2-[(8-amino-7-{[4-substituted-2-sulfonatophenyl]diazenyl}-1-hydroxy-3,6-disulfonaphthalen-2-yl)diazenyl]-4-[(4-chloro-6-{[3-(substituted)phenyl](ethyl)amino}-heteromonocycl-2-yl)amino]arylsulfonic acid, potassium and sodium salts

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:

2010-08-05 till2010-11-05

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:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

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)

Limit test:

no

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

- Name of test material (as cited in study report): FAT 40'849/A TE
- Substance type: colouring dye
- Physical state: blue powder
- Analytical purity: 76.9% of all coloured components
- Lot/batch No.: TZ 5463 / BOP 03-09
- Expiration date of the lot/batch: 2014-06-30
- 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 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: 7 weeks
- Weight at study initiation: Males: 176g to 199g (mean 184g); Females: 144g to 162g (mean 155g)
- 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 (e.g. ad libitum): Pelleted standard Harlan Teklad 2914C rodent maintenance diet was available ad libitum.
- Water (e.g. ad libitum): Community tap-water from Itingen was available ad libitum in water bottles.
- Pre-Randomization Period: 1 d
- Acclimation period: 6 d
- Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.
- Randomization: Randomly allocated to groups by body weight.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22 ± 3 °C
- Humidity (%):30 - 70%). A value for humidity was above these ranges on one occasion, following room cleaning, which was considered not
to have any influence on the study. Therefore, these data are not reported but are retained at Harlan Laboratories Ltd.
- 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:

DOSING SOLUTIONS PREPARATION
- Rate of preparation of dosing solution (frequency): dose formulations were prepared weekly,
FAT 40849/A TE were weighed into a glass beaker on a tared Mettler balance. A small amount of vehicle and 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.
- Stability of Dose Formulations: For at least 8 days, based upon the results of stability analyses performed during Harlan Laboratories study C93547 (non-GLP).
- Storage of Dose Formulations: The dose formulations were prepared weekly and stored at room temperature (20 ± 5 °C).

VEHICLE
- Justification for use and choice of vehicle (if other than water): none (water)
- Concentration in vehicle: 0, 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage): Dose volume 10mL/kg bw
- Purity: bidistilled

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

After experimental start and during week 3, 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. Samples after experimental start of about 2 g of each concentration were taken to confirm stability (4 hour and 8 days at room temperature).
The samples for dose formulation analysis 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.
Additionally, duplicate samples were stored frozen at -20 ± 5 °C.
The analysis was performed by Harlan Laboratories Ltd. using a HPLC method provided by the Sponsor.

The identity of FAT 40849/A TE was confirmed by its retention time which was similar to that measured in the working standards. The test item content in all samples ranged from 101.3% to 107.9% and was therefore within the accepted range of ±20% of the nominal content. In addition, the homogeneous distribution of FAT 40849/A TE in bidistilled water was demonstrated. The application formulations were considered to be stable for 4 hours and 8 days when kept at room temperature.

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:

28d

Frequency of treatment:

daily, 7d/week

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 300 and 1000mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

Group 1: 0 mg/kg/day 5 males and 5 females
Group 2: 100 mg/kg/day 5 males and 5 females
Group 3: 300 mg/kg/day 5 males and 5 females
Group 4: 1000 mg/kg/day 5 males and 5 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:The dose levels were selected based on a previous non-GLP dose range finding toxicity study in Wistar rats, Harlan Laboratories study C93593
- Rationale for animal assignment (if not random): Randomly allocated to groups by body weight.
- Rationale for selecting satellite groups: no satellite groups
- 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.
Histological examinations were performed on organs and tissues from all control and high dose animals, and all gross lesions from all animals. Additionally, from the animals of the low and middle dose groups, the kidneys were examined histopathologically 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 see table 1 were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals were observed for clinical signs once daily during acclimatization as well as twice daily on days 1 – 3 and once daily thereafter during the treatment period.
Weekly Behavioural 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: once during pre-randomization phase and once weekly during the acclimatization and treatment periods,


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: recorded once during the acclimatization period and weekly thereafter. Results for groups
- 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: Body weight gain in %

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


OPHTHALMOSCOPIC EXAMINATION: see table 1


HAEMATOLOGY: Yes
- Time schedule for collection of blood: After 4 Weeks
- Anaesthetic used for blood collection: Yes, 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
- 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.
- Parameters checked in table No.3 were examined.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 4 weeks
- 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.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.
- Animals fasted: Yes , in metabolism cages
- 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 talbe 1
- Battery of functions tested: reflexes / grip strength / locomotor activity

OTHER: Vaginal Smear for Estrus Stage
Vaginal smears were taken during week 3-4 over four days from all females, and the stage of estrus was evaluated, if possible.

