin/Globulin ratio, Sodium, Potassium, Chloride, Calcium, Inorganic phosphorus, Gamma glutamyltranspeptidase, Aspartate aminotransferase, Alanine aminotransferase, Creatinine, Triglycerides, Total cholesterol and Total Bilirubin.

URINALYSIS: Yes
- Time schedule for collection of urine: Non-recovery test and control animals- during week four; Recovery animals-during week six.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- The following parameters were examined: Volume, Ketones, Specific gravity, Bilirubin, pH, Urobilinogen, Protein, Reducing Substances, Glucose and Blood.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Days 7, 13, 20 nd 26 at the same time each day.
- Dose groups that were examined: All dose groups
- Battery of functions tested: sensory activity, grip strength and motor activity

Sacrifice and pathology:
GROSS PATHOLOGY: Yes

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.

Thyroid Hormone Assessment
At termination, blood samples were taken from the exsanguination procedure and the plasma from each animal was stored at -20°C. As no treatment-related effect on the pituitary-thyroid axis was identified, these samples were discarded.

Oestrus Cycle Assessment
At termination, a vaginal smear was taken from all females and the stage of oestrus was 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, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Spleen, Testes, Thymus

HISTOPATHOLOGY: Yes

Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin except where stated:

Adrenals♦, Aorta (thoracic), Bone & bone marrow (femur including stifle joint), Bone & bone marrow (sternum)♦, Brain (including cerebrum, cerebellum and pons)♦, Caecum♦, Colon♦, Duodenum♦, Epididymides ♦~, Eyes♦*, Gross lesions♦, Heart♦, Ileum♦, Jejunum♦, Kidneys♦, Liver♦, Lungs (with bronchi)♦#, Lymph nodes (cervical and mesenteric)♦, Mammary gland♦, Muscle (skeletal), Oesophagus, Ovaries♦, Pancreas, Pituitary♦, Prostrate♦, Rectum♦, Salivary glands (submaxillary), Sciatic nerve♦, seminal vesicles (with coagulating glands and fluids)♦, skin (hind limb), Spinal cord (cervical, mid thoracic and lumbar)♦, Spleen♦, Stomach♦, Testes♦~, Thymus♦, Thyroid/parathyroid♦, Trachea♦, Urinary bladder♦, Uterus & Cervix♦, Vagina♦.
*: Eyes fixed in Davidson's fluid.
~: Preserved in Bouin's fluid then transferred to Industrial Methylated Spirits (IMS) approximately 48 hours later.
#: Lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative.

All tissues were despatched to the Test Site: Propath UK Ltd, Willow Court, Netherwood Road, Rotherwas, Hereford, UK for processing. The tissues marked ♦ from all non-recovery control and 1000 mg/kg/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. Any macroscopically observed lesions were also processed together with the liver and spleen from all 30 and 300 mg/kg/day dose group animals.
Other examinations:
Since there were indications of treatment-related stomach, intestinal tract, mesenteric lymph node and kidney changes, examination was subsequently extended to include similarly prepared sections of the stomach, intestinal tract, mesenteric lymph nodes and kidneys from all animals in the other treatment groups.
Microscopic examination was conducted by the Study Pathologist. All findings were entered into the ROELEE Pathology computerisation system for tabulation and report production.
Statistics:
Data were processed to give group mean values and standard deviations where appropriate.
Where appropriate, quantitative data were analysed by the ProvantisTM Tables and Statistics Module. For each varaiable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogenity of means assessed using ANOVA or ANCOVA and Bartletts test. The transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data of the Shirley Test for non-parametric data. If no dose response was found, but the data showed non-homogenity 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 present as follows:

P < 0.01 **
P < 0.05 *
p ≥ 0.05 (not significant)

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No toxicologically significant clinical signs were detected.
Mortality:
no mortality observed
Description (incidence):
No toxicologically significant clinical signs were detected.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No treatment-related effects in bodyweight development were detected.
Food efficiency:
no effects observed
Description (incidence and severity):
No adverse effect on food consumption or food efficiency was detected for treated animals when compared to controls throughout the treatment period.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No intergroup differences were detected.
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No toxicologically significant effects were detected in the haematological parameters measured.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No toxicologically significant effects were detected in the blood chemical parameters measured.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No toxicologically significant effects were detected in the urinalytical parameters measured.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
See details below
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No toxicologically significant effects were detected in the organ weights measured.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No toxicologically significant macroscopic abnormalities were detected.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See details below
Histopathological findings: neoplastic:
no effects observed
Details on results:
NEUROBEHAVIOUR:
Behavioural Assessments: There were no treatment-related changes in the behaviourial parameters measured.
Functional Perfomrance Test: There were no toxicologically significant changes in the functional parameters measured.
Sensory Reactivity Assessment: There were no treatment-related changes in sensory reactivity.

