C-C1

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 March 2007 to 17 August 2007

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:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3050 Repeated Dose 28-Day Oral Toxicity Study

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese guidelines

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(Date of inspection: 30 August 2005 Date of Signature: 21 November 2005)

Limit test:

no

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

- Name of test material (as cited in study report): C-C1.

- Physical state: Dark blue powder.

- Analytical purity: 96.3%.

- Lot/batch No.: MB-1.

- Storage condition of test material: Room temperature in the dark over silica gel.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, United Kingdom.

- Age at study initiation: Approximately 5 to 8 weeks old.

- Weight at study initiation: 146 to 183 g (males), 133 to 161 g (females).

- Fasting period before study: No data.

- Housing: The animals were housed in groups of five by sex in polypropylene grid-floor cages suspended over trays lined with absorbent paper.

- Diet: A pelleted diet (Rodent 5LF2 (Certified) Diet, BCM IPS Limited, London, UK), ad libitum.

- Water: Mains drinking water, ad libitum.

- Acclimation period: 7 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2ºC.

- Humidity (%): 55 ± 15%.

- Air changes (per hr): At least 15 air changes per hour.

- Photoperiod (hrs dark / hrs light): Twelve hours continuous light and twelve hours darkness.

IN-LIFE DATES: From: Day 0 To: Day 28 (non-recovery group) and Day 42 (recovery group).

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: For the purpose of this study the test material was prepared at the appropriate concentrations as a solution in Distilled water. The stability and homogeneity of the test material formulations were determined by the testing facility. Results showed that the formulations to be stable for at least 14 days. Formulations were therefore prepared weekly and stored at 4ºC in the dark.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Summary; The concentration of C-C1 in the test material formulations was determined spectrophotometrically.

Samples; The test material formulations were diluted with water to give a final, theoretical test material concentration of approximately 0.01 mg/ml.

Standards; Standard solutions of test material were prepared in water at a nominal concentration of 0.01 mg/ml.

Procedure; The sample and standard solutions were analysed by spectrophotometrically.

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 performed in triplicate.

Stability determinations; The test material formulations were sampled and analysed initially and then after storage at approximately + 4ºC in the dark for fourteen days.

Verification of test material formulation concentrations; The test material formulations were sampled and analysed within two days of preparation.

Results; Mean concentration found was in the range of 100 - 110% of nominal concentrations. The results showed the formulation to be stable for at least 14 days.

Duration of treatment / exposure:

28 days.

Frequency of treatment:

Once daily.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
1000 mg/kg/day
Basis:
nominal in water

Doses / concentrations 2

Remarks:

Doses / Concentrations:
300 mg/kg/day
Basis:
nominal in water

Doses / concentrations 3

Remarks:

Doses / Concentrations:
150 mg/kg/day
Basis:
nominal in water

Doses / concentrations 4

Remarks:

Doses / Concentrations:
25 mg/kg/day
Basis:
nominal in water

No. of animals per sex per dose:

5 animals per sex per dose and the control group.
Additional 5 animals per sex at the highest dose group and control group were used for recovery group.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were chosen based on the results of the range-finding study performed. See the summary of the range-finding study in 'any other information on materials and methods incl. tables'.

- Rationale for selecting satellite groups: random.

- Post-exposure recovery period in satellite groups: 14 days.

Positive control:

No.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS / DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: 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 and on public holidays).
- Cage side observations checked: Overt signs of toxicity, ill-health and behavioural change.

BODY WEIGHT: Yes
- Time schedule for examinations: 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: Yes
Weekly food consumption was recorded for each cage group.

FOOD EFFICIENCY: Yes
- Food efficiency (the ratio of bodyweight gain / food consumption) was calculated.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water intake was observed daily for each cage group by visual inspection of the water bottles for any overt changes. Water intake was measured and recorded daily during Week 3 for each cage group and possible intergroup differences were detected. Water consumption was, therefore, measured and recorded for each cage group during the remaining study period.

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
Haematological and blood chemical investigations were performed on Day 28 for non-recovery group and on Day 42 for recovery group. Blood samples were obtained from the lateral tail vein.
- Anaesthetic used for blood collection: No.
- Animals fasted: No.
- How many animals: All non-recovery animals and all recovery animals.
- Parameters checked:
(Haematology) Hb, RBC, Hct, MCH, MCV, MCHC, WBC, APTT, Neut, Lymph, Mono, Eos, Bas, CT, PLT, Meth and Retic.
(Clinical Chemistry) Urea, Glucose, Tot.Prot, Albumin, A/G, Na+, K+, Cl-, Ca++, P, gamma-GT, ASAT, ALAT, AP, Creat, Tri, Chol and Bili.