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, treatment-related

Description (incidence and severity):

one female died (1000mg/kg bw/d

Mortality:

mortality observed, treatment-related

Description (incidence):

one female died (1000mg/kg bw/d

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

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):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

coloration effect of the test item without toxicological relevance

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

bluish dicoloration of multiple organs, minimal deposits of fine granular brownish pigment in the tubular epithelium of the kidneys in animals of both sexes at 1000 mg/kg bw/day.

Details on results:

CLINICAL SIGNS AND MORTALITY
All animals, with the exception of one female in group 4, survived their scheduled treatment period.

Female no 36 (group 4 at 1000 mg/kg bw/day) spontaneously died on day 7 of treatment period, shortly after oral application. This animal showed dark feces at clinical observations and bluish discoloration of the whole animal and incompletely collapsed lungs at necropsy. Due to single incidence of this death along with acute onset after application, in absence of specific clinical signs or specific macroscopical findings at necropsy and in presence of bluish discoloration in the lungs and trachea as well as a slight interstitial edema of the lungs at histopathology, this mortality was considered to be due to accidental misgavage and not test item-related.


BODY WEIGHT AND WEIGHT GAIN
No changes were noted in absolute body weights and relative body weights (body weight gains) in animals of both sexes at 100, 300 and 1000 mg/kg bw/day during acclimatization and treatment period compared to the respective controls.


FOOD CONSUMPTION
No changes were noted in the mean absolute (g/animal/day) and relative (g/ kg bw/day) food consumption in animals of both sexes at 100, 300 and 1000 mg/kg bw/day during acclimatization and treatment period compared to the respective controls.


OPHTHALMOSCOPIC EXAMINATION
No findings were noted during weekly behavioral observations in animals of group 1 to 4 during acclimatization and treatment (weeks 1-3).


HAEMATOLOGY
No test item-related findings of toxicological relevance were noted in hematology parameters in males and females at any dose level tested.

In animals at 1000 mg/kg bw/day the following findings were noted:
• Elevated methemoglobin in males and females at 1000 mg/kg bw/day with statistical significance of p<0.05. Values stayed marginally above ranges of historical control data. In absence of formation of Heinz bodies, this finding was considered not to indicate changes in methemoglobin levels of any toxicological relevance. It is assumed, that the method of spectroscopic measurement of hemoglobin derivates with the hemoximeter OSM3 interferes with absorption spectra of the test item FAT 40849/A TE being present in plasma samples. Therefore, elevated methemoglobin values were considered to be an artefact and of no toxicological relevance.

The remainder of findings was considered to be not related to the treatment with the test item:
• Decreased absolute large unstained cells (LUC) in males at 100, 300 and 1000 mg/kg bw/day with statistical significance of p<0.05. Values stayed well within ranges of historical control data. Statistical significance was considered to be caused by high values in the control group, only.
• Elevated prothrombin time in females at 300 mg/kg bw/day with statistical significance
of p<0.05. Values stayed well within ranges of historical control data and the finding was not noted in any other dose group or in males. Therefore it was considered to be within ranges of normal biological variance and not related to the treatment with the test item.


CLINICAL CHEMISTRY
No test item-related findings of toxicological relevance were noted in biochemistry parameters in males and females at any dose level tested.

In animals at 100, 300 and 1000 mg/kg bw/day the following findings were noted:
• Decreased total bilirubin in animals of both sexes in a dose-dependent distribution (statistical significance of p<0.05 and p<0.01was noted in males at 100 and 300 mg/kg bw/day and no statistical significance respectively statistical significance of p<0.05 was noted in females at 100 and 300 mg/kg bw/day). Measurement of total bilirubin with a Hitachi 917 analyzer, Roche Diagnostics in animals of both sexes at 1000 mg/kg bw/day was not possible due to a bluish discoloration of the plasma samples. It is assumed, that the method of calorimetric assay of total bilirubin with the Hitachi 917 analyzer, Roche Diagnostics (nominal wavelength of 570 nm for total bilirubin) interferes with absorption spectra of the test item FAT 40849/A TE being present in plasma samples which showed a noticeable bluish color in the high dose group. Therefore, decreased bilirubin values were considered to be an artefact and of no toxicological relevance.

