Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-01-08 till 2009-06-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline-conform study under GLP without deviations

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Guidelines for Screening Toxicity Testing of Chemicals: Testing Methods for New Substances, enacted July 13, 1974, amended December 5, 1986
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- public name of test material:
Reaction mass of 6,13-dichloro-3,10-bis{[2-({[(2-chloroethyl)sulfonyl]alkanoyl}amino)ethyl]-amino}- polycarboheterocyclo 4,11-disulfonic acid, mono and/or disodium salt and 6,13-dichloro-3-{[2-({[(2-chloroethyl)sulfonyl]alkanoyl}amino) ethyl]amino}-10-[(2-{[4-(ethenylsulfonyl)alkanoyl]amino}ethyl)amino] polycarboheterocyclo -4,11-disulfonic acid, mono and/or di sodium salt
- Physical state: solid, dark blue powder
- Analytical purity: approx. 86.6%
- Lot/batch No.: VER 2108 BOP 02/07
- Expiration date of the lot/batch: November 30, 2012
- Storage condition of test material: At room temperature at about 20 °C

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS: Rat, HanRcc: WIST(SPF)
- Source: Harlan Laboratories Ltd., Laboratory Animal Services, Wölferstrasse 4, 4414 Füllinsdorf / Switzerland
- Age at study initiation: ca. 7 weeks at delivery
- Weight at study initiation: at Acclimatization: Males: 177 g to 198 g (mean 189 g) Females: 139 g to 156 g (mean 146 g)
- Fasting period before study:
- Housing:
- Diet (e.g. ad libitum): Pelleted standard Kliba Nafag 3433 (batch no. 61/08) rat / mouse maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum
- Water (e.g. ad libitum): Community tap-water from Itingen was available ad libitum in water bottles.
- Acclimation period: 7d
Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used.
- Randomization: Computer-generated random algorithm
- Accommodation: In groups of five in Makrolon type-4 cages with wire mesh tops and standard softwood bedding (‘Lignocel’ Schill AG, 4132 Muttenz / Switzerland).



ENVIRONMENTAL CONDITIONS
- Temperature (°C): temp. range: 22 ± 3 °C
- Humidity (%): relative humidity range: 30 - 70%)
- Air changes (per hr): Air-conditioned with 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): There was 12-hour fluorescent light/12-hour dark cycle with music during the light period.






Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
bidistilled
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared weekly
- Mixing appropriate amounts with (Type of food): test material was weighed into a glass beaker on a tared Mettler balance. Thereafter the remaining vehicle was added. The mixtures were stirred using a magnetic stirrer and stored at room temperature (15 - 25 °C). Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

The dose formulations were stored in glass beakers at room temperature (20 ± 5 °C).
Based upon the results of dose formulation analyses performed during a non-GLP dose range finding study (Harlan Laboratories Study C19567), the stability of the test item formulations was considered to be sufficient to justify weekly preparation. These analyses were repeated under GLP for this study.


VEHICLE
- Justification for use and choice of vehicle (if other than water): water
- Concentration in vehicle: 0, 5, 20 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
After experimental start, samples of the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of homogeneity and concentration and stability. Samples of about 2 g of each concentration were taken during week 3 after commencement of dosing to confirm homogeneity and concentration.
The samples were delivered at ambient temperature to the Analytical Department and then stored at -20 ± 5 °C until analysis. The analysis was performed using a HPLC method provided by the Sponsor. The test item was used as analytical standard.

This analytical part was conducted at Harlan Laboratories Ltd., Itingen, Switzerland under GLP¬compliant conditions to verify the identity of the test item test material administered and to determine the content, homogeneity and stability of application formulations.
Several application formulations were prepared and representative analytical samples were collected and dispatched to the analytical laboratories internally. The test item concentrations were determined by HPLC coupled to a UV detector and quantified with the area under the peak.
The identity of test material was confirmed by its peak retention times which were similar to that measured in the working standards. The test item content in all samples was found to be within the accepted range of ±20% of the nominal content. In addition, the homogeneous distribution of test material in bi-distilled water was demonstrated. The application formulations were considered to be stable for at least 7 days when kept at room temperature.
In conclusion, the results obtained within this part confirm the correct preparation and storage of application formulations during the conduct of this study.
Duration of treatment / exposure:
28 d
Frequency of treatment:
once daily, 7d/wk
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 50, 200 and 1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
Group 1: 0 mg/kg/day 10 males 10 females
Group 2: 50 mg/kg/day 5 males 5 females
Group 3: 200 mg/kg/day 5 males 5 females
Group 4: 1000 mg/kg/day 10 males 10 females
Reserve animals:
Group 10: 1 male and 1 female
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:The dose levels were selected based on a previous dose range finding toxicity study in Wistar rats (Harlan Laboratories Study C19567).
- Rationale for animal assignment (if not random):Computer-generated random algorithm
- Rationale for selecting satellite groups:Toxicity testing (termination after 28 treatment days) resp Recovery testing (termination after an additional 14-day recovery period)
- Post-exposure recovery period in satellite groups: 14 d
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked (see table 1) were included.

