Registration Dossier

Diss Factsheets

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Some information in this page has been claimed confidential.

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-08-17 till 2010-11-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Swiss GLP

Test material

Constituent 1
Details on test material:
- Name of test material (as cited in study report): FAT 40850/A TE
- Substance type: textile dyestuff
- Physical state: red powder
- Analytical purity: ca. 63 % of all colored components
- Lot/batch No.: BOP 07-09
- Expiration date of the lot/batch: 2014-07-31
- Storage condition of test material: At room temperature at about 20 °C
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: TZ 5978 / BOP 07-09
- Expiration date of the lot/batch: 31-Jul-2014

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In the original container at room temperature (15 - 25 °C)
- Stability of the test substance in the solvent/dispersant/vehicle/test medium: Up to 8 days at room temperature (15 - 25 °C)

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Rationale: Recognized by international guidelines as a recommended test system.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS: Rat, RccHanTM: WIST(SPF)
- Source:Harlan Laboratories B.V. Kreuzelweg 53 5961 NM Horst / Netherlands
- Age at study initiation:Ca. 7 weeks
- Weight at study initiation: Males: 182 to 207 g, Females: 148 to 168 g
- Fasting period before study: no data
- Housing:In groups of five in Makrolon type-4 cages with wire mesh tops and standard softwood bedding including paper enrichment
- Diet: Pelleted standard Harlan Teklad 2914C rat / mouse maintenance diet was available ad libitum
- Water:Community tap-water from Itingen was available ad libitum in water bottles.
- Acclimation period: 5 days. Under test conditions after health examination. Only animals without any visible signs of illness were used for the study
- Pre-Randomization Period: 1 day
- Randomization: Randomly allocated to groups by body weight
- Group 10: reserve, not used in the test


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Values outside of these ranges occasionally occurred, usually following room cleaning, and are considered not to have any influence on the study.
- Air changes (per hr):Air-conditioned with 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light):12-hour fluorescent light/12-hour dark cycle with music during the light period.

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
Rationale for Method: Administration by gavage is a common and accepted route of exposure for studies of this type.
Vehicle:
water
Remarks:
bidistilled
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

DOSING SOLUTIONS PREPARATION
- Rate of preparation of dosing preparation (frequency):The dose formulations were prepared weekly.
FAT 40850/A TE was weighed into a glass beaker on a tared Mettler balance and a part of the vehicle (approx. one third) was added, stirred and the
remaining vehicle was added (except the first preparation, when in the low dose the total volume of the 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.
- Stability of dose formulations: stable in application formulations when kept four hours and eight days under room temperature due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean.
- Storage of dose formulations: in glass beakers at room temperature (15 - 25 °C).


VEHICLE
- Justification for use and choice of vehicle (if other than water): none (water)
- Concentration in vehicle: 0 mg/mL, 10 mg/mL, 30 mg/mL and 100 mg/mL
- Amount of vehicle (if gavage): Dose Volume: 10 mL/kg body weight
- Purity: bidistilled water
- Dose Volume: 10 mL/kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analysis was performed by Harlan Laboratories Ltd. using a HPLC method provided by the Sponsor. After experimental start and during week 3, duplicate 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. Duplicate samples of about 2 g of each concentration were taken to confirm stability (4 h and 8 days). The samples were delivered to the analytical laboratory (Harlan Laboratories Ltd., Analytics, Itingen / Switzerland) at room temperature (20 ± 5 °C) and stored there at -20 ± 5 °C until analysis. The application formulations investigated during the study were found to comprise FAT 40850/A in the range of 93.3 % to 111.3 % and, thus, the required content limit of ±20 % with reference to the nominal concentration was met. The homogeneous distribution of FAT 40850/A in the preparations was approved because single results found did not deviate more than 5.6 % (<15 %) from the corresponding mean. The following acceptance criteria will be applied to analytical results:
sample contents should be within a range of ±20 % of nominal content. Formulations will be considered homogeneous if the maximum deviation from mean calculated from top, middle and bottom samples is not more than 15 %. The results obtained from storage stability samples should not deviate more than 10 % fromtime-zero reference (content or mean of homogeneity samples).
Duration of treatment / exposure:
28 d
Frequency of treatment:
daily, 7d/week
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Low dose
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Middle dose
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
High dose
No. of animals per sex per dose:
5
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 C93571 (non-GLP).
- Rationale for animal assignment (if not random):Randomly allocated to groups by body weight.
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random):Sacrifice: After 4 Weeks. All animals were weighed and necropsied. Descriptions of all macroscopical abnormalities were recorded. All animals were anesthetized by intraperitoneal injection of pentobarbitone and killed by exsanguination. Slides of all organs and tissues 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. Additionally, the kidneys of both sexes were examined from the animals of the low and middle dose groups to establish a no-effect level.
Positive control:
none

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked in table No1 were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:The animals were observed for clinical signs once daily during acclimatization as well as daily on days 1 - 28 (twice daily during days 1 - 3) during the treatment period.