The remainder of findings was considered to be not related to the treatment with the test item:
• Decreased albumin in males at 1000 mg/kg bw/day (with p<0.01). Values were well within ranges of historical control data, not acompanied by changes in hematocrit indicating water imbalances or increases in biochemistry parameters such as urea, ASAT, ALAT indicating changes in liver or kidney function and not noted in any other dose group or females. This finding was therefore considered to be within ranges of normal biological variance.
• Increased creatinine in females at 1000 mg/kg bw/day (with p<0.05). Values were well within ranges of historical control data, not acompanied by changes in biochemistry such as urea or electrolytes indicating changes in kidney function and not noted in any other dose group or males. This finding was therefore considered to be within ranges of normal biological variance.


URINALYSIS
No changes of toxicological relevance were noted in urinalysis parameters at any dose group tested. In animals at 1000 mg/kg bw/day the only finding was light green, grey or deep blue color of the urine which was considered to be due to a dyeing effect of the test item. In absence of any other findings at urinalysis and no kidney-related findings in biochemistry and no histopathological findings indicating degeneration or inflammatory lesions of the kidneys, the finding of bluish discoloration of the urine was considered to be not adverse.


NEUROBEHAVIOUR
Functional Observational Battery (Neurotoxicity Screen)
No findings were noted in the functional observational battery performed at week 4.
Grip Strength
No changes were noted in grip strength values of the fore- and hindlegs in test item treated animals of both sexes at 100, 300 and 1000 mg/kg bw/day compared to the respective controls.
Locomotor Activity
No changes were noted in the locomotor activity in test item treated animals of both sexes at 100, 300 and 1000 mg/kg bw/day compared to the respective controls. A single statistical significance of p<0.05 in males at 100 mg/kg bw/day showing an increase in the locomotor activity at the first timepoint of measurement was considered to be within ranges of normal biological variance.


ORGAN WEIGHTS
No test item-related changes in organ weights were noted at any dose level tested.
The single finding of slightly increased absolute and relative liver weights in mid dose females at 300 mg/kg bw/day with statistical significance of p<0.05 were not distributed dose-dependently, not noted in males and not accompanied by specific microscopic findings and therefore considered to be within ranges of normal biological variance and not related to the treatment with the test item. Additionally, one female of this dose group showed a diaphragmatic hernia which was considered to be congenital and in consequence an increase in individual liver weight leading to an increase of liver weight in summary data of this dose group.


GROSS PATHOLOGY
Macroscopically, no test item related findings were noted in males at 100 mg/kg bw/day.
Bluish discoloration of multiple organs (mucosa of the gastrointestinal tract, kidneys, thymus, mesenteric lymphnodes, testes, prostrate, uterus and vagina) were noted in a dose-dependent distribution in animals of both sexes at 1000 and 300 mg/kg bw/day and in one organ (kidneys) in a single female at 100 mg/kg bw/day.

In animals at 1000 mg/kg bw/day the following test item-related findings were noted:
• bluish discoloration of the mucosa of stomach, duodenum, jejunum, ileum, caecum, colon and rectum in 5/5 males and 4/4 females at scheduled necropsy
• bluish discoloration of the kidneys in 5/5 males and 4/4 females at scheduled necropsy
• bluish discoloration of testes (5/5 males), prostrate (1/5 males),
• bluish discoloration of the thymus in 5/5 males and 1/4 females at scheduled necropsy
• bluish discoloration in the mesenteric lymphnodes in 5/5 males and 4/4 females at scheduled necropsy
• bluish discoloration of uterus and vagina in 3/4 females at scheduled necropsy
• bluish discoloration of the whole animal in 1/1 female at unscheduled necropsy on day 7 (spontaneous decedent no. 36)

In animals at 300 mg/kg bw/day the following test item-related findings were noted:
• bluish discoloration of the stomach in 3/5 males and 1/5 females
• bluish discoloration of the kidneys in 5/5 males and 5/5 females
• bluish discoloration of the testes in 1/5 males In animals at 100 mg/kg bw/day the following test item-related findings were noted:
• bluish discoloration of the kidneys in 1/5 females

The remainder of macroscopic findings noted, such as foci in the lungs, nodule in the liver, reddish discoloration of the pancreas, pelvic dilation in the kidneys, enlarged mandibular lymphnodes, diaphragmatic hernia or watery fluid in the uterus were sporadically distributed between treated and untreated animals. Due to nature, incidence and distribution, these findings were considered to be within ranges of normal background findings which might be seen in rats of this age and strain.

