4-[[5-(anilino)carbonyl-2-methoxyphenyl]azo]-3-hydroxynaphthalene-2-carboxamide

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:

Experimental start date 30 January 2002. Experimental completion date: 19 December 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3050, Repeated Dose 28-Day Oral Toxicity Study in Rodents, July 2000.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-[[5-(anilino)carbonyl-2-methoxyphenyl]azo]-3-hydroxynaphthalene-2-carboxamide

EC Number:

260-152-0

EC Name:

4-[[5-(anilino)carbonyl-2-methoxyphenyl]azo]-3-hydroxynaphthalene-2-carboxamide

Cas Number:

56396-10-2

Molecular formula:

C25H20N4O4

IUPAC Name:

4-[[5-(anilino)carbonyl-2-methoxyphenyl]azo]-3-hydroxynaphthalene-2-carboxamide

Test material form:

solid: particulate/powder

Remarks:

(magenta)

Details on test material:

For all studies conducted on substance, where identifier was ST463, purity was stated as >98 - 99.9% (depending on batch)

Specific details on test material used for the study:

Identity: ST463
Chemical name: 4- [[5-(anilino)Carbonyl- 2-mehoxyphenyl]azo]-3-
hydroxynaphthttene-2-carboxamde
Appearance: Magenta powder
Storage conditions: Ambient temperature
Lot number: 6558
Expiry: october 2002
Purity: >99.9 %
Samples received: 1st August 2000 and 15 October 2001

Test animals

Species:

rat

Strain:

other: Crl:CD (SD)IGS BR

Details on species / strain selection:

The abino rat was chosen as the test species as it has been shown to be a suitable model for his type of study and is the species recommended in the test guidelines.The strain Crl :CD(SD)IGS BR of rat used was chosen on he account of the availability of background data.

Sex:

male/female

Details on test animals or test system and environmental conditions:

A total of 46 Crl:CD°(SD)IGS BR rats(23 males and 23 females), 35± 2 days old was received from Charles River (UK) Limited, Manston Road, Margate, Kent, England. selected rats were approximately 6 weeks old when treatment commenced and bodyweight ranges of 183.1 - 208.0 g (males) and 135.4 - 152.1 g (females) were recorded at the start of treatment.

on arrival 40 animals were randomly allocated to the four treatment groups, such that each group contained 5 males and 5 female rats. Each cage was made of stainless steel with stainless steel grid floors. Cages and undercage trays holding absorbent paper were changed at appropriate intervals. At the start of treatment individual bodyweights were within ±20 % of the group mean for each sex.

Animal room temperature and relative humidity were set and generally maintained at 19 - 23°C and 40- 70% respectively during the study. However on occasion a slight deviation outside this range occured resulting in an overall range of 52 - 88% for humidity. These parameters were monitored using a 7 day chart recorder. Air exchange was maintained such that a minimum of 15 air changes per hour occured and lightning was controlled to provide 12 hours continuous artifical light in each 24-hour period.

All rats had free access to tap water via polycarbonate bottles with sