1,3,5-Naphthalenetrisulfonic acid, 7-[2-[2-[2-[5-cyano-4-methyl-2,6-bis[(4-sulfophenyl)amino]-3-pyridinyl]diazenyl]-4-(2-naphthalenyl)-5-thiazolyl]diazenyl]-, lithium sodium salt (1:?:?)

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:

25 August 2005 and 22 Janaury 2006 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Meets the criteria for classification as Reliable without restriction according to Klimisch et al (1997)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

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)

Test guideline 2

Guideline:

other: 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.

GLP compliance:

yes

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

471-980-9

EC Name:

-

Cas Number:

1016986-95-0

Molecular formula:

Hill formula: C42 H24 Lim N9 Nan O15 S6 m+n=5 3=

IUPAC Name:

7-{2-[2-(2-{5-cyano-4-methyl-2,6-bis[(4-sulfophenyl)amino]pyridin-3-yl}diazen-1-yl)-4-(naphthalen-2-yl)-1,3-thiazol-5-yl]diazen-1-yl}naphthalene-1,3,5-trisulfonic acid trilithium hydride disodium hydride

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): FSM-003B
- Physical state: Black Powder
- Analytical purity: 85.6%
- Lot/batch No.: CE-501-2
- Date received: 23 August 2005
- Storage condition of test material: room temperature in the dark, ubder nitrogen 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
- Strain: Sprague-Dawley Crl:CD® (SD) BR strain rats
- Source: Charles River (UK) Limited, Margate, Kent.
- Age at study initiation: Approximately 6 to 8 weeks old.
- Weight at study initiation: At the start of treatment the males weighed 217 to 262g and the females weighed 165 to 211g.
- Housing: The animals were housed in groups of five by sex in polypropylene grid-floor cages suspended over trays lined with absorbent paper. The animals were allowed free access to food and water.
- Diet (e.g. ad libitum): Pelleted diet (Rodent 5LF2 (Certified) Diet).
- Water (e.g. ad libitum): Mains drinking water was supplied from polycarbonate bottles attached to the cage.
- Acclimation period: 15 days

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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: Dried Arachis oil BP

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration in the test material formulations was determined spectrophotometrically. The test material formulations were extracted with methanol to give a final, theoretical test material concentration of approximately 0.01 mg/ml. Standard solutions of test material were prepared in methanol at a nominal concentration of 0.01mg/ml. The standard and sample solutions were analysed spectrophotometrically using the following conditions:

Spectrophotometer : Perkin-Elmer Lambda 20
Wavelength : λmax at ~ 602 nm
Cell path length : 1 cm
Reference medium : methanol

The test material formulations were sampled and analysed within three days of preparation.

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.


Conclusion
The analytical method was considered sufficiently accurate and valid in terms of linearity, specificity and accuracy for the purposes of the study.

Duration of treatment / exposure:

Test duration: 28 days

Frequency of treatment:

Dosing regime: 7 days/week

Doses / concentrations

Remarks:

Doses / Concentrations:

Basis:

No. of animals per sex per dose:

Male: 5 animals at 0 mg/kg bw/day
male: 5 animals at 0 mg/kg/bw/day (recovery group control)
Male: 5 animals at 25 mg/kg bw/day
Male: 5 animals at 150 mg/kg bw/day
Male: 5 animals at 500/400 mg/kg bw/day*
Male: 5 animals at 750/600 mg/kg bw/day*
Male: 5 animals at 750/600 mg/kg/bw/day (recovery group)*
Female: 5 animals at 0 mg/kg bw/day
Female: 5 animals at 0 mg/kg/bw/day (recovery group control)
Female: 5 animals at 25 mg/kg bw/day
Female: 5 animals at 150 mg/kg bw/day
Female: 5 animals at 500/400 mg/kg bw/day*
Female: 5 animals at 750/600 mg/kg bw/day*
Female: 5 animals at 750/600 mg/kg/bw/day (recovery group)*


*Due to a deterioration in physical health of the animals at the highest dose level, it was considered necessary to stop treatment at this dose level on Day 9 and recommence treatment on Day 10 at a reduced dose level of 600 mg/kg/day. To sustain integrity of the study, the 500 mg/kg/day dose level was also reduced on Day 10 to 400 mg/kg/day (see Any other Information on materials and Methods section for further information).

The above concentations refer to dose of active.
 

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were chosen on the basis of the results from a 14-day rangefinder study.
- Rationale for animal assignment (if not random): random
- Post-exposure recovery period in satellite groups: 14 days

Positive control:

None

Examinations

Observations and examinations performed and frequency:

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 dosing and one hour after dosing at weekends. During the treatment-free period, animals were observed twice daily, morning and afternoon (once daily at weekends). All observations were recorded.