DETAILED CLINICAL OBSERVATIONS: yes
- Time schedule: once before administration and once weekly (week 1-3)
The animals were observed for clinical signs once before commencement of administration as well as twice daily on days 1 to 3, once daily on days 4-28 (treatment period), and once daily during days 1 to 14 (recovery period).

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

BODY WEIGHT: Yes
Body weights were recorded weekly during acclimatization, treatment and recovery periods and before necropsy, using an on-line electronic recording system consisting of a Mettler balance connected to the Harlan Laboratories computer

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

FOOD EFFICIENCY:
- Body weight gain : data: relative food consumption (g/kg body weight/day)

OPHTHALMOSCOPIC EXAMINATION: Yes , see table 1

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 4 weeks (satellite A and B), after 6 weeks satellite B
Blood samples were drawn from the retro-orbital plexus from all animals under light isoflurane anesthesia. The animals were fasted in metabolism cages for approximately 18 hours before blood sampling but allowed access to water ad libitum. The samples were collected early in the working day to reduce biological variation caused by circadian rhythms. Blood samples were drawn from the retro-orbital plexus using a micro-hematocrit glass capillary tube.
- How many animals: all animals
- Parameters checked in table 3 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 4 weeks (satellite A and B), after 6 weeks satellit B
Blood samples were drawn from the retro-orbital plexus from all animals under light isoflurane anesthesia. The animals were fasted in metabolism cages for approximately 18 hours before blood sampling but allowed access to water ad libitum. The samples were collected early in the working day to reduce biological variation caused by circadian rhythms. Blood samples were drawn from the retro-orbital plexus using a micro-hematocrit glass capillary tube.
- How many animals: all animals
- Parameters checked in table 4 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine:Urine was collected during the 18 hours fasting period into a specimen vial, using a metabolism
cage.
- Parameters checked in table 5 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes, see table 1
- Time schedule for examinations:During week 4, relevant parameters from a modified Irwin screen test were evaluated in all animals.
- Dose groups that were examined: see table 1
- Battery of functions tested: reflexes / grip strength / locomotor activity

OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, see table 2
HISTOPATHOLOGY: Yes, see table 2
Statistics:
The following statistical methods were used to analyze body weight, grip strength, locomotor activity, clinical laboratory data, organ weights and ratios as well as macroscopic findings:
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was 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 Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Minor differences in the mean locomotor activity, but considered not to be related to the test item
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
some discolaration, considerdto be related to the test item
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
Viability / Mortality
All animals survived until scheduled necropsy.
Detailed Clinical Observations (Weekly)
No findings of toxicological relevance were noted during the Detailed Clinical Observations (weeks 1-3).


BODY WEIGHT AND WEIGHT GAIN
After the treatment period, no test item-related differences in the mean body weights or mean body weight gain values were noted at any dose level. Differences noted during recovery in the mean body weights of females previously treated with the test item were, in the absence of changes during treatment, considered to be unrelated to the test item.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
The mean daily food consumption of the test item-treated rats compared favorably with those of the controls.


OPHTHALMOSCOPIC EXAMINATION
General Cageside Observations (Daily)
Dark feces was noted in males and females of all test item-treated groups. The speed of onset and the mean severity of this finding were related to dose. Although this is a test item-related finding, it is considered to be common following oral administration of dyestuffs and of no toxicological relevance.
No clinical findings of adverse nature were seen at any dose level.


SUMMARY Clinical Laboratory Investigations
Hematology / Clinical Biochemistry / Urinalysis
After four weeks’ treatment, no differences of toxicological relevance were noted at any dose
level. All differences remained within the ranges of the historical control data, were doseunrelated,
were largely due to aberrant control values, or were not accompanied by concomitant
changes in related parameters and therefore considered to be incidental.
After two weeks’ recovery, none of the observed differences were considered to be late effects.

HAEMATOLOGY
After four weeks’ treatment, the differences noted in a small number of parameters attained statistical significance when compared with the respective control values, but remained within the ranges of the historical control data, were dose-unrelated, were largely due to aberrant control values, or were not accompanied by concomitant changes in related parameters and therefore considered to be incidental.
After two weeks’ recovery, none of the observed differences had been seen at the end of the treatment period, and remained within the ranges of the historical control data. These were not considered to be late effects.