Microscopic Findings
No test item-related microscopic findings were noted in animals at 100 and 300 mg/kg bw/day.
Test item-related findings were minimal deposits of fine granular brownish pigment in the tubular epithelium of the kidneys in animals of both sexes at 1000 mg/kg bw/day.
In animals at 1000 mg/kg bw/day the following test item-related findings were noted:
• Deposits of fine granular brownish pigment in the tubular epithelium of the kidneys in 5/5 males and 4/5 females, grade 1
In absence of tubular degeneration or inflammatory lesions in the kidneys, these pigment deposists were considered to be not adverse.

In the spontaneous decedent female no. 36 at 1000 mg/ kg bw/day the following findings were
noted:
• Autolysis
• Bluish blood serum in tissues of all organs
• Focal bluish coloration in the trachea
• Diffuse bluish coloration with focal bluish granules in the alveoli of the lungs

Macroscopically bluish coloration in the trachea and alveoli of the lungs along with acute onsetof death after application and no specific clinical signs, this spontaneous death was considered to be caused by accidental misgavage and not test item-related.

The remainder of microscopic findings noted, such as fatty change in the liver, extramedullary hemopoiesis in the spleen, inflammatory cell infiltration of the stomach, fatty replacement in the bone marrow and other findings were distributed evenly between treated and untreated animals. Due to nature, incidence and distribution, these findings were considered to be within ranges of normal background findings which might be seen in rats of this age and strain.


HISTOPATHOLOGY: NON-NEOPLASTIC


OTHER FINDINGS
Vaginal Smear for Estrus Stage
The incidence and pattern of the estrus cycles were comparable in test item-treated females and controls.

Thyroid Hormone Analysis
In absence of changes in the absolute and/or relative organ weights of the pituitary and/or thyroid gland (in conjunction with changes in liver weights) and no respective macroscopic or microsopic changes at necropsy, effects on the pituitary-thyroid axis were unlikely and therefore an analysis of thyroid hormones was not performed.

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: the highest dose level tested. a no-observed-effect-level (NOEL) of FAT 40849/A TE could not be established

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

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	143	145-	145-	143-
ST.DEV.	6	7	7	5
MINIMUM	136	138	136	139
MAXIMUM	151	155	153	152
N	5	5	5	5
ACCLIMATIZATION
Day 1      MEAN	154	154-	155-	155-
ST.DEV.	7	3	6	5
MINIMUM	147	151	144	149
MAXIMUM	162	158	160	161
N	5	5	5	5
TREATMENT
Day 1      MEAN	168	165-	171-	171-
ST.DEV.	5	4	6	4
MINIMUM	163	159	165	167
MAXIMUM	176	170	179	177
N	5	5	5	5
Day 8      MEAN	181	180-	190-	190-
ST.DEV.	9	10	3	4
MINIMUM	169	170	185	187
MAXIMUM	190	195	193	196
N	5	5	5	4
Day 15     MEAN	194	195-	205-	200-
ST.DEV.	9	12	5	6
MINIMUM	179	185	199	194
MAXIMUM	203	214	210	208
N	5	5	5	4
Day 22     MEAN	204	203-	220-	217-
ST.DEV.	10	15	5	3
MINIMUM	187	190	213	215
MAXIMUM	214	228	225	221
N	5	5	5	4
Day 28     MEAN	215	215-	228-	231-
ST.DEV.	17	19	12	9
MINIMUM	192	199	214	220
MAXIMUM	236	246	246	241
N	5	5	5	4