URINALYSIS: Yes
- Time schedule for collection of urine: During week 4 (non-recovery group) and during week 6 (recovery group).
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: Volume, specific gravity, pH, protein, glucose and ketones.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
Behavioural assessment: Prior to the start of treatment and on Days 3, 10, 17 and 22.
Functional Performance Tests: During Week 4.
Sensory Reactivity: During Week 4.

- Dose groups that were examined: All animals.


- Parameters examined:
Behavioural assessment: 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 and 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 thirty minutes for each animal. The time in seconds each animal was active and mobile was recorded for the overall thirty minute 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 and finger approach.

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 (Rhone Mérieux, Dagenham, Essex, UK) 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, liver, brain, ovaries, epididymides, spleen, heart, testes, kidneys and thymus.

HISTOPATHOLOGY: Yes
Samples of the following tissues were preserved from all animals and preserved in buffered 10% formalin.
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. Lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative), lymph nodes (cervical and mesenteric), muscle (skeletal), oesophagus, ovaries, pancreas, pituitary, prostate, rectum, salivary glands (submaxillary), sciatic nerve, seminal vesicles, skin (hind limb), spinal cord (cervical), spleen, stomach, testes, thymus, thyroid/parathyroid, trachea, urinary bladder and uterus.

All tissues were despatched to the external facility for processing. The tissues (except aorta, bone & bone marrow, eyes, muscle, oesophagus, pancreas, pituitary, salivary glands and skin) from all non-recovery control and 1000 mg/kg/day A.I 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 25, 150 and 300 mg/kg/day. Since there were indications of treatment-related kidney, intestines, lungs, cervical/mesenteric lymph nodes and stomach changes, examination was subsequently extended to include similarly prepared sections of the kidneys, intestines, lungs, cervical/mesenteric lymph nodes and stomach from all animals in the other treatment groups.

Other examinations:

Not performed.

Statistics:

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) were calculated as follows:

p < 0.01 **
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):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

no effects observed

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

Description (incidence and severity):

non-adverse effects

Histopathological findings: neoplastic:

no effects observed

Details on results:

MORTALITY: No deaths.

CLINICAL SIGNS: Blue/dark staining on the cage tray liners and blue/dark faeces was evident throughout the treatment period for animals of either sex treated with 1000, 300 and 150 mg/kg/day A.I. Associated episodes of generalised blue fur staining and blue stained tail was also evident in animals of either sex treated with 1000 and 300 mg/kg/day A.I and in females treated with 150 mg/kg/day A.I from Day 5 onwards.
Staining of faeces and on cage tray liners remained evident in recovery animals of either sex treated with 1000 mg/kg/day A.I throughout the treatment free period.

BODY WEIGHT AND WEIGHT GAIN: No adverse effects observed.

FOOD CONSUMPTION AND FOOD EFFICIENCY: No adverse effects observed.

WATER CONSUMPTION: No overt differences.

HAEMATOLOGY: Females treated with 1000 mg/kg/day A.I showed a statistically significant increase in lymphocyte count. No such effects were detected in males treated with 1000 mg/kg/day A.I or animals of either sex treated with 300, 150 or 25 mg/kg/day A.I.
The intergroup differences detected in total leucocyte count and mean cell haemoglobin concentration were considered to be of no toxicological importance (see Justification of No Observed Effect Level).

CLINICAL CHEMISTRY: No toxicologically significant effects seen.

URINALYSIS: No toxicologically significant changes seen.

NEUROBEHAVIOUR: No toxicologically significant changes seen.

ORGAN WEIGHTS: No toxicologically significant changes seen.

GROSS PATHOLOGY: Discolouration was detected in the majority of tissues in animals of either sex treated with 1000, 300 and 150 mg/kg/day A.I. Discolouration of the majority of tissues continued to be evident for recovery 1000 mg/kg/day A.I animals following cessation of treatment.
The findings recorded at necropsy were considered to be of no toxicological importance (see Justification of No Observed Effect Level).


