Black HM 2482

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:

From October 27th to December 22nd, 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2004

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)

Version / remarks:

July 27, 1995

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Directive 96/54/EC, September 30, 1996

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Japanese Guidelines for Screening, Toxicity Testing of Chemicals : Testing Methods for new Substances, enacted July 13, 1974, amended December 5, 1986

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

400-920-6

EC Name:

-

Cas Number:

89857-06-7

Molecular formula:

C50 H53 N11 O14 S

IUPAC Name:

5'-[2-(7-{2-[4-(2-{1'-[3-(dimethylazaniumyl)propyl]-6'-hydroxy-4'-methyl-2'-oxo-1',2'-dihydro-1λ⁵-[1,3'-bipyridin]-1-ylium-5'-yl}diazen-1-yl)phenyl]diazen-1-yl}-8-hydroxy-6-sulfonatonaphthalen-2-yl)diazen-1-yl]-6'-hydroxy-3,4'-dimethyl-2'-oxo-1',2'-dihydro-1λ⁵-[1,3'-bipyridin]-1-ylium bis(2-hydroxypropanoate)

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Recognized by the international guidelines as the recommended test system.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Test system: Rat, HanBrl:WIST (SPF)
- Source: RCC Ltd, Laboratory Animal Sciences, CH-4414 Füllinsdorf / Switzerland
- Age at study initiation: 6 weeks
- Weight at study initiation: males at 139-161 g (mean 151 g); females at 115-133 g (mean 124 g)
- Housing: In groups of five in Makrolon type-4 cages with wire mesh tops and standardized softwood bedding ('Lignocel' Schill AG, CH-41 32 Muttenz/Switzerland).
- Diet: Pelleted standard Provimi Kliba 3433 (batch no. 54103) rat maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugs/Switzerland), ad libitum
- Water: Community tap-water from ltingen, Switzerland, ad libitum. A bacteriological, chimical and contaminant analyses were performed.
- Acclimation period: 7 days. Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.
- Identification: Cage card and individual tattoo
- Randomization: computer-generated random algorithm

DETAILS OF FOOD AND WATER QUALITY:
- Food quality: The feed batch was analyzed for contaminants.
- Water quality: A bacteriological, chimical and contaminant analyses were performed.
None of the contaminants analyzed in the water and diet is considered to have been present at a concentration which would have affected the validity of the results.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3°C
- Humidity: 30-70 %
- Air changes: 10-15 per hour
- Photoperiod: 12 hours fluorescent light / 12 hours dark, music during the light period

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

Accidental oral ingestion is a possible route of human exposure.

Vehicle:

water

Remarks:

bidistilled water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item formulations were prepared weekly.
Test item was weighed into a glass beaker on a tared Mettler balance and the vehicle added. The mixtures were prepared using a magnetic stirrer and stored at room temperature (17-23 °C).
Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.
Dose volume: 10 ml/kg/ b.w

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration, homogeneity and stability (after 2 hours and 7 days) of the dose formulations were determined in samples taken after experimental start. Concentration and homogeneity of the dose formulations were determined in samples taken during week 3 of the treatment.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

200 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Total number of animals per group:
Groups 1 and 4: 10 males; 10 females
Groups 2 and 3: 5 males; 5 females

Total number of animals: 30 males and 30 females

Control animals:

yes, concurrent vehicle

Details on study design:

Rationale for dose level selection: based upon the results of a non-GLP 5-day dose-range-finding study in which test item was administered by gavage to 2 rats per group and sex.

Examinations

Observations and examinations performed and frequency:

MORTALITY/VIABILITY
Observations for mortality/viability were recorded twice daily.

CAGE SIDE OBSERVATIONS: daily
The animals were observed for clinical signs once before commencement of administration; twice daily on days 1-3; as well as once daily on days 4-28 and once daily during days 29-42 (recovery).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly
The animals were observed in their home cages, outside their home cages in a standard arena and in the hand. These observations were performed in random sequence once before commencement of administration and once weekly (weeks 1-3) thereafter.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly
Body weights were recorded weekly during pretest, treatment and recovery and before necropsy, using an on-line electronic recording system consisting of a Mettler balance connected to the computer.