HISTOPATHOLOGY: NON-NEOPLASTIC

Stomach: A greater incidence and generally higher grades of severity of agglomeration of secretion in the mucosa, more especially adjacent to the limiting ridge, mucous cell hypertrophy/hyperplasia, higher grades of severity of submuscosal inflammatory cells, and vacuolation/acanthosis/hyperkeratonisis of the epithelium of the limiting ridge were seen in relation to treatment for animals of wither sex treated with 1000 mg/kg/day. Mucous cell hypertrophy/hyperplasia and vacuolation/acanthosis/hyperkeratosis of the limiting ridge epithelium were also seen occasionally among animals of either sex treated with 300 mg/kg/day or at 30 mg/kg/day.
Treatment-related conditions were observed to have regressed among recovery 1000 mg/kg/day animals of either sex following an additional fourteen days without treatment.

Intestinal Tract: Accumulations of blue pigment were seen in the lamina propria or lymphoid tissue of the ileum and caecum of animals of either sex treated with 1000 mg/kg/day, but not at any other dose level. The colon of males at this dose level was similarly affected but to a lesser extent and not further evaluated at lower dose levels. Pigment accumulations were considered to be the test material and there were no associated degenerative or inflammatory changes.
There was no evidence of regression of the condition among recovery 1000 mg/kg/day animals following completion of the recovery period.

Mesenteric Lymph Node: Accumulations of blue pigment, considered to be the test material, were seen in animals of either sex treated with 1000 mg/kg/day. There were no associated degenerative or inflammatory changes. Animals from the remaining treatment levels were not similarly affected.
There was no evidence of regression if the condition among recovery 1000 mg/kg/day animals following an additional fourteen days without treatment and grades of severity of blue pigment accumulation were generally higher than non-recovery animals treated at 1000 mg/kg/day.

Kidneys: Accumulations of blue pigment, considered to be the test material, were seen in the renal proximal tubular epithelium of animals of either sex treated with 1000 mg/kg/day or at 300 mg/kg/day but not at 30 mg/kg/day. There were no associated degenerative or inflammatory changes although associated karyomegaly was seen for one female treated with 1000 mg/kg/day.
A higher incidence of globular accumulations of eosinophilic material was also observed in the tubular epithelium of males treated with 1000 mg/kg/day, 300 mg/kg/day or at 30mg/kg/day. This finding is consistent with the presence of hydrocarbon nephropathy, which results from the excessive accumulation of α2-Microglobulin in renal proximal tubular epithelial cells. α2-Microglobulin is found only in the proximal tubular epithelium of adult male rats.
There was no evidence of regression of blue pigment accumulation among recovery 1000 mg/kg/day animals of either sex following completion of the recovery period, with grades of severity of the condition being generally higher than those among non-recovery 1000 mg/kg/day animals of either sex with two associated instances of karyomegaly.

Effect levels

open allclose all
Dose descriptor:
NOEL
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Remarks on result:
not measured/tested
Remarks:
Effect level not specified (migrated information)
Dose descriptor:
NOAEL
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Remarks on result:
not measured/tested
Remarks:
Effect level not specified (migrated information)

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

The administration of H-CB Sodium Salt by oral gavage at dose levels of 30, 300 and 1000 mg/kg/day for a period of twenty-eight consecutive days resulted in treatment related effects in animals of either sex from all treatment groups.

The visible signs of blue fur staining were detected in animals of either sex treated with 1000 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 a greater incidence and generally higher grades of severity of agglomeration of secretion in the mucosa, more especially adjacent to the limiting ridge, mucous cell hypertrophy/hyperplasia, higher grades of severity of submucosal inflammatory cells and vacuolation/acanthosis/hyperkeratosis of the epithelium of the limiting ridge in animals of either sex treated with 1000 mg/kg/day. Mucous cell hypertrophy/hyperplasia and vacuolation/acanthosis/hyperkeratosis of the limiting ridge epithelium were also seen in animals of either sex treated with 300 and 30 mg/kg/day. Treatment-related conditions were observed to have regressed among recovery 1000 mg/kg/day animals following an additional fourteen days without treatment.

Accumulations of blue pigment were seen in the intestinal tract, mesenteric lymph nodes and kidneys of animals of either sex treated with 1000 mg/kg/day and in the kidneys of 300 mg/kg/day animals. There were no associated degenerative or inflammatory changes and although there was no evidence of regression in recovery animals following fourteen days without treatment the effects were not considered to be adverse.

Further microscopic abnormalities were detected in the kidneys. A higher incidence of globular accumulations of eosinophilic material was also observed in the tubular epithelium of males treated with 1000, 300 and 30 mg/kg/day. Accumulations of globular eosinophilic material in the tubular epithelium 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 is not indicative of haxard to human health.