Prior to the start of treatment and on Days 6, 13, 20 and 26, all surviving animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on all surviving non-recovery animals during Week 4, together with an assessment of sensory reactivity to different stimuli. Observations were carried out from at least two hours after dosing on each occasion.

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

Sacrifice and pathology:

On completion of the dosing period, or in the case of recovery group animals, at the end of the treatment-free period, all surviving 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 were removed from animals that were killed either at the end of the dosing period or at the end of the treatment-free period. They were dissected free from fat and weighed before fixation:

Adrenals
Brain
Epididymides
Heart
Kidneys
Liver
Ovaries
Prostate
Spleen
Seminal vesicles
Testes
Thymus
Uterus

Histopathology

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

Aorta (thoracic)
Bone & bone marrow (femur including stifle joint)
Eyes Skin (hind limb)
Muscle (skeletal)
Oesophagus
Pancreas
Pituitary
Salivary glands (submaxillary)


Samples of the tissues below were removed from all animals and preserved in buffered 10% formalin. Tissues from all non-recovery control and high dose group animals, together with those animals dying during the study, were prepared as paraffin blocks, sectioned at nominal thickness of 5 μm and stained with haematoxylin and eosin for subsequent microscopic examination.

Adrenals
Bone & bone marrow (sternum)
Brain (including cerebrum, cerebellum and pons)
Caecum
Colon
Duodenum
Epididymides
Heart
Gross lesions
Ileum
Jejunum
Kidneys
Liver
Lymph nodes (cervical and mesenteric)
Ovaries
Spinal cord (cervical)
Spleen
Prostate
Stomach
Sciatic nerve
Seminal vesicles
Rectum
Testes
Thymus
Thyroid/parathyroid
Lungs (with bronchi)
Trachea
Urinary bladder
Uterus

Any macroscopically observed lesions were also processed together with the liver and spleen from all 400, 150 and 25 mg/kg/day dose group animals.

Since there were indications of treatment-related changes in the liver, kidney, urinary bladder, adrenal gland, colon, stomach, thymus, mesenteric lymph node, bone marrow and seminal vesicles, examination was subsequently extended to include sections of these tissues from all animals in the remaining groups.

Other examinations:

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 percentage of time 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:

Blink reflex
Finger approach
Grasp response
Pupil reflex
Startle reflex
Toe pinch
Tail pinch
Touch escape
Vocalisation

Statistics:

If appropriate data were processed to give group mean values and standard deviations. Where appropriate, quantitative data was 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).

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

See details in results section

Mortality:

mortality observed, treatment-related

Description (incidence):

See details in results section

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

See details in results section

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

See details in results section

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

See details in results section

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

effects observed, treatment-related

Description (incidence and severity):

See details in results section

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

See details in results section

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

See details in results section

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

See details in results section

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See details in results section

Histopathological findings: neoplastic:

no effects observed

Details on results:

Mortality

One intermediate (II) dose male was found dead on Day 4 and 4 males and 1 female treated with the highest dose level were killed in extremis on Day 12. Another male from the high dose group was terminated on Day 14. Two further males treated at the highest dose level were killed in extremis on Day 26.


Clinical Observations

Incidents of increased salivation were detected up to one hour after dosing in high dose level animals of either sex from Day 8. Hunched posture was also evident for either sex animals treated with the highest dose level from Day 8, together with isolated findings of tip-toe gait, dehydration and tail elevation.


The physical health of the high dose animals continued to deteriorate following the reduction of the highest dose level, resulting in the termination of three high dose females and two high dose males on Day 12. Another male from the high dose group showed clinical signs, including dehydration, hunched posture and pilo-erection, contributing to its early sacrifice on Day 14. Clinical signs were still evident in the remaining high dose animals and two further males were terminated on Day 26. Following cessation of treatment, a complete regression in these signs was apparent.


Isolated incidents of hunched posture, noisy and decreased respiration were detected for animals of either sex treated with the intermediate (II) dose. No such effects were detected for animals of either sex treated with 150 or 25 mg/kg/day.


Behavioural Assessment

Weekly open field arena observations confirmed the signs detected during the daily clinical observations, together with a higher incidence of urination / defecation at the high and intermediate (II) dose levels during the first two weeks of the treatment period.


Bodyweight

Reduced bodyweight gains with actual bodyweight losses were evident for high dose animals during Week 1, contributing to the reductions in dose levels at the two highest doses. Following the reduction, males continued to show reduced bodyweights gains during Week 2 and Week 3 and actual bodyweight losses were still evident. A complete regression of symptoms was evident for recovery high dose animals during the fourteen day treatment period.