FOOD CONSUMPTION: Yes
The food consumption was recorded once during the pretest period and weekly thereafter, using an on-line electronic recording system consisting of a Mettler balance connected to the computer.

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
atter 4 weeks - 08 December 2003 (Allocation A and B)
after 6 weeks - 22 December 2003 (Allocation B)
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes, 18 hours before blood sampling but allowed access to water ad libitum.
- How many animals: not specified
- Parameters checked in table [No. 1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
atter 4 weeks - 08 December 2003 (Allocation A and B)
after 6 weeks - 22 December 2003 (Allocation B)
- Animals fasted: Yes, 18 hours before blood sampling but allowed access to water ad libitum
- How many animals: not specified
- Parameters checked in table [No.2] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: during the 18-hour fasting period into a specimen vial
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [No.3] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes / No / Not specified
- Time schedule for examinations: During week 4
- Dose groups that were examined: all
- Battery of functions tested: functional observational battery / grip strength / motor activity

Sacrifice and pathology:

NECROPSY: Yes (see table 4)
Sacrifice:
after 4 weeks (allocation A)
after 6 weeks (allocation B)
All animals were weighed and necropsied. Descriptions of all macroscopic abnormalities were recorded. All animals surviving to scheduled necropsy were anesthetized by intraperitoneal injection of sodium pentobarbitone and killed by exsanguination.

HISTOPATHOLOGY: Yes (see table 4, slides of all organs and tissues listed in boldface type)
Because treatment-related morphologic changes were detected in the organs of high-dose animals, the same organs (kidney, liver) from animals of the mid- and low-dose groups were examined.

HISTOTECHNIQUE
All organ and tissue samples, as defined under Histopathology, were processed, embedded and cut at an approximate thickness of 2 to 4 micrometers, and stained with hematoxylin and eosin.

ABSOLUTE AND RELATIVE ORGAN WEIGHTS
- The following organ weights were recorded on the scheduled dates of necropsy: Adrenals, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Spleen, Testes, Thymus.
The organ to terminal body weight ratios as well as organ to brain weight ratios were determined. The determination of the terminal body weight was performed immediately prior to necropsy.

Statistics:

The following statistical methods were used to analyze the grip strength, locomotor activity, body weight, organ weights and ratios, as well as:
-The Dunnett-test (many to one ttest) based on a pooled variance estimate were applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
-The Steeltest (many-one rank test) were applied instead of the Dunnett-test when the data can not be assumed to follow a normal distribution.
-Fisher's exact-test were applied to the macroscopic findings.
The following statistical methods were used for statistical analysis of clinical laboratory data:
-Quantitative data were analyzed by a one-way analysis of variance (ANOVA) when the variances are considered homogeneous according to Bartlett. Alternatively, if the variances are considered to be heterogenous (p<0.05), a non-parametric Kruskal-Wallis test was used. Treated groups were compared to the control groups using Dunnett's test if the ANOVA was significant at the 5% level and by Dunn's test in the case of a significant Kruskal-Wallis test (p<0.05).
-Ordinal data such as urine sediment were analyzed using the Kruskal-Wallis test. lf this test was significant (p<0.05), comparisons were made between the control group and each of the treatment groups using Dunn's test.
References :
C.W. Dunnett: A Multiple Comparison Procedure for Comparing Several Treatments with a Control, J. Amer. Stat. Assoc. 50, 1096-1 121 (1955).
S.C. Gad and C.S. Weil: Statistics and Experimental Design for Toxicologists. The Telford Press, Caldwell, New Jersey, 43-45 (1986).
W.H. Kruskal and W.A. Wallis: Use of ranks in one-criterion variance analysis, Journal of the American Statistical Association , 47 , 583-621 (1952).
O.J. Dunn: Multiple comparisons using rank sums, Technometrics 6, 241-252 (1964).
R.G. Miller: Simultaneous Statistical Inference, Springer Verlag, New York (1981).