Applicant's summary and conclusion

Conclusions:
The oral administration of H-CB Sodium Salt to rats by gavage for a period of twenty-eight consecutive days at dose levels of 30, 300 and 1000 mg/kg/day resulted in treatment-related effects in animals of either sex at all dose levels. A 'No Observed Effect Level' (NOEL) or a 'No Observed Adverse Effect Level' (NOAEL) was therefore not established.

Executive summary:

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

i) Commission Directive 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 Environment Protection Agency (EPA) Health Effects Test Guidelines, OPPTS 870.3050 Repeated Dose 28 -Day Oral Toxicity Study in Rodents, July 2000.

MethodsThe test material was administered by gavage to three groups, each of five make and five female Wistar HanTM:HsdRccHanTM:WIST strain rats, for twenty-eight consecutive days, at dose levels of 30, 300 and 1000 mg/kg/day (incorporating a correction factor for 94.8% 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 deveolpment, 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 clinical signs were detected.

Functional observations

Behaviourial Assessments There were no treatment-related changes in the bahaviourial parameters measured.

Functional Performance Test There were no toxicologically significant changes in the functional parameters measured.

Sensory reactivity AssessmentThere were no treatment-related changes in sensory activity.

Bodyweight No treatment-related effects in bodyweight development were detected.

Food Consumption No adverse effect on food consumption or food efficiency was detected for treated animals when compared to controls throughout the treatment period.

Water consumption No intergroup differences were detected.

Haematology No toxicologically significant effects were detected in the haematological parameters measured.

Blood Chemistry No toxicologically significant effects were detected in the blood chemical parameters measured.

Urinalysis No toxicologically significant effects were detected in the urinalytical parameters measured.

Pathology:

Necropsy No toxicologically significant macroscopic abnormalities were detected.

Organ weights No toxicologically significant effects were detected in the organ weights measured.

Histopathology The following treatment-related changes were observed:

Stomach: A greater incidence and generally higher grades of severity of agglomeration of secretion in the mucosa, more especially adjacent to the limiting ridge, mucous cell hypertrophy/hyperplasia, higher grades of severity of submuscosal inflammatory cells, and vacuolation/acanthosis/hyperkeratonisis of the epithelium of the limiting ridge were seen in relation to treatment for animals of wither sex treated with 1000 mg/kg/day. Mucous cell hypertrophy/hyperplasia and vacuolation/acanthosis/hyperkeratosis of the limiting ridge epithelium were also seen occasionally among animals of either sex treated with 300 mg/kg/day or at 30 mg/kg/day.

Treatment-related conditions were observed to have regressed among recovery 1000 mg/kg/day animals of either sex following an additional fourteen days without treatment.

Intestinal Tract: Accumulations of blue pigment were seen in the lamina propria or lymphoid tissue of the ileum and caecum of animals of either sex treated with 1000 mg/kg/day, but not at any other dose level. The colon of males at this dose level was similarly affected but to a lesser extent and not further evaluated at lower dose levels. Pigment accumulations were considered to be the test material and there were no associated degenerative or inflammatory changes.

There was no evidence of regression of the condition among recovery 1000 mg/kg/day animals following completion of the recovery period.

Mesenteric Lymph Node: Accumulations of blue pigment, considered to be the test material, were seen in animals of either sex treated with 1000 mg/kg/day. There were no associated degenerative or inflammatory changes. Animals from the remaining treatment levels were not similarly affected.

There was no evidence of regression if the condition among recovery 1000 mg/kg/day animals following an additional fourteen days without treatment and grades of severity of blue pigment accumulation were generallyhigher than non-recovery animals treated at 1000 mg/kg/day.

Kidneys: Accumulations of blue pigment, considered to be the test material, were seen in the renal proximal tubular epithelium of animals of either sex treated with 1000 mg/kg/day or at 300 mg/kg/day but not at 30 mg/kg/day. There were no associated degenerative or inflammatory changes although associated karyomegaly was seen for one female treated with 1000 mg/kg/day.

A higher incidence of globular accumulations of eosinophilic material was also observed in the tubular epithelium of males treated with 1000 mg/kg/day, 300 mg/kg/day or at 30mg/kg/day. This finding is consistent with the presence of hydrocarbon nephropathy, which results from the excessive accumulation of α2-Microglobulin in renal proximal tubular epithelial cells. α2-Microglobulin is found only in the proximal tubular epithelium of adult male rats.

There was no evidence of regression of blue pigment accumulation among recovery 1000 mg/kg/day animals of either sex following completion of the recovery period, with grades of severity of the condition being generally higher than those among non-recovery 1000 mg/kg/day animals of either sex wuth two associated instances of karyomegaly.

Conclusion

The oral administration of H-CB Sodium Salt to rats by gavage for a period of twenty-eight consecutive days at dose levels of 30, 300 and 1000 mg/kg/day resulted in treatment-related effects in animals of either sex at all dose levels. A 'No Observed Effect Level' (NOEL) or a 'No Observed Adverse Effect Level' (NOAEL) was therefore not established.