Salts of 9-[2-(ethoxycarbonyl)phenyl]-3,6-bis(ethylamino)-2,7-dimethylxanthenium chloride and 4-{[1-hydroxy-6-({[5-hydroxy-6-(phenyldiazo)-7-sulfonaphthalen-2-yl]carbamoyl}amino)-3-sulfonaphthalen-2-yl]diazo}benzoic acid (2:1)

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2008-09-11 to 2008-10-16

Reliability:

1 (reliable without restriction)

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

Preparation of the Test Item
The test item was formulated in vehicle at concentrations of 5, 35, and 100 mg/mL in the Central Dispensary of LAB Research Ltd. Formulations were prepared as appropriate to allow their use within 72 hr pending refrigerated storage, according to stability assessment results.
Analytical control (concentration, homogeneity) of formulations was performed in the Analytical Laboratory of LAB Research Ltd. on Days 0 and 27.
The measured concentrations ranged from 90 to 96% (Day 0), and 100 to 105 % (Day 27) of nominal concentrations, results considered suitable for the study purposes. Assessment of test item stability in this vehicle, under the conditions employed on the study (LAB study code 08/612-316AN) indicated up to 4-hour stability at room temperature and up to 72-hour stability when stored refrigerated, at concentrations from approximately 1 to 100 mg/mL in 1% methylcellulose formulations, with a recovery within the acceptable range of 100 ± 10% (105-93 %).

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

rat, Wistar Crl:(WI)BR (SPF-Quality)
males and females, nulliparous and non-pregnant
young adult rats, 7 - 8 weeks old

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 1% aqueous methylcellulose

Details on oral exposure:

The test item was administered by oral gavage for 28 consecutive days. Control animals were treated concurrently and received the vehicle only. A
constant treatment volume of 10 mL/kg body weight was applied in all groups. The actual treatment volume was calculated according to the most
recent body weight. Animals were not treated on the day of necropsy. Duration of treatment: 28 days.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Assessment of test item stability in this vehicle, in the conditions employed on the study indicated up to 4-hour stability at room temperature and up to 72-hour stability when stored refrigerated, at concentrations from approximately 1 to 100 mg/mL in 1% methylcellulose formulations, with a recovery within the acceptable range of 100 ± 10% (actual range 105-93 % of nominal). Analytical control of dosing solutions was conducted on Days 0 and 27. The measured concentrations ranged from 90 to 96% (Day 0), and from 100 to 105 % (Day 27) of nominal concentrations, results considered suitable for the study purposes.

Duration of treatment / exposure:

28 consecutive days by oral gavage
treatment volume of 10 mL/kg body weight

Frequency of treatment:

once daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 50, 350, 1000 mg/kg bw/day (m/f)
Basis:
other: nominal via gavage

No. of animals per sex per dose:

5 animals per group and sex

Control animals:

yes, concurrent vehicle

Details on study design:

Justification of the dose selection:
Dose level selection based on results of an acute oral toxicity study and a 14-day oral dose range finding toxicity study in CRL:(WI)BR rats. In the acute study, F 213 Red caused mortality in 1/3 female rats of each of the 2 groups administered the limit dose 2000 mg/kg bw. In the 14-day preliminary study performed at 62.5, 250 and 1000 mg/kg bw/day, no mortality occurred; red discoloration of the faeces was noted in all animals as of Day 2/3, and pink urine in male animals, as of Day 9 of the treatment, at all dose levels. There were no adverse effects or other systemic changes that could be ascribed to F 213 Red at up to 1000 mg/kg bw, daily for 14 days.

Randomisation / Animals Assignment:
All animals were sorted according to body weight by computer and divided into weight ranges. There was an equal number of animals from each weight group in each of the experimental groups assigned by randomisation. The grouping was controlled by SPSS/PC software, according to the actual body weight verifying the homogeneity and variability among the groups and cages.

Positive control:

no data

Examinations

Observations and examinations performed and frequency:

Observations:
Clinical observations for signs of ill health or reaction to treatment were made once daily. Detailed clinical examination was made before the first treatment, then once weekly during the study. A functional observation battery was conducted during the last week of the treatment period (Day 26).
Body weight and food consumption were measured weekly. Clinical pathology examinations and gross necropsy were conducted at the end of the treatment period (Day 28). The absolute and relative organ weights of adrenals, brain, epididymides, heart, liver, kidney, spleen, testes, and thymus were determined. A full histopathological examination was performed on the selected list of preserved organs and tissues of the animals of Groups 1 (Control) and 4 (High dose), and on abnormal tissues from Low and Mid dose groups.
Analytical control (concentration, homogeneity) of formulations was performed in the Analytical Laboratory of LAB Research Ltd. on Days 0 and 27.