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The following clinical signs were noted for different time periods after onset of test item administration: soft feces in both males and females treated with 200 and 1000 mg/kg/day and fecal discoloration (dark feces) in animals of both sexes treated with 50, 200 and 1000 mg/kg/day. These findings were considered to represent passive test item- and administration-related effects but were judged to be of no adverse character. Breathing noises were audible for some days in one female treated with 1000 mg/kg/day. This transient finding was considered to be of no toxicological relevance.

No test item-related changes were noted at any dose level in the mean fore- and hind-limb grip strength values when compared with the controls. The mean hindlimb grip strength of the females treated with 200 mg/kg/day was signficantly greater (p<0.05) than that of the control females, but in the absence of a dose-response relationship, this finding was considered to be incidental.

The mean locomotor activity of the males treated with 1000 mg/kg/day was significantly reduced (p<0.01)during the second measurement interval (15-30 minutes) and significantly reduced (p<0.05) during the overall measurement interval (0-60 minutes), when compared with the control males. Females treated with 1000 mg/kg/day had significantly reduced mean locomotor activity (p<0.05) during the first measurement interval (0-15 minutes). These slight reductions were considered to be minor test item-related but non-adverse changes. The locomotor activity of rats treated with 50 mg/kg/day and 200 mg/kgiday were considered to be unaffected when compared with the controls.

Mortality:

no mortality observed

Description (incidence):

All animals survived until their respective scheduled necropsy.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean body weights of the males treated with 200 mg/kg/day and 1000 mg/kg/day were slightly lower than those of the control males from day 8 of treatment onwards although
dose-unrelated. These minor differences were considered incidental. The mean body weights of females of all groups compared favorably.
The mean body weight gain values were significantly reduced in males treated with 200 mg/kg/day (p<0.05) on day 15 and in males treated with 1000 mg/kg/day on days 8 (p<0.01)
und 15 (p<0.05). All body weight gain values were less than those of the control males during the treatment period; this finding persisted during the recovery period (in males previously treated with 1000 mg/kg/day) although a slight improvement was noted. ln femates treated with 1000 mg/kg/day, the mean body weight gain was significantly reduced on day 8 (p<0.05) when compared with the controls. Thereafter the mean body weight gain improved to levels comparable to those of the controls. During recovery, the mean body weight gain of females previously treated with 1000 mg/kg/day was generally similar to that of the controls.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The mean daily food consumption values of the test item-treated rats compared favorably with or exceeded those of the controls during the treatment and recovery phases.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

After 4 Weeks
Significantly increased mean absolute reticulocyte counts were noted in males treated with 1000 mg/kg/day (p<0.05) when compared to the controls. In the absence of commensurate findings (i.e. anemia) in selected parameters in males (e.g. red blood cells, haemoglobin, haematocrit), this change was considered to be incidental and not test item-related.
Significantly decreased mean erythrocyte counts were noted in females treated with 200 mg/kg/day (p<0.05) and a significantly decreased mean relative haematocrit value (p<0.05) was noted in females treated with 200 mg/kg/day and 1000 mg/kg/day when compared to the controls. As the reference values of the control group were at the upper limit of the historical control data and the values of the treated groups were comparable to the historical control data, these findings were considered to be incidental and not test item-related.
Males and females treated with 1000 mg/kg/day had significantly prolonged mean relative prothrombin times (p<0.05) and the same males had significantly abbreviated mean activated partial thromboplastin times (p<0.05) when compared to the controls. The transient effect of increased mean relative prothrombin times was considered to be of no toxicological relevance. As very high mean activated partial thromboplastin times were noted in the male control group (compared to the historical control data), the decrease in mean activated partial thromboplastin times was considered to be incidental and not test item-related.
After 6 Weeks
Significantly increased mean corpuscular volume was noted in females treated with 1000 mg/kg/day (p<0.05) when compared to the controls. In the absence of an effect in males, this finding was considered to be incidental and not test item-related.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