Weekly Behavioral Observations
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
- Time schedule for examinations:Body weights were recorded once during the pre-randomization phase, once weekly during the acclimatization and treatment periods and once before necropsy,

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, the food consumption was recorded once during the acclimatization period and weekly thereafter
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes, body weight gain in %

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): not applicable

OPHTHALMOSCOPIC EXAMINATION: Yes, see table 1

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 4 weeks
- Anaesthetic used for blood collection: Yes (identity) Blood samples were drawn from the retro-orbital plexus from all animals under light isoflurane anesthesia
- Animals fasted: Yes, in metabolism cages for approximately 18 hours before blood sampling but allowed access to water ad libitum
- How many animals: all
- Parameters checked in table No.3 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:after 4 weeks
- Animals fasted: Yes, Yes, in metabolism cages for approximately 18 hours before blood sampling but allowed access to water ad libitum
- How many animals: all
- Parameters checked in table No.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.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table No.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:
Vaginal Smear for Estrus Stage
Vaginal smears were taken over four days from all females, and the stage of estrus was evaluated, if possible (During Week 3 and 4)
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:
effects observed, non-treatment-related
Description (incidence and severity):
Red stained feces were noted in males and females treated with the test item for four weeks at all dose levels. Otherwise, no test item-related findings were noted at the daily observation. No abnormalities were recorded at the weekly behavioral observations in all animals at any dose level.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
In the males treated with 1000 mg/kg/day, a statistically significant decrease in white blood cell (WBC) count was noted in correlation with significantly decreased absolute lymphocyte value.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
In the males treated with 1000 mg/kg/day, a significant decrease in phosphorus and potassium concentration was noted. Due to the occurrence in the high dose group, these effects are considered to be test item-related, although potassium levels are within the range of historical data. Furthermore, the total protein level was increased in correlation with increased albumin and increased albumin/globulin ratio. As these findings were noted only in the males and there was no dose response relationship, these changes were considered not to be of adverse nature.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
No significant differences of absolute organ weights were noted in the males. Significantly increased (p<0.05) liver/body weight ratio was noted in males of all treatment groups compared to control animals. Furthermore, in males treated with 1000 mg/kg/day, the kidney/brain weight ratio was significantly increased (p<0.05). In the females, no significant differences in absolute and relative organ weights were noted. (see Table 1).
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Red staining of kidneys and/or lymphnodes and/or stomach was noted in several male and female animals at all dose levels, and is considered not to be indicative of systemic toxicity. Otherwise, no test tem-related findings were noted at necropsy. See Table 2.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Relevant microscopic findings were recorded only in the kidneys. Deposits of a fine granular red pigment were recorded in the tubular epithelium at minimal to slight severity in both sexes treated with 1000 mg/kg/day and in females treated with 300 mg/kg/day. The deposits of red pigment were in some cases associated with increased severity of hyaline droplets in males treated with 1000 mg/kg/day and minimal to slight vacuolar degeneration of tubular epithelium in females treated with 1000 mg/kg/day.
Details on results:
CLINICAL SIGNS AND MORTALITY
All animals survived the scheduled treatment period.

BODY WEIGHT AND WEIGHT GAIN
No effects on the body weight or body weight gain were noted in male and female animals during four weeks of treatment.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
No test item-related difference in the mean daily food consumption was noted at any dose level.

OPHTHALMOSCOPIC EXAMINATION
Daily Observations
Red stained feces were noted in males and females at all dose levels (from day 8 at 100 mg/kg/day, from day 5 at 300 mg/kg/day and from day 3 at 1000 mg/kg/day). Otherwise, no test item-related findings were noted at the daily observation.
In one female of the control group, red scabs around the left eye were noted from day 6 up to the end of treatment. In one female treated with 1000 mg/kg/day, hunched posture and dyspnoe were noted on day 14 of treatment. These findings were considered to be incidental.

Weekly Behavioral Observations
No abnormalities were recorded at the weekly behavioral observations in all animals at any dose level.