Sacrifice and pathology:

At the end of the study all of the animals were sacrificed and a detailed gross necropsy was performed.

Other examinations:

Following completion of necropsy the organs investigated were fixed in 10% buffered formaldehyde solution. The eyes with the optic nerve and the testes were preserved in modified Davidson fixative. Full histology was performed on the preserved organs or tissues of the animals of the Group 1 (control) and Group 4 and gross lesions of groups 2 and 3. Organs or representative samples were embedded into paraffin. The sections were stained with haematoxylin and eosin, and examined with light microscope. Histological evaluation was carried out according to the 9/2001. (III.30.) EüM-FVM joint decree of the Minister of Health and the Minister of Agriculture and Regional Development which corresponds to the OECD GLP ENV/MC/CHEM (98) 17).

Statistics:

Statistical analysis was performed on the following data sets using SPSS PC + software:
- body weight
- haematology
- clinical chemistry
- organ weight
The heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the obtained result was positive, Duncan's Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was assessed by a Kolmogorov-Smirnov test. In case of a not normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was applied. If there was a positive result, the intergroup comparisons were performed using a Mann-Whitney U-test. Frequency of clinical observations, mean daily food consumption by groups and sex, frequency of pathological and histopathological findings by sex and dose were calculated.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

not specified

Ophthalmological findings:

not specified

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:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

Body Weight
No adverse effects were noted on the mean body weight and body weight
gain values in the treated groups compared to control animals following daily
administration of F 213 Red at dose levels of up to and including
1000 mg/kg bw/day.

Food Consumption
There were no test item related differences in the mean daily food intake in
any test item treated groups (50, 350, or 1000 mg/kg bw/day, male or female)
when compared to the control.

Haematology
No toxicologically relevant differences between the control and
test item-treated groups, or any adverse effects of F 213 Red on haematology
parameters in the male and female animals.
Statistically significant variations were noted only on occasion compared to control
animals.
Evaluation of the mean and individual results in correlation with the control
and historical haematology data did not reveal any test-item related cause of
these variations. The differences observed between the control and treated
groups were considered to be incidental or individual findings, not related to treatment and generally remained within the historical control ranges with no toxicological significance.

Clinical Chemistry
No relevant changes in the examined clinical chemistry parameters that
could be attributed to F 213 Red administration.
Statistically significant changes were noted with regard to some parameters in the
treated animals compared to control. Although occasionally these changes were statistically significant, no dose related response was observed, and/or there was no consistent reaction in the male and female groups. These changes were neither considered toxicologically significant, nor indicated a test item related etiology.

Necropsy
Test article-related findings were observed in the digestive contains, stomach,
small intestine, colon, caecum, rectum, salivary gland and tail at necropsy.
Red coloured digestive content was observed in 6/10 Low Dose, 5/10 Mid
dose and 9/10 High Dose animals.
Diffuse mucosal red, or pink discoloration of the stomach, small intestine
and/or colon, caecum, rectum was seen in 8/10 Low dose, 8/10 Mid Dose and
10/10 High Dose rats.
Diffuse pink discoloration of the salivary gland was noted in 8/10 High Dose
animals. In addition, pink coloured tail was recorded in 2 High Dose males
(401 and 402).
Focal/multifocal/diffuse pale areas or mottling of the lungs seen cross all
groups were regarded as agonal. All other changes were considered to be
incidental.

In summary, daily oral gavage of F 213 Red for 28-days to the Wistar rat at
dose levels of 50, 350 and 1000 mg/kg was associated with red coloured
digestive content, diffuse red discoloration in the stomach, small intestine,
colon, caecum and rectum when rats were examined macroscopically.
Additionally, diffuse pink discoloration of salivary gland was noted at a dose
level of 1000 mg/kg.

Organ Weight
No toxicologically significant changes in organ weight values
noted after F 213 Red administration daily for 28 days, at up to and including
1000 mg/kg bw/day.
Statistically significant differences in the thymus and/or epididymes weights
were observed in male animals at all dose levels tested when compared
to the control values. In females there no statistically significant organ
weight differences were noted. Thymus weights (absolute and/or relative to the body and
brain weights) were below the control value.
As these changes were within the historical range and not correlated with pathological findings, they were considered incidental and not related to treatment.
All other examined organ weights (absolute and relative to the body and brain
weights) were similar in the control and test item treated groups.