After 4 Weeks
Males treated 1000 mg/kg/day had significantly increased mean urea levels (p<0.01) when compared to the controls. As the value lay well within the historical control values and in the absence of a similar effect in females, this finding was considered to be incidental and not test item-related.
Significantly decreased mean alkaline phosphatase levels were noted in males treated with 1000 mg/kg/day (p<0.05) and significantly decreased mean potassium levels were noted in males treated with 200 mg/kg/day (p<0.01 ) when compared to the controls. Males treated with 50 mg/kg/day had significantly increased mean chloride levels (p<0.01). In the absence of changei ai other dose levels or in females, these findings were considered to be incidental and not test item-related.
Significantly decreased mean glutamate dehydrogenase levels were noted in females treated with 200 mg/kg/day (p<0.01) and significantly decreased mean calcium levels were noted in females treated with t00O mg/kg/day (p<0.05) when compared to the controls. ln the absence of changes at other dose levels or in males, these findings were considered to be incidental and not test item-related.
After 6 Weeks
Males treated with 1000 mg/kg/day had significantly increased mean urea levels (p<0'05) when compared to the controls. As the value lay well within the historical control values and in the absence of a similar effect in females, this finding was considered to be incidental and not test item-related.
Significantly decreased mean alanine aminotransferase levels (p<0.05) and creatine kinase levels (p<0.01) were noted in females treated with 1000 mg/kg/day when compared to the control. Females treated with 1000 mg/kg/day had significantly decreased mean chloride levels (p<0.05) when compared to the controls. In the absence of similar observations in males, these findings were considered to be incidental and not test item-related.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

After 4 weeks
Significantly decreased mean urine volumes were noted in males and females treated with 200 mg/kg/-day ((p<0.05 or p<0.01) and in males treated with 1000 mg/kgiday (p<0.01). This finding was considered to be the consequence of an osmonephrotic change due to elevated renal-protein levels resulting in increased tubular urine resorption. This reversible, test item related effect was considered to be of no adverse nature.
Significantly increased mean erythrocyte counts were noted in the urine of males treated with 200 and 100 mg/kg/day (p<0.05) and in the urine of females treated with 1000 mg/kg/day (p<0.01) when compared to the controls. Males treated with 200 mg/kg/day (p<0.05) and females treated with 1000 mg/kg/day (p<0.01) had significantly increased mean leucocyte counts when compared to the controls. Based on available data, increased leucocyte counts must be suspected in males treated with 1000 mg/kg/day as well but due to the test item related discoloration of the urine, eight out of ten samples could not be tested for leucocytes. These findings were considered to be a consequence of renal cell damage and were considered to be test item-related.
Urine color was brown and red-brown in all males treated with 1000 mg/kg/day and in eight of ten females treated with 1000 mg/kg/day. The other females treated with 1000 mg/kg/day had yellow-brown and red urine. The urines of all control males and females had normal color. This change in urine color was considered to be a passive, joint effect of the test item (dye) and the haematuria.
Significantly increased mean relative urine density was noted in males treated with 1000 mg/kg/day (p<0.05) when compared to the controls. Females treated with 200 mg/kg/day had significantly increased mean urine protein levels (p<0.01) and females treated with 50 mg/kg/day had significantly increased mean bilirubin levels (p<0.01) when compared to the controls. As no changes were noted at other dose levels, these findings were considered to be incidental and not test item-related.