Food Consumption

Slight reductions in dietary intake were evident for high dose animals during the first two weeks of treatment, with recovery detected thereafter. Food efficiencies were also reduced.


Water Consumption

Substantial increases in water consumption were detected for animals of either sex treated with the high and intermediate (II) dose levels throughout the treatment period, with the effects still evident for surviving recovery high dose animals during the first week without treatment.


Urinalysis

Increased urine volume of reduced specific gravity was detected for animals of either sex treated with the high and intermediate (II) dose levels, prior to the end of the treatment period.

Blood Chemistry

Increases in cholesterol were evident for animals of either sex treated with 600, 400 and 150 mg/kg/day. Females treated with 600 and 400 mg/kg/day showed elevated bilirubin, and creatinine levels were elevated for 600 mg/kg/day males. Males treated with 600 and 400 mg/kg/day also showed reductions in plasma chloride.


Organ Weights

Males treated with the highest dose level showed an increase in relative liver weights. Elevated liver weights, both absolute and relative to terminal bodyweights were also apparent for males treated with 400 and 150 mg/kg/day. Animals of either sex treated with the highest dose also showed an increase in relative kidney weights in comparison to controls.


Necropsy

Treatment-related abnormalities were confined to raised limiting ridge of the gastric epithelia at 600, 400 and 150 mg/kg/day. Dark and black discolouration of various organs and black contents present in the gastro-intestinal tract were also prevalent at these dose levels, for both interim death andterminal kill animals.



Histopathology


LIVER: Centrilobular hepatocyte enlargement was observed in relation to treatment for animals of either sex treated with 600 mg/kg/day. Generalised hepatocyte enlargement was seen for four animals that were killed in extremis. Centrilobular hepatocyte enlargement was also observed for animals of either sex treated with 400 mg/kg/day.


KIDNEY: Hypertrophy and basophilia of the collecting duct epithelium occasionally with associated dilatation of tubules and higher grades of severity of isolated groups of basophilic tubules were seen as a consequence of treatment for animals of either sex treated with 600 mg/kg/day and for all females and for one male treated with 400 mg/kg/day. Partial regression of treatment-related renal pathology was observed following completion of the fourteen day recovery period.



URINARY BLADDER: Hyperplasia of the transitional epithelium was observed for two interim death males and for two interim death females treated with 600 mg/kg/day. Epithelial hyperplasia was observed for one recovery group female control and for one recovery 600 mg/kg/day female following completion of the recovery period.


ADRENAL GLANDS: Vacuolation of the cortical zona glomerulosa was observed in relation to treatment for four male and one female premature death animal treated with 600 mg/kg/day. Animals from remaining treatment levels were not similarly affected. One male from the recovery 600 mg/kg/day dose group was also observed with vacuolation following completion of the fourteen day recovery period.



COLON: Mucosal hypertrophy and basophilia were observed for three premature death males and for one premature death female treated with the high dose level.

STOMACH: Acanthosis/hyperkeratosis of the limiting ridge, agglomeration of secretion in mucosal cells, and superficial mucosal atrophy/basophilia were seen as a consequence of treatment for animals of either sex treated with 600 and 400 mg/kg/day, and for females treated with 150 mg/kg/day.Three males were variously affected at the 150 mg/kg/day dose level.

Regression of gastric changes was observed among surviving recovery high dose animals.


THYMUS: Lymphoid atrophy was observed in relation to treatment for five males and for two females treated with 600 mg/kg/day, all of which were interim deaths. Animals from the remaining treatment levels were not similarly affected, but the condition was observed for one surviving recovery high dose female.


MESENTERIC LYMPH NODE: Sinus histiocytosis was observed for two males and for three females treated with the high dose, four of these animals died prematurely. The condition was not observed among animals from the remaining treatment or recovery group animals.


BONE MARROW: Generally higher grades of severity of adipose infiltration of the marrow, indicative of marrow hypoplasia, were seen among animals of either sex treated with 600 mg/kg/day but not at any other treatment level.



SEMINAL VESICLES: Reduced secretory content was observed in the seminal vesicles of four males treated with the high dose, three of which died prematurely, and in two males treated with the intermediate (II) dose, one of which died prematurely. A similar effect was not observed at the remaining dose levels.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (nominal)

Basis for effect level:

other: original NCD unit is mg/kg/day.