HAEMATOLOGY
In the males treated with 1000 mg/kg/day, a distinctive and statistically significant (p<0.01) decrease in white blood cell count (WBC) was noted. This was in correlation with significantly decreased (p<0.01) absolute lymphocyte value. Both values were below the range of historical data.

Further, significant changes in differential WBC count were noted in males treated with 1000 mg/kg/day such as decrease in neutrophils, eosinophils, basophils, monocytes and large unstained cells. However, these values stayed within the historical data even though at the lower limit. Furthermore, in males treated with 300 mg/kg/day, lymphocytes were significantly decreased, but also within the range of historical data for rats of this age, strain and sex. Further, statistically significant changes in males in hemoglobin, hematocrit, mean corpuscular hemoglobin concentration (MCHC) and hemoglobin concentration distribution width (HDW) are all within the historical control data, and, therefore, considered not to be treatment-related. There were no test item-related changes in hematological parameters detected in the females of all dose groups. The few significantly different parameters noted in females treated with 300 mg/kg/day only, were within the historical data range (HDW slightly higher) with no dose response-relationship and, therefore, considered not to be treatment-related.

CLINICAL CHEMISTRY
No adverse test item-related findings in clinical biochemistry parameters were noted. In the males treated with 1000 mg/kg/day, significantly decreased alanine aminotransferase (ALAT) and alkaline phosphatase (ALP) levels were detected. A decrease in these parameters was considered to be within the range of biological variance. In the same animals, a decrease in potassium and phosphorus concentrations was noted. Due to the occurrence in the high dose group, these effects are considered to be test item-related, although potassium levels are within the range of historical data. Furthermore, the total protein level was increased in correlation with increased albumin and increased albumin/globulin ratio. As these findings were noted only in males and as there was no dose response relationship, these changes were considered not to be of adverse nature. There were no changes considered to be test item-related in clinical biochemistry parameters in the females of all dose groups. The few significantly different parameters only in females treated with 300 mg/kg/day, are all within the range of historical data and considered not to be treatment related.

URINALYSIS
No test item-related effects were noted in male and female animals of all dose groups compared to control group.

NEUROBEHAVIOUR
Functional Observational Battery (Screen). No findings were evident during the functional observational battery performed during week 4 of treatment.

Grip Strength
No differences compared to the control group were recorded in the grip strength of male and female animals at any dose level.

Locomotor Activity
There were no effects on the locomotor activity observed in male and female animals of all groups.

ORGAN WEIGHTS (see table 1)
In the males, significantly increased (p<0.05) liver/body weight ratio was noted in all treatment groups. The liver/brain weight ratio as well as the absolute mean liver weight was slightly (nonsignificantly) increased in males of all treatment groups (see table below). The kidney/brain weight ratio was significantly (p<0.05) increased in the males treated with 1000 mg/kg/day. No significant changes were noted in the absolute and relative organ weights of the females.

GROSS PATHOLOGY (see table 2)
Test item-related dark red discoloration of the kidney was noted in four males treated with 300 mg/kg/day and all males treated with 1000 mg/kg/day. In the males treated with 1000 mg/kg/day, the mesenteric lymphnodes showed also a light red discoloration. In the females, dark red discoloration of the kidneys was noted in four out of five animals treated with 100 mg/kg/day and all animals treated with 300 mg/kg/day and 1000 mg/kg/day, respectively. In the females treated at 1000 mg/kg/day, light red discoloration of the lymphnodes was also noted in all animals and discolored (light red) stomach in one animal. Furthermore, pelvic dilation was noted in one control male and red foci in the thymus were recorded in three control males as well as two males treated with 300 mg/kg/day.

HISTOPATHOLOGY: NON-NEOPLASTIC (see table 3)
Relevant microscopic findings were recorded only in the kidneys. The remainder of further findings recorded was within the range of normal background lesions which may be recorded in animals of this strain and age.

Kidneys
Deposits of a fine granular red pigment were recorded in the tubular epithelium at minimal to slight severity in both sexes of group 4 and in females of group 3. The deposits of red pigment were sometimes associated with increased severity of hyaline droplets in males of group 4 and minimal to slight vacuolar degeneration of tubular epithelium in females of group 4.

OTHER FINDINGS
Vaginal Smear for Estrus Stage: No special findings were noted concerning the estrous stage of the female animals.

Thyroid Hormone Analysis
No analysis of the thyroid hormone levels was performed, as there were neither alterations in the organ weights nor macroscopic or microscopic changes in the thyroid.