After 6 Weeks
Significantly increased mean erythrocyte counts were noted in females treated with 1000 mg/kg/day (p<0.05) when compared to the controls. This finding was considered to be an ongoing consequence of the renal cell damage.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

After 4 Weeks
Significantly increased kidneyto-body weight ratios were noted in males treated with 200 mg/kg/day (p<0.01) and 1OOO mg/kg/day (p<0.01) and in females treated with 1000 mg/kg/day (p<0.05) when compared to the controls. These changes were considered to be test item-related.
The mean absolute thymus weight and mean thymus-to-brain weight ratio noted in males treated with 200 mg/kg/day were significantly reduced (both p<0.05) when compared with the control males, but the thymusto-body weight ratios were generally comparable. The liver-to-body weight ratio of females treated with 50 mg/kg/day was significantly reduced (p<0.05) when compared with controls. As no clear dose-response relationship was evident, these differences were considered to be unrelated to the treatment with the test item.
Males treated with 200 mg/kg/day and 1000 mg/kg/day had significantly increased braintobody weight ratios (p<0.01) when compared to the controls. These changes were considered to be body weight effects and not a direct result of the test item.
After 6 Weeks
No test item-related differences were noted in the mean absolute or relative organ weights alter 2 weeks' recovery. The significantly elevated liver-to-body weight ratio noted in males treated previously with 1000 mg/kg/day (p<0.05) was, in the absence of microscopical changes and similar findings after the end of the treatment period, considered to be incidental.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At the end of the treatment and following recovery periods, no test item-related gross lesions were observed. The macroscopic findings recorded were considered to be within the range of normal background lesions which may be seen in rats of this strain and age in oral toxicity studies and were considered incidental, reflecting the usual individual variability.
Evidence of treatment-induced findings consisted of an increased incidence and severity of hyaline droplets in the kidneys of males at 50, 200 and, 1000 mg/kg/day when compared with controls. This finding did not exceed control group incidences/severities in the recovery
groups. Tubular cell swelling, including vacuolated cytoplasm of renal tubular cells could be found in a single male at 1000 mg/kglday and one female at 200 mg/kg/day and 5 females at 1000 mg/kg/day. This finding could still be observed in two recovery males treated previously with 1000 mg/kg/day.
Evidence for tubular cell damage could be found in both sexes. Tubular cell necrosis was recorded in two males treated with 1000 mg/kg/day. This finding resolved in the recovery group. Increased incidence (5/5) of tubular basophilia could be observed In recovery females.
Minimal to slight degree of mainly centrilobular hepatocellular hypertrophy was recorded in single males treated with 200 mg/kg/day and single females treated with 1000 mg/kg/day.
Bile duct proliferation exceeding control group incidence (0/0) was observed in recovery males (0/0) previously treated with 1000 mg/kg/day.

Effect levels

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

haematology

organ weights and organ / body weight ratios

urinalysis

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

NOAEL = 2000 mg/kg bw/day

Executive summary:

The repeated oral toxicity of test item was assessed following OECD Guideline 407, Repeated Dose 28 -Day Oral Toxicity in Rodents. In this subacute toxicity study, test item was administered daily by oral gavage to SpF-bred Wistar rats of both sexes at dose levels of 50, 200 and 1000 mg/kg body weight/day for a period of 28 days. A control group was treated similarly with the vehicle, bidistilled water, only.
The groups comprised 5 animals per sex, which were sacrificed after 28 days of treatment. Additional 5 rats per sex and group were used at 0 and 1000 mg/kg. These animals were treated for 28 days and then allowed a 14-day treatment-free recovery period after which they were sacrificed.
Clinical signs, outside cage observation, food consumption and body weights were recorded periodicaliy during pretest, the treatment and recovery periods. Functional observational battery, locomotor activity and grip strength were performed during week 4.
At the end of the dosing and the treatment-free recovery period, blood samples were withdrawn for hematology and plasma chemistry analyses. Urine samples were collected for urinalyses. All animals-were killed, necropsied and examined post mortem. Histological examinations were performed on organs and tissues from all control and high dose animals, and all gross lesions from all animals. From the animals of the low and middle dose groups, the kidneys and livers were examined to establish a no-effect level.
Results
MORTALITY / VIABILITY
All animals survived untiltheir respective scheduled necropsy.
CLINICAL SIGNS
No test item-related effects were noted during the weekly behavioral observations (weeks 1-3).
FUNCTIONAL OBSERVATIONAL BATTERY
No test item-related effects were noted during the weekly behavioral observations (weeks 1-3) and during the functional observational battery (week 4).
Grip Strength
No test item-related changes were noted at any dose level in the mean fore- and hind-limb grip strength values when compared with the controls.
Locomotor Activity
The mean locomotor activity was slightly reduced in both sexes treated with 1000 mg/kg/day. These slight reductions were considered to be minor, but non-adverse test item-related changes.