CLINICAL CHEMISTRY
After four weeks’ treatment, males and females treated with 1000 mg/kg/day had elevated glucose levels (p<0.05 and p<0.01, respectively) and elevated total bilirubin (p<0.01 and p<0.05, respectively) when compared with the controls. The latter difference exceeded the upper limit of the respective historical control data. These isolated differences were considered to be unrelated to the test item treatment.
The mean triglyceride level of females treated with 1000 mg/kg/day was significantly lower (p<0.01) than that of the control females, but remained within the lower limit of the historical control data.
Some differences were incurred by outliers in individual control animals, or by general differences in the control values when compared with the historical control data. For example, although the mean triglyceride level for males treated with 1000 mg/kg/day was markedly lower than that of the control males, this was due to a single outlier in the control group (male no. 3).
Males treated with 1000 mg/kg/day and 200 mg/kg/day had significantly elevated potassium levels (p<0.01 and p<0.05, respectively) but the differences were largely considered to be due to a low control value, and were unrelated to the treatment. All other differences were doseunrelated or were not accompanied by concomitant changes in related parameters and therefore considered to be incidental.
After two weeks’ recovery, there was a small number of differences when compared with the controls. All remained within the ranges of the historical control values, and none of the differences were seen after the treatment period. Hence they were considered to be incidental.
There were no differences of toxicological relevance in the clinical biochemistry parameters of females.

URINALYSIS
After four weeks’ treatment, the differences noted in a small number of urinalysis parameters were significantly higher than those of the respective control values, but remained within the ranges of the historical control data or were not accompanied by concomitant changes in related parameters and therefore considered to be incidental. Staining of the urine samples was considered to be related to the excretion of the test item via the renal pathway.
After two weeks’ recovery, there were no differences of toxicological relevance when compared with the controls.

NEUROBEHAVIOUR
Functional Observational Battery
No findings of toxicological relevance were noted during the Functional Observational Battery (week 4).
Grip Strength
The mean fore- and hindlimb grip strength of the test item-treated rats compared favorably with those of the respective controls.
Locomotor Activity
Males treated with 1000 mg/kg/day had significantly elevated locomotor activity during the 0-10 minute measurement interval (p<0.05). No other differences were noted in males, and females at this dose were unaffected.
At 50 mg/kg/day, the mean locomotor activity was significantly elevated in females from 10-20 minutes (p<0.05) and from 20-30 minutes (p<0.05). A similar difference was noted in the total locomotor activity (0-60 minutes, p<0.05) of the females. In the absence of similar changes at 200 mg/kg/day, these differences were considered to be incidental.


ORGAN WEIGHTS
The organs from satellite A and B animals were weighed before fixation and recorded on the scheduled dates of necropsy. Relative organ weights were calculated on the basis of the body weight and brain weight.
The terminal body weight was recorded immediately prior to necropsy and the organ to terminal
body weight ratios as well as organ to brain weight ratios were determined.


GROSS PATHOLOGY
Organ Weights
(See Summary Tables on p. , Individual Tables on p. )
No test item-related effects upon the mean absolute or relative organ weights were noted.
At 1000 mg/kg/day, the mean absolute and relative organ weights compared favorably with those of the control rats after fours weeks of treatment.
At 200 mg/kg/day, the mean absolute and relative adrenal weights of males were significantly reduced (p<0.05) when compared with the controls. This finding was considered to be unrelated to the test item as the control value was skewed by a single outlying value (male no. 2). Females at this dose level had significantly elevated (p<0.05) liver-to-body weight ratio and significantly reduced (p<0.05) thymus-to-body weight ratio. The mean spleen-to-brain weight ratio of females was significantly reduced (p<0.05) after four weeks of treatment. These findings, in the absence of changes at the higher dose, were considered to be incidental.

At 50 mg/kg/day, the mean absolute adrenal weight was significantly reduced (p<0.05) when compared with the controls. This finding was considered to be unrelated to the test item as the control value was skewed by a single outlying value (male no. 2).
After the recovery period, the mean absolute liver weights and mean absolute spleen weights were significantly reduced (p<0.05 and p<0.01, respectively) in females previously treated with 1000 mg/kg/day. The mean brain-to-body weight ratio was significantly elevated (p<0.01) in these females, whereas the mean heart-, liver-, and spleen-to-brain weight ratios were all significantly reduced (p<0.01, p<0.05 and p<0.01, respectively) when compared with controls. These differences were not seen at the end of the treatment period and were not considered to be late effects of the test item.