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

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	172	166-	169-	168-
ST.DEV.	7	3	7	8
MINIMUM	166	162	160	158
MAXIMUM	184	169	179	178
N	5	5	5	5
ACCLIMATIZATION
Day 1      MEAN	186	180-	188-	183-
ST.DEV.	8	4	7	3
MINIMUM	179	176	178	178
MAXIMUM	199	186	196	187
N	5	5	5	5
TREATMENT
Day 1      MEAN	222	220-	225-	220-
ST.DEV.	8	6	6	5
MINIMUM	215	215	217	215
MAXIMUM	236	229	233	228
N	5	5	5	5
Day 8      MEAN	261	258-	261-	257-
ST.DEV.	8	12	2	8
MINIMUM	254	240	259	245
MAXIMUM	272	271	264	264
N	5	5	5	5
Day 15    MEAN	290	290-	294-	289-
ST.DEV.	9	19	5	14
MINIMUM	277	263	287	270
MAXIMUM	299	310	301	306
N	5	5	5	5
Day 22    MEAN	318	316-	320-	317-
ST.DEV.	11	21	9	15
MINIMUM	301	286	310	298
MAXIMUM	328	338	330	334
N	5	5	5	5
Day 28   MEAN	337	338-	340-	337-
ST.DEV.	12	29	10	18
MINIMUM	316	299	329	317
MAXIMUM	345	367	351	357
N	5	5	5	5

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

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

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, a no-observed-effect-level (NOEL) of FAT 40849/A TE could not be established. The no-observed-adverse-effect-level (NOAEL) was set at 1000 mg/kg bw/day, the highest dose level tested.

Executive summary:

Oral administration of FAT 40849/A TE to Wistar rats of both sexes at doses of 100, 300 and 1000 mg/kg bw/day for a period of 28 days resulted in no test item-related deaths, no adverse clinical signs during daily and weekly (weeks 1-3) observations, no effects on the functional observational battery including grip strength and locomotor activity, no effects on the mean food consumption or body weight development, no effects on the estrous stage of female animals, no adverse effects on hematology, clinical biochemistry or urinalysis parameters, no differences in mean absolute and relative organ weights, and no adverse macroscopical or microscopical findings.

One spontaneous death in a high dose female shortly after oral application was considered to be not test item-related, but caused by misgavage due to single incidence of this death along with acute onset after application, no specific clinical signs nor specific macroscopical findings at necropsy and due to bluish discoloration in the lungs and trachea as well as a slight interstitial edema of the lungs at histopathology.

Test item-related findings were generally restricted to clinical signs of bluish discoloration of the feces in animals of both sexes at 100, 300 and 1000 mg/kg bw/day along with general discoloration in animals at 1000 mg/kg bw/day, green to grey or blue discoloration of the urine in animals at 1000 mg/kg bw/day, macroscopically bluish discoloration of multiple organs such as mucosa of the gastrointestinal tract, kidneys, thymus, mesenteric lymphnodes, testes, prostrate, uterus and vagina with a dose-dependent distribution in animals at 100, 300 and 1000 mg/kg bw/day and microscopic findings of minimal deposits of fine granular brownish pigment in the tubular epithelium of the kidneys (in absence of any tubular degeneration or inflammatory lesions) in animals at 1000 mg/kg bw/day. These findings were considered to be a non-adverse dyeing effect of the test item.

No test item-related findings of toxicological relevance were noted in hematology parameters in males and females at any dose level tested. Nevertheless, in animals at 1000 mg/kg bw/day elevated methemoglobin was noted. Values stayed marginally above ranges of historical control data. In absence of formation of Heinz bodies, this finding was considered not to indicate changes in methemoglobin levels of any toxicological relevance. It is assumed, that the method of spectroscopic measurement of hemoglobin derivates with the hemoximeter OSM3 interferes with absorption spectra of the test item FAT 40849/A TE being present in plasma samples. Therefore, elevated methemoglobin values were considered to be an artefact and of no toxicological relevance.

No test item-related findings of toxicological relevance were noted in biochemistry parameters in males and females at any dose level tested. In animals of both sexes at 100, 300 mg/kg bw/day decreased total bilirubin was noted in a dose-dependent distribution. Measurement of total bilirubin with a Hitachi 917 analyzer, Roche Diagnostics in animals of both sexes at 1000 mg/kg bw/day was not possible, due to a bluish discoloration of the plasma samples. It is assumed, that the method of calorimetric assay of total bilirubin with the Hitachi 917 analyzer, Roche Diagnostics (nominal wavelength of 570 nm for total bilirubin) interferes with absorption spectra of the test item FAT 40849/A TE being present in plasma samples which showed a noticeable bluish colour in the high dose group. Therefore, decreased bilirubin values were considered to be an artefact and of no toxicological relevance.