Effect levels

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
gross pathology
histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
System:
urinary
Organ:
kidney
Treatment related:
yes

Any other information on results incl. tables

Table 1: Changes in Mean Absolute Organ Weight and Mean Relative Organ Weight

(Organ/Body Weight (bw) or Organ/Brain Weight (brw) Ratio) as Compared to Controls

Organ/ Ratio

Males

Females

100 mg/kg/day

300 mg/kg/day

1000 mg/kg/day

100 mg/kg/day

300 mg/kg/day

1000 mg/kg/day

Liver weight

+15.3% (-)

+15.4% (-)

+15.8% (-)

 

 

 

Liver/bw ratio

+9.7% (*)

+9.7% (*)

+7.6% (*)

 

 

 

Liver/brw ratio

+16.1% (-)

+14.7% (-)

+14.5% (-)

 

 

 

Kidney weight

 

 

+16.1% (-)

 

 

 

Kidney/ bw ratio

 

 

+7.4% (-)

 

 

 

Kidney/ brw ratio

 

 

+14.6% (*)

 

 

 

*/**/- : DUNNETT-Test based on pooled variance sig. at 5% (*), 1% (**) or not sig.(-)

Table 2:Incidence of Main Macroscopic Findings

Findings/Incidence

Group 1

Group 2

Group 3

Group 4

Terminal Sacrifice

5 M

5 F

5 M

5 F

5 M

5 F

5 M

5 F

Kidneys: Discoloration, red

0

0

0

4

4

5

5

5

Mesenteric lymph node: Discoloration, red

0

0

0

0

0

0

5

5

Stomach: Discoloration, red

0

0

0

0

0

0

0

1

Table 3 Incidence and Mean Severity Grade of Main Findings in Kidneys

Finding

Group 1

Group 2

Group 3

Group 4

Incidence / Mean Severity

5 M

5 F

5 M

5 F

5 M

5 F

5 M

5 F

Deposits, red pigment

-

-

-

-

-

5/1.0

5/1.0

5/1.4

Vacuolar degeneration

-

-

-

-

-

-

-

4/1.8

Hyaline droplets

2/1.0

-

1/1.0

-

2/1.0

-

3/2.0

-

Males Body Weights Summary

 

Group 1

0 mg/kg

Group 2

100 mg/kg

Group 3

300 mg/kg

Group 4

 1000 mg/kg

PRE-RANDOMIZATION

 

 

 

 

Day 1      MEAN

178

 176-

180-

176-

ST.DEV.

7

 6

 7

 6

MINIMUM

169

 168

 172

 166

MAXIMUM

188

 183

 190

 182

N

5

5

5

5

ACCLIMATIZATION

 

 

 

 

Day 1      MEAN

194

 195-

195-

193-

ST.DEV.

6

 8

 8

 6

MINIMUM

186

 182

 189

 187

MAXIMUM

203

 205

 207

 200

N

5

5

5

5

TREATMENT PERIOD

 

 

 

 

Day 1      MEAN

220

 225-

225-

228-

ST.DEV.

9

 9

 11

 5

MINIMUM

205

 215

 218

 222

MAXIMUM

231

 239

 243

 234

N

5

5

5

5

Day 8      MEAN

251

 257-

260-

266-

ST.DEV.

13

 12

 16

 10

MINIMUM

232

 249

 249

 253

MAXIMUM

265

 278

 287

 278

N

5

5

5

5

Day 15    MEAN

279

 284-

286-

297-

ST.DEV.

17

 16

 22

 12

MINIMUM

255

 266

 262

 283

MAXIMUM

299

 308

 321

 315

N

5

5

5

5

Day 22    MEAN

305

 309-

312-

323-

ST.DEV.

20

 20

 27

 16

MINIMUM

273

 284

 282

 309

MAXIMUM

325

 340

 354

 350

N

5

5

5

5

Day 28   MEAN

320

 326-

327-

341-

ST.DEV.

22

 25

 29

 21

MINIMUM

283

 292

 295

 323

MAXIMUM

340

 361

 372

 376

N

5

5

5

5

*/**/- : DUNNETT-Test based on pooled variance sig. at 5% (*), 1% (**) or not sig.(-)

Females Body Weights Summary

 

Group 1

0 mg/kg

Group 2

100 mg/kg

Group 3

300 mg/kg

Group 4

1000 mg/kg

PRE-RANDOMIZATION

 

 

 

 

Day 1      MEAN

145

 146-

148-

145-

ST.DEV.