Macroscopic Findings
After four weeks of treatment, dark discoloration of several organs (tongue, stomach, jejunum, ileum, cecum, colon, rectum, kidneys, and mesenteric lymph nodes) was noted at several dose levels and considered to be related to the test item.
Other macroscopical findings (accentuated lobular pattern in one male at 1000 mg/kg/day, reddish discoloration of the pancreas in one male at 200 mg/kg/day, foci on the mandibular lymph nodes of one male treated with 200 mg/kg/day) occurred at low incidence and were considered to be typical background changes unrelated to the treatment with the test item.
After two weeks of recovery, dark discoloration of the kidneys persisted in all animals and was considered to be effects of the test item. Dark red foci were noted on the stomach of one male, an isolated dark brown focus was noted on the lungs of one male and one female, reddish discoloration of the ileum was seen in one female, and a minor ear injury was recorded in one female.


HISTOPATHOLOGY: NON-NEOPLASTIC
All organ and tissue samples (as defined in the table 2) were processed, embedded and cut at an approximate thickness of 2 to 4 micrometers and stained with hematoxylin and eosin.
Slides of all organs and tissues (as defined in the table 2) which were collected at scheduled sacrifices from all animals of the control and high-dose groups and all gross lesions from all animals were examined by the study pathologist. Organ and tissue samples taken from animals which died spontaneously or which were killed in extremis were evaluated similarly to those organs taken from animals of the high-dose group.
As test item-related morphologic changes were detected in the organs of high-dose animals, the same organs (kidneys) from animals of the mid- and low-dose groups were examined.
A description of all abnormalities is included in the pathology report. Attempts were made to correlate gross observations with microscopic findings.

Microscopic Findings
No microscopical findings of toxicological relevance were noted in animals necropsied after four weeks of treatment. After two weeks of recovery, blue pigment (engulfed in pulmonary phagocytes) were noted in three animals. All lesions recorded during the microscopic investigation were within the range of background alterations that may be recorded in this type of study, and in rats of this strain and age.

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Body Weight Males

 

Group 1

Group 2

Group 3

Group 4

 

0 mg/kg

50 mg/kg

200 mg/kg

1000 mg/kg

ACCLIMATIZATION

 

 

 

 

Day 1  MEAN

189

 188

188

188

ST.DEV.

7

 7

 6

 7

MINIMUM

177

 179

 181

 178

MAXIMUM

198

 197

 194

 196

N

10

5

5

10

TREATMENT PERIOD

 

 

 

 

Day 1   MEAN

233

 231

229

229

ST.DEV.

8

 8

 6

 8

MINIMUM

218

 221

 221

 213

MAXIMUM

241

 240

 236

 242

N

10

5

5

10

Day 8  MEAN

269

 271-

261-

267

ST.DEV.

11

 11

 8

 11

MINIMUM

244

 261

 252

 246

MAXIMUM

281

 287

 271

 287

N

10

5

5

10

Day 15 MEAN

298

 299

288

298

ST.DEV.

15

 15

 10

 15

MINIMUM

269

 287

 277

 276

MAXIMUM

318

 323

 301

 326

N

10

5

5

10

Day 22 MEAN

323

 319

311

318-

ST.DEV.

19

 16

 10

 17

MINIMUM

290

 305

 300

 292

MAXIMUM

356

 347

 326

 345

N

10

5

5

10

Day 28 MEAN

342

 332

325

336

ST.DEV.

21

 19

 13

 20

MINIMUM

309

 319

 309

 313

MAXIMUM

381

 366

 343

 369

N

10

5

5

10

RECOVERY PERIOD

 

 

 

 

 

Day 1  MEAN

323

---

---

318

ST.DEV.

19

---

---

14

MINIMUM

293

---

---

298

MAXIMUM

346

---

---

331

N

5

0

0

5

Day 8   MEAN

358

---

---

348

ST.DEV.

24

---

---

22

MINIMUM

322

---

---

320

MAXIMUM

391

---

---

379

N

5

0

0

5

Day 14  MEAN

374

---

---

361

ST.DEV.

25

---

---

21

MINIMUM

338

---

---

337

MAXIMUM

408

---

---

393

N

5

0

0

5

Body Weight Females

 

Group 1

Group 2

Group 3

Group 4

 

0 mg/kg

50 mg/kg

200 mg/kg

1000 mg/kg

ACCLIMATIZATION

 

 

 

 

Day 1   MEAN

147

145

145

147

ST.DEV.