2

Dose descriptor:

NOEL

Effect level:

25 mg/kg bw/day (nominal)

Basis for effect level:

other: original NCD unit is mg/kg/day.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Oral administration of the test material to rats for a period of up to twenty-eight consecutive days at dose levels of up to 600 mg/kg/day (reduced from 750 mg/kg/day from Day 10) resulted in toxicologically significant effects at 600 and 400 mg/kg/day. The effects detected at 150 mg/kg/day were considered not to represent an adverse health effect, therefore, the “No Observed Adverse Effect Level” (NOAEL) was considered to be 150 mg/kg/day.

No toxicologically significant effects of treatment were detected at 25 mg/kg/day and the ‘No Observed Effect Level’ (NOEL) was, therefore, considered to be 25 mg/kg/day.

Executive summary:

INTRODUCTION

 

The study was designed to investigate the systemic toxicity of the test material and complied with the OECD Guidelines for Testing of Chemicals No. 407 "Repeated Dose 28 Day Oral Toxicity Study in Rodents" (adopted 27 July 1995). The study also covered the requirements of 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.

 

METHOD

 

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 up to twenty-eight days, at dose levels of 25, 150, 500 and 750 mg/kg/day (incorporating a correction factor for 85.6% purity). 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 (750 mg/kg/day) or the vehicle alone for up to twenty-eight consecutive days and then maintained without treatment for a further fourteen days. Due to a deterioration in physical health of the animals at the highest dose level, it was considered necessary to stop treatment at this dose level on Day 9 and recommence treatment on Day 10 at a reduced dose level of 600 mg/kg/day. To sustain integrity of the study, the 500 mg/kg/day dose level was also reduced on Day 10 to 400 mg/kg/day.

 

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

One intermediate dose male was found dead on Day 4 and four males and one female treated with the highest dose level were killedin extremison Day 12. Another male from the high dose group was terminated on Day 14. Two further males treated at the highest dose level were killedin extremison Day 26.

 

There were no further unscheduled deaths attributed to test material toxicity.

 

Clinical Observations

 

Incidents of increased salivation were detected up to one hour after dosing in high dose level animals of either sex from Day 8. Hunched posture was also evident for either sex animals treated with the highest dose level from Day 8, together with isolated findings of tiptoe gait, dehydration and tail elevation. The physical health of the high dose animals continued to deteriorate following the reduction of the highest dose level, resulting in the termination of three high dose females and two high dose males on Day 12. Another male from the high dose group showed clinical signs, including dehydration, hunched posture and pilo-erection, contributing to its early sacrifice on Day 14. Clinical signs were still evident in the remaining high dose animals and two further males were terminated on Day 26. Following cessation of treatment, a complete regression in these signs was apparent.

 

Isolated incidents of hunched posture, noisy and decreased respiration were detected for animals of either sex treated with the intermediate (II) dose. No such effects were detected for animals of either sex treated with 150 or 25 mg/kg/day.

 

Behavioural Assessment

Weekly open field arena observations confirmed the signs detected during the daily clinical observations, together with a higher incidence of urination / defecation at the high and intermediate dose levels during the first two weeks of the treatment period. No such effects were detected for animals of either sex treated with 150 or 25 mg/kg/day.

 

Functional Performance Tests

No treatment-related effects were detected.

 

Sensory Reactivity Assessments

No treatment-related effects were detected.

 

Bodyweight

Reduced bodyweight gains with actual bodyweight losses were evident for high dose animals during Week 1, contributing to the reductions in dose levels at the two highest doses. Following the reduction, males continued to show reduced bodyweights gains during Week 2 and Week 3 and actual bodyweight losses were still evident. A complete regression of symptoms was evident for recovery high dose animals during the fourteen day treatment period. No adverse effect on bodyweight gains was evident for animals of either sex treated 400, 150 or 25 mg/kg/day.

 

Food Consumption

Slight reductions in dietary intake were evident for high dose animals during the first two weeks of treatment, with recovery detected thereafter. Food efficiencies were also reduced. No such effects were detected for animals of either sex from the remaining dose groups.

 

Water Consumption

 Substantial increases in water consumption were detected for animals of either sex treated with the high and intermediate dose levels throughout the treatment period, with the effects still evident for surviving recovery high dose animals during the first week without treatment. No overt differences in water intake were detected for animals of either sex treated with 150 or 25 mg/kg/day, in comparison to controls.

Urinalysis

Increased urine volume of reduced specific gravity was detected for animals of either sex treated with the high and intermediate (II) dose levels, prior to the end of the treatment period. No such effects were detected for animals from the remaining treatment groups.

 

Haematology

No treatment-related effects were detected.