HISTOPATHOLOGY: The following treatment-related changes were detected:
KIDNEY: Accumulations of blue pigment in the proximal tubular epithelium of the kidneys were seen as a consequence of treatment in animals of either sex treated with 1000 mg/kg/day A.I or at 300 mg/kg/day A.I, and for females only treated with 150 mg/kg/day A.I. Blue pigment accumulation was not seen among animals treated with 25 mg/kg/day A.I.
There was no evidence to suggest that accumulations of pigment had reduced among recovery 1000 mg/kg/day A.I animals of either sex following an additional fourteen days without treatment.
STOMACH: Agglomeration of secretion, mucous cell hyperplasia, and mucosal basophilia/atrophy were observed in relation to treatment in the glandular stomach of animals of either sex treated with 1000 mg/kg/day A.I and for males only treated with 300 mg/kg/day A.I, 150 mg/kg/day A.I, or 25 mg/kg/day A.I. Isolated instances of these conditions were seen among females at the remaining dose levels but not convincingly as a consequence of treatment. Acanthosis and hyperkeratosis of the limiting ridge was also seen as an effect of treatment in animals of either sex treated with 1000 mg/kg/day A.I and among males at the remaining dose levels.
All conditions were observed to have regressed among recovery 1000 mg/kg/day A.I group animals following completion of the recovery period.
INTESTINAL TRACT: Accumulations of blue pigment were seen in the lamina propria at all levels of the intestinal tract except in the duodenum for animals of either sex treated with 1000 mg/kg/day A.I. Blue pigment accumulations were observed only in the jejunum of animals of either sex treated with 300 mg/kg/day A.I or at 150 mg/kg/day A.I and not at any level of the intestinal tract for animals of either sex treated with 25 mg/kg/day A.I.
Blue pigment accumulation at any level of the intestinal tract was not observed to have regressed among recovery group animals of either sex following completion of an additional fourteen days without treatment. Furthermore, the duodenum was observed to also have accumulations in the lamina propria among recovery 1000 mg/kg/day A.I animals of either sex.
LUNGS: Accumulations of blue pigment were seen within alveolar macrophages in three males and in two females treated with 1000 mg/kg/day A.I. The distribution of pigment accumulations suggested that they may have resulted from accidental instillation/inhalation of the test material during dosing, although a systemic effect of treatment cannot be entirely excluded. A similar effect was not seen among animals of either sex at any other treatment level.
One recovery male and two recovery females treated with 1000 mg/kg/day A.I showed similar pulmonary changes and in addition, the two females had extensive areas of inflammation and macrophage infiltration suggesting mis-dosing to be a factor in pigment accumulation in these instances. The Inflammatory lesions among recovery animals were considered not to be an adverse reaction specifically to the pigment accumulation.
CERVICAL LYMPH NODE: Accumulations of blue pigment were seen in relation to treatment for females only treated with 1000 mg/kg/day A.I and for two females treated with 300 mg/kg/day A.I.
There was evidence of a regression of pigment accumulation among recovery 1000 mg/kg/day A.I females although accumulations were also present among two males in this group.
MESENTERIC LYMPH NODE: Accumulations of blue pigment were seen in relation to treatment for animals of either sex treated with 1000 mg/kg/day A.I, but not at any other treatment level.
There was no evidence of regression of blue pigment accumulation among recovery 1000 mg/kg/day A.I animals following an additional fourteen days without treatment.
Blue pigment accumulations in various tissues in this study were attributable to the test material or to a metabolite thereof. The accumulations at the levels seen did not elicit an inflammatory response or any other tissue reaction suggesting the material to be inert at the levels of accumulation encountered. It is not uncommon for pigment accumulations of this type to persist beyond a fourteen day recovery period.

Effect levels

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The oral administration of C-C1 at dose levels of 25, 150, 300 and 1000 mg/kg/day A.I for a period of twenty-eight consecutive days resulted in treatment related effects in animals of either sex treated with 1000, 300 and 150 mg/kg/day A.I and in males treated with 25 mg/kg/day A.I. A “No observed Effect Level” (NOEL) for males has therefore not been established. No such changes were detected in females at 25 mg/kg/day A.I and the ‘No Observed Effect Level’ (NOEL) for females was, therefore, considered to be 25 mg/kg/day A.I.
In the absence of an inflammatory response or any other tissue reaction the pigment accumulation detected was regarded of no toxicologically importance. The gastric changes observed in this study regressed 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 A.I.

Executive summary:

Introduction. The study was designed to investigate the systemic toxicity of 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).

iv)                USAEnvironmental 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 three 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 96.3% 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.

All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed.

Results.

Mortality.There were no unscheduled deaths during the study.

Clinical Observations.Blue/dark staining on the cage tray liners and blue/dark faeces was evident throughout the treatment period for animals of either sex treated with 1000, 300 and 150 mg/kg/day A.I. Associated episodes of generalised blue fur staining and blue stained tails was also evident in animals of either sex treated with 1000 and 300 mg/kg/day A.I and in females treated with 150 mg/kg/day A.I. 

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

Staining of faeces and on cage tray liners remained evident in recovery animals of either sex treated with 1000 mg/kg/day A.I throughout the treatment free period.

Functional Observations.

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

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

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

Bodyweight.No adverse effect on bodyweight development was detected.

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

Water Consumption.No intergroup differences were detected.

Haematology.No toxicologically significant effects were detected.

Blood Chemistry.No toxicologically significant effects were detected.

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

Organ Weights.No toxicologically significant effects were detected.