4

 4

 4

5

MINIMUM

143

 141

 141

141

MAXIMUM

154

 151

 151

156

N

10

5

5

10

TREATMENT PERIOD

 

 

 

 

Day 1  MEAN

171

167

167

165

ST.DEV.

6

 5

 5

10

MINIMUM

164

 162

 162

144

MAXIMUM

180

 173

 173

180

N

10

5

5

10

Day 8  MEAN

187

186

186

184

ST.DEV.

9

 8

 8

10

MINIMUM

173

 174

 174

164

MAXIMUM

200

 195

 195

198

N

10

5

5

10

Day 15 MEAN

206

203

203

200

ST.DEV.

8

 9

 9

9

MINIMUM

194

 190

 190

183

MAXIMUM

216

 212

 212

215

N

10

5

5

10

Day 22 MEAN

218

211

211

212

ST.DEV.

9

 10

 10

11

MINIMUM

203

 199

 199

193

MAXIMUM

231

 222

 222

228

N

10

5

5

10

Day 28 MEAN

224

221

221

221

ST.DEV.

11

 13

 13

11

MINIMUM

206

 198

 198

202

MAXIMUM

240

 233

 233

236

N

10

5

5

10

RECOVERY PERIOD

 

 

 

 

Day 1   MEAN

223

---

---

203

ST.DEV.

10

---

---

13

MINIMUM

210

---

---

193

MAXIMUM

231

---

---

220

N

5

0

0

5

Day 8   MEAN

242

---

---

222

ST.DEV.

13

---

---

12

MINIMUM

226

---

---

211

MAXIMUM

253

---

---

240

N

5

0

0

5

Day 14  MEAN

253

---

---

232

ST.DEV.

9

---

---

13

MINIMUM

243

---

---

221

MAXIMUM

262

---

---

249

N

5

0

0

5

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, a no-observed-effect-level (NOEL) for test material could not be established, but the no-observed-adverse-effect-level (NOAEL) was considered to be 1000 mg/kg body weight/day.
Executive summary:

In this subacute toxicity study, test material was administered daily by oral gavage to SPF-bredWistar 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 weresacrificed.

Clinical signs, outside cage observation, food consumption and body weights were recordedperiodically 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 withdrawnfor hematology and plasma chemistry analyses. Urine samples were collected for urinalyses. Allanimals were killed, necropsied and examined post mortem. Histological examinations wereperformed on organs and tissues from all control and high dose animals, and all gross lesionsfrom all animals.

From the animals of the low and middle dose groups, kidneys were examined to establish a no-effect level.

Mortality / Viability

All animals survived until scheduled necropsy.

Clinical Signs (Daily and Weekly)

Dark feces were observed with dose-related speed of onset and mean severity. Although this wasa test item-related finding, it was considered to be common following oral administration ofdyestuffs and of no toxicological relevance.

No findings of toxicological relevance were noted during the Detailed Clinical Observations(weeks 1-3).

Functional Observational Battery

No findings of toxicological relevance were noted during the Functional Observational Battery(week 4).

Grip Strength

The mean fore- and hindlimb grip strength of the test item-treated rats compared favorably with those of the respective controls.

Locomotor Activity

Minor differences in the mean locomotor activity were considered to be unrelated to the test item.

Food Consumption

The mean daily food consumption of the test item-treated rats compared favorably with those of the controls.

Body Weights

No test item-related differences in the mean body weights or mean body weight gain values werenoted at any dose level.

Clinical Laboratory Investigations

Hematology / Clinical Biochemistry / Urinalysis

After four weeks’ treatment, no differences of toxicological relevance were noted at any doselevel. All differences remained within the ranges of the historical control data, were dose-unrelated, were largely due to aberrant control values, or were not accompanied by concomitantchanges in related parameters and therefore considered to be incidental.

After two weeks’ recovery, none of the observed differences were considered to be late effects.

Organ Weights

No test item-related effects upon the mean absolute or relative organ weights were noted.

Macroscopic / Microscopic Findings

After four weeks of treatment, dark discoloration of several organs (tongue, stomach, jejunum,ileum, cecum, colon, rectum, kidneys, and mesenteric lymph nodes) was noted macroscopicallyat various dose levels and considered to be related to the test item.

After two weeks of recovery, persistent dark discoloration of the kidneys was noted in previouslytreated rats.

Microscopically, no findings of toxicological relevance were noted in animals necropsied after four weeks of treatment. After two weeks of recovery, blue pigment (engulfed in pulmonaryphagocytes) were noted in three animals.

CONCLUSION

Based on the results of this study, a no-observed-effect-level (NOEL) for test material could

not be established, but the no-observed-adverse-effect-level (NOAEL) was considered to be

1000 mg/kg body weight/day.