Histopathology
In the organs of animals subjected to histopathological examination there
were no lesions that could be ascribed to F 213 Red administration.
Minor focal alveolar emphysema and focal atelectasis were noted
sporadically in the lungs, correlated with the macroscopically visible pale
areas and dark focuses. Dilatation of the uterine horns was observed in two
females (1000 mg/kg); in addition, peribiliary fibrosis in the
liver (Control) occurred. These findings were considered
incidental.
No morphological evidence of acute or subacute pathological changes
(degeneration, inflammation, ulcer etc.) of gastrointestinal tract was observed,
in spite of macroscopically visible red discoloration of the mucosa. The
cardiovascular, immune, hematopoietic, skeletal and muscular systems, the
male and female reproductive system or the central or peripheral nervous
system did not show any microscopic lesions. The structure and the cell
morphology of the endocrine glands (pituitary, thyroid, parathyroid and
adrenal glands, and Langerhans- islets) was the same in the control and
treated animals.

In summary, F 213 Red administered by oral gavage daily for 28 days to male
and female rats, at dose levels up to and including 1000 mg/kg bw/day, did
not cause any toxic or other test item related lesions detectable by histological
examination of investigated organs in the experimental animals.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: No adverse toxicity effects, but colouration of faeces and urine.

2

Dose descriptor:

NOEL

Effect level:

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

Sex:

male/female

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

not applicable

Applicant's summary and conclusion

Conclusions:

F 213 Red administered daily by oral gavage for 28 days in Wistar rats did not lead to any toxicologically significant adverse effects at dose levels of 50, 350 or 1000 mg/kg bw/day.
The faeces of all animals, and the urine of males at 350 mg/kg bw/day, or of all animals at 1000 mg/kg bw/day were coloured red. At necropsy the gastrointestinal tract was coloured red, as were the intestinal contents in all treatment groups. Additionally, diffuse pink discoloration of salivary gland was noted at a dose level of 1000 mg/kg. There were no adverse findings at macroscopic or microscopic examinations.

Executive summary:

F 213 Red caused no mortality at up to and including 1000 mg/kg bw/day in CRL:(WI)BR rats.

No toxicologically significant systemic clinical changes were noted following administration of F 213 Red by oral gavage, daily for 28 days. Day 0 was regarded as the first day of treatment. Red faeces were observed in animals’ cages for both sexes, in the bedding, at all the dose levels tested as of Day 1 (females at 50 mg/kg bw/day, and both males and females at 350 and 1000 mg/kg bw/day) or Day 2 (males at 50 mg/kg bw/day). At 1000 mg/kg bw/day, both the male and female animals eliminated apparently increased volumes of pink/reddish urine in the cage bedding for 19 out of 28 treatment days, commencing on Day 9. At 350 mg/kg bw/day, this

clinical sign was noted in the male animals only as commencing on Day 10, for 18 out of 28 days.

These changes were ascribed to elimination of F 213 Red or its metabolites through faeces and/or urine; moreover, in the absence of any clinical pathology alterations, they were not considered to be an adverse effect.

The behaviour and the general condition of the test animals were normal during the study. There was no treatment-related effect on motor activity or in the functional observation battery tests across groups of treated male or female animals and no findings indicative for neurotoxicity were observed.

There were no toxicologically significant changes in body weight, body weight gain or animal feed intake between the control and test item treated groups.

Minor variations were noted in the clinical pathology parameters (haematology, coagulation and clinical chemistry). Although occasionally these changes were statistically significant (potentially due to high values of certain parameters noted in the control animals compared to the historical range, i.e. for MPV, APPT, or PT values), no dose related response was observed, the results were within the historical range, and/or there was no consistent reaction in the male and female groups. These changes were neither considered toxicologically significant, nor indicated a test item related etiology.

At necropsy, the gastrointestinal tract was coloured red, as were the intestinal contents in all treatment groups. Additionally, diffuse pink discoloration of salivary gland was noted at a dose level of 1000 mg/kg. There were no adverse findings at macroscopic or microscopic examination. There were no toxicologically significant changes in organ weight values, although differences, on occasion attaining statistical significance, were observed in the thymus and/or epididymes weight at all the dose levels tested.

As these changes were within the historical range, had low magnitude and were not correlated with pathological findings, they were considered incidental and not related to treatment.

F 213 Red administered daily by oral gavage for 28 days in Wistar rats did not lead to any toxicologically significant adverse effects at dose levels of 50, 350 or 1000 mg/kg bw/day. The faeces of all animals, and the urine of males at 350 mg/kg bw/day, or

of all animals at 1000 mg/kg bw/day were coloured red. At necropsy the gastrointestinal tract was coloured red, as were the intestinal contents in all treatment groups. Additionally, diffuse pink discoloration of salivary gland was noted at a dose level of 1000 mg/kg. There were no adverse findings at macroscopic or microscopic examination. In conclusion, in the conditions of this study, the no observed adverse effect level (NOAEL) for F 213 Red is considered 1000 mg/kg bw/day, and the no observed effect level (NOEL), 50 mg/kg bw/day.