FOOD CONSUMPTION
The mean daily food consumption values of the test item-treated rats compared favorably with or exceeded those of the controls during the treatment and recovery phases.

BODY WEIGHT
Slightly lower body weight gains were noted for the males treated with 200 mg/kg/day and for both sexes treated with 1000 mg/kg/day. During recovery, lower body weight gains were noted in males treated previously with 1000 mg/kg/day but not in females. These effects resulted in generally but not significantly lower absolute body weights in males treated with 200 and 1000 mg/kglday (from day 8 of treatment onwards). The effects on body weight and body weight gain were considered to represent test item-related effects.

CLINICAL LABORATORY INVESTIGATIONS
Hematology
No test item-related differences were noted at any dose level.
Clinical Biochemistry
No test item-related differences were noted at any dose levels.
Urinalysis
The noted significant effects on erythrocyte and leucocyte counts in urine, urine volumes and urine color in males and females treated with 200 and 1000 mg/kg/day were considered to be test item-related.
The marked increase in erythrocyte counts in males treated with 200 and 1000 mg/kg/day and females treated with 1000 mg/kg/day and the increase in leucocyte counts in males treated with 2OO mg/kg/day and females (and supposedly males) treated with 1000 mg/kg/day were considered to be a consequence of renal cell damage. At the end of the reoovvery period, mean erythrocyte counts in females treated with 1000 mg/kg/day were still
significantly increased. The decrease in urine volume in males and females treated with 200 mg/kg/day and in males treated with 1000 mg/kg/day was considered to be the consequence of an osmonephrotic change due to elevated renal protein levels resulting in increased tubular urine resorption. This reversible effect was considered to be of no adverse nature. The change in urine color to brown and red-brown or red noted in all males and females treated with 1000 mg/kg/day was considered to be a passive, joint effect of the test item (dye) and the haematuria.

ORGAN WEIGHTS
The increased kidney-to-body weight ratio noted in males and females treated with 1000 mg/kg/day and in males treated with 200 mg/kg/day was considered to be a test item-related effect. No test item-related differences were noted in the mean absolute or relative organ weights after 2 weeks' recovery.

MACROSCOPIC / MICROSCOPIC FINDINGS
At necropsy, no test item-related macroscopic findings were recorded.
Microscopically, evidence for tubular cell damage could be found in both sexes. These adverse changes consisted of tubular cell necrosis in males treated with 1000 mg/kg/day in the 4 week necropsy group and tubular basophilia in females of the recovery group previously treated with 1000 mg/kg/day. Tubular basophilia can be understood as sequel to tubular damage.
Further treatment-related findings were judged as non adverse and consisted of increased incidence and severity of hyaline droplets in the kidneys of male animals in all treated groups, tubular cell swelling in a single male treated with 1000 mg/kglday and in females treated with 200 (one single animal) and 1000 mg/kg/day. This finding could still be observed after the recovery period in two males previously treated with 1000 mg/kg/day. The occurrence of hyaline droplets was considered to be a typical male rat specific effect. In addition, minimal to slight degree, mainly centrilobular hepatocellular hypertrophy could be observed in single males treated with 200 mg/kg/day and single females treated with 1000 mg/kg/day. Increased bile duct proliferation could be observed in male recovery animals.
Hepatocellular hypertrophy and bile duct proliferation were deemed to represent an adaptive effect.

Based on the results of this study, 200 mg/kg body weigh/day of test item was established as the no-observed-adverse-effect-level (NOAEL).