5

 5

 7

 4

MINIMUM

139

 140

 141

 139

MAXIMUM

152

 153

 159

 149

N

5

5

5

5

ACCLIMATIZATION

 

 

 

 

Day 1      MEAN

156

 160-

160-

154-

ST.DEV.

5

 2

 5

 5

MINIMUM

149

 158

 155

 148

MAXIMUM

162

 164

 168

 161

N

5

5

5

5

TREATMENT PERIOD

 

 

 

 

Day 1      MEAN

169

 170-

173-

172-

ST.DEV.

4

 5

 4

 2

MINIMUM

165

 161

 168

 169

MAXIMUM

175

 173

 178

 175

N

5

5

5

5

Day 8      MEAN

182

 183-

183-

182-

ST.DEV.

6

 3

 5

 5

MINIMUM

173

 178

 176

 177

MAXIMUM

189

 185

 189

 189

N

5

5

5

5

Day 15    MEAN

191

 192-

192-

192-

ST.DEV.

9

 8

 7

 9

MINIMUM

179

 181

 184

 182

MAXIMUM

204

 204

 199

 203

N

5

5

5

5

Day 22    MEAN

201

 206-

194-

200-

ST.DEV.

10

 8

 5

 9

MINIMUM

186

 196

 187

 187

MAXIMUM

210

 219

 200

 213

N

5

5

5

5

Day 28   MEAN

207

 209-

206-

204-

ST.DEV.

13

 7

 7

 10

MINIMUM

196

 198

 200

 189

MAXIMUM

228

 217

 215

 215

N

5

5

5

5

*/**/- : DUNNETT-Test based on pooled variance sig. at 5% (*), 1% (**) or not sig.(-)

Applicant's summary and conclusion

Conclusions:
The no-observed-adverse-effect-level (NOAEL) of FAT 40850/A was established at 300 mg/kg/day.
Executive summary:

The 28-day oral repeat dose study with FAT 40850/A was carried out in Wistar rats according to OECD guideline 407 and EU method B.7 at doses of 100, 300 and 1000 mg/kg/day. The study was performed under GLP-conditions. A control group was treated similarly with the vehicle, bidistilled water, only. Each group comprised 5 animals per sex which were terminated after 28 days of treatment. Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during the acclimatization and treatment periods. Functional observational battery, locomotor activity and grip strength were assessed during week 4. At the end of the treatment 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.

The following test item related findings were noted:

- Red stained feces, noted in males and females at all dose levels (100, 300 and 1000 mg/kg/day) were considered to be caused by a dyeing effect of the test item but not to be adverse. Red discoloration was also detected macroscopically in multiple organs, like kidneys, mesenteric lymphnodes and stomach.

- A significant decrease in the white blood cell count and in lymphocytes (absolute) with values below ranges of historical control data in males at 1000 mg/kg/day was possibly related to the treatment with the test item. The lymphocyte value in males treated with 300 mg/kg/day was also decreased and there seemed to be a tendency in reduction of lymphocytes (absolute) with an effect on total WBC in the highest dose group. In the absence of histopathologigal changes e.g. in the spleen, bone marrow, thymus or lymphnodes and no findings in the females, this effect is considered not to be adverse.

- A decrease in potassium and phosphorus concentrations was noted in males at 1000 mg/kg/day and, as occurred in the high dose group, is considered to be test item-related, although potassium levels are within the range of historical data. Furthermore, the total protein level was increased in correlation with increased albumin and increased albumin/globulin ratio. As these findings were noted only in males and as there was no dose response relationship, these changes were considered not to be of adverse nature.

- The increase in liver weights is considered to be caused by an adaptive reaction but, in absence of further findings, e.g. pathohistological changes in the liver, not to be of adverse nature.

- Changes in kidney weights in males at 1000 mg/kg/day are in correlation with macroscopic and microscopic findings. Histologically, treatment related findings consisted of minimal to slight deposits of a red pigment in the kidneys of animals of both sexes treated with 1000 mg/kg/day and in females treated with 300 mg/kg/day. The pigment was red fine granular and located in the tubular epithelium, and sometimes associated with increased severity of hyaline droplets in males treated with 1000 mg/kg/day and minimal to slight vacuolar degeneration of tubular epithelium in females of the same dose group. The deposits of red pigment were considered to be not adverse itself, but the secondary reactions consisting of increased severity of hyaline droplets and vacuolar degeneration of tubular epithelium were deemed to be adverse. Based on the results of this study, a no-observed-effect-level (NOEL) of FAT 40850/A could not be established. The no-observed-adverse-effect-level (NOAEL) was established at 300 mg/kg/day.