Blood Chemistry

Increases in cholesterol were evident for animals of either sex treated with 600, 400 and 150 mg/kg/day. Females treated with 600 and 400 mg/kg/day showed elevated bilirubin, and creatinine levels were elevated for 600 mg/kg/day males. Males treated with 600 and 400 mg/kg/day also showed reductions in plasma chloride. No such effects were detected for animals of either sex treated with 25 mg/kg/day.

 

Organ Weights

Males treated with the highest dose level showed an increase in relative liver weights. Elevated liver weights, both absolute and relative to terminal bodyweights were also apparent for males treated with 400 and 150 mg/kg/day. Animals of either sex treated with the highest dose also showed an increase in relative kidney weights in comparison to controls. No such effects were detected at 25 mg/kg/day or in the recovery high dose animals after the fourteen day treatment free period.

 

Necropsy

Treatment-related abnormalities were confined to raised limiting ridge of the gastric epithelia at 600, 400 and 150 mg/kg/day. Dark and black discolouration of various organs and black contents present in the gastro-intestinal tract were also prevalent at these dose levels, for both interim death and terminal kill animals. No such effects were detected at 25 mg/kg/day.

 

Histopathology

 

The following treatment-related changes were observed:

 

LIVER: Centrilobular hepatocyte enlargement was observed in relation to treatment for animals of either sex treated with 600 mg/kg/day. Generalised hepatocyte enlargement was seen for four animals that were killedin extremis. Centrilobular hepatocyte enlargement was also observed for animals of either sex treated with 400 mg/kg/day.

 

KIDNEY: Hypertrophy and basophilia of the collecting duct epithelium occasionally with associated dilatation of tubules and higher grades of severity of isolated groups of basophilic tubules were seen as a consequence of treatment for animals of either sex treated with 600 mg/kg/day and for all females and for one male treated with 400 mg/kg/day. Partial regression of treatment-related renal pathology was observed following completion of the fourteen day recovery period.

 

URINARY BLADDER: Hyperplasia of the transitional epithelium was observed for two interim death males and for two interim death females treated with 600 mg/kg/day. Epithelial hyperplasia was observed for one recovery group female control and for one recovery 600 mg/kg/day female following completion of the recovery period.

ADRENAL GLANDS: Vacuolation of the cortical zona glomerulosa was observed in relation to treatment for four male and one female premature death animal treated with 600 mg/kg/day. Animals from remaining treatment levels were not similarly affected. One male from the recovery 600 mg/kg/day dose group was also observed with vacuolation following completion of the fourteen day recovery period.

COLON: Mucosal hypertrophy and basophilia were observed for three premature death males and for one premature death female treated with the high dose level.

STOMACH: Acanthosis/hyperkeratosis of the limiting ridge, agglomeration of secretion in mucosal cells, and superficial mucosal atrophy/basophilia were seen as a consequence of treatment for animals of either sex treated with 600 and 400 mg/kg/day, and for females treated with 150 mg/kg/day. Three males were variously affected at the 150 mg/kg/day dose level. Regression of gastric changes was observed among surviving recovery high dose animals.

 

THYMUS: Lymphoid atrophy was observed in relation to treatment for five males and for two females treated with 600 mg/kg/day, all of which were interim deaths. Animals from the remaining treatment levels were not similarly affected, but the condition was observed for one surviving recovery high dose female.

 

MESENTERIC LYMPH NODE: Sinus histiocytosis was observed for two males and for three females treated with the high dose, four of these animals died prematurely. The condition was not observed among animals from the remaining treatment or recovery group animals.

 

BONE MARROW: Generally higher grades of severity of adipose infiltration of the marrow, indicative of marrow hypoplasia, were seen among animals of either sex treated with 600 mg/kg/day but not at any other treatment level.

 

SEMINAL VESICLES: Reduced secretory content was observed in the seminal vesicles of four males treated with the high dose, three of which died prematurely, and in two males treated with the intermediate (II) dose, one of which died prematurely. A similar effect was not observed at the remaining dose levels. One recovery control male showed a similar effect which was not observed in either of the surviving recovery high dose males.

 

 

Conclusion

 

Oral administration of the test material to rats for a period of up to twenty-eight consecutive days at dose levels of up to 600 mg/kg/day (reduced from750 mg/kg/day from Day 10) resulted in toxicologically significant effects at 600 and 400 mg/kg/day. The effects detected at 150 mg/kg/day were considered not to represent an adverse health effect, therefore, the “No Observed Adverse Effect Level” (NOAEL) was considered to be 150 mg/kg/day.

 

No toxicologically significant effects of treatment were detected at 25 mg/kg/day and the ‘No Observed Effect Level’ (NOEL) was, therefore, considered to be 25 mg/kg/day.