Necropsy.Discolouration was detected in the majority of tissues in animals of either sex treated with 1000, 300 and 150 mg/kg/day A.I. No such effects were detected for animals of either sex treated with 25 mg/kg/day A.I. Discolouration of the majority of tissues continued to be evidentfor recovery 1000 mg/kg/day A.I animals following cessation of treatment.

Histopathology.The following treatment-related changes were detected:

KIDNEY:Accumulations of blue pigment in the proximal tubular epithelium of the kidneys were seen as a consequence of treatment in animals of either sex treated with 1000 mg/kg/day A.I or at 300 mg/kg/day A.I, and for females only treated with 150 mg/kg/day A.I. Blue pigment accumulation was not seen among animals treated with 25 mg/kg/day A.I.

There was no evidence to suggest that accumulations of pigment had reduced among recovery 1000 mg/kg/day A.I animals of either sex following an additional fourteen days without treatment. 

STOMACH: Agglomeration of secretion, mucous cell hyperplasia, and mucosal basophilia/atrophy were observed in relation to treatment in the glandular stomach of animals of either sex treated with 1000 mg/kg/day A.I and for males only treated with 300 mg/kg/day A.I, 150 mg/kg/day A.I, or 25 mg/kg/day A.I. Isolated instances of these conditions were seen among females at the remaining dose levels but not convincingly as a consequence of treatment. Acanthosis and hyperkeratosis of the limiting ridge was also seen as an effect of treatment in animals of either sex treated with 1000 mg/kg/day A.I and among males at the remaining dose levels.

All conditions were observed to have regressed among recovery 1000 mg/kg/day A.I group animals following completion of the recovery period.

INTESTINAL TRACT: Accumulations of blue pigment were seen in the lamina propria at all levels of the intestinal tract except in the duodenum for animals of either sex treated with 1000 mg/kg/day A.I. Blue pigment accumulations were observed only in the jejunum of animals of either sex treated with 300 mg/kg/day A.I or at 150 mg/kg/day A.I and not at any level of the intestinal tract for animals of either sex treated with 25 mg/kg/day A.I.

Blue pigment accumulation at any level of the intestinal tract was not observed to have regressed among recovery group animals of either sex following completion of an additional fourteen days without treatment. Furthermore, the duodenum was observed to also have accumulations in the lamina propria among recovery 1000 mg/kg/day A.I animals of either sex. 

LUNGS: Accumulations of blue pigment were seen within alveolar macrophages in three males and in two females treated with 1000 mg/kg/day A.I. The distribution of pigment accumulations suggested that they may have resulted from accidental instillation/inhalation of the test material during dosing, although a systemic effect of treatment cannot be entirely excluded. A similar effect was not seen among animals of either sex at any other treatment level.

One recovery male and two recovery females treated with 1000 mg/kg/day A.I showed similar pulmonary changes and in addition, the two females had extensive areas of inflammation and macrophage infiltration suggesting mis-dosing to be a factor in pigment accumulation in these instances. The Inflammatory lesions among recovery animals were considered not to be an adverse reaction specifically to the pigment accumulation.

CERVICAL LYMPH NODE: Accumulations of blue pigment were seen in relation to treatment for females only treated with 1000 mg/kg/day A.I and for two females treated with 300 mg/kg/day A.I.

There was evidence of a regression of pigment accumulation among recovery 1000 mg/kg/day A.I females although accumulations were also present among two males in this group.

MESENTERIC LYMPH NODE: Accumulations of blue pigment were seen in relation to treatment for animals of either sex treated with 1000 mg/kg/day A.I, but not at any other treatment level.

There was no evidence of regression of blue pigment accumulation among recovery 1000 mg/kg/day A.I animals following an additional fourteen days without treatment.

Blue pigment accumulations in various tissues in this study were attributable to the test material or to a metabolite thereof. The accumulations at the levels seen did not elicit an inflammatory response or any other tissue reaction suggesting the material to be inert at the levels of accumulation encountered. It is not uncommon for pigment accumulations of this type to persist beyond a fourteen day recovery period.

Conclusion.The oral administration of C-C1at dose levels of 25, 150, 300 and 1000 mg/kg/day A.I for a period of twenty-eight consecutive days resulted in treatment related effectsin animals of either sex treated with 1000, 300 and 150 mg/kg/day A.I and in males treated with 25 mg/kg/day A.I. A “No observed Effect Level” (NOEL) for males has therefore not been established. No such changes were detected in females at 25 mg/kg/day A.I and the ‘No Observed Effect Level’ (NOEL) for females was, therefore, considered to be 25 mg/kg/day A.I.

In the absence of an inflammatory response or any other tissue reaction the pigment accumulation detected was regarded of no toxicologically importance. The gastric changes observed in this study regressed 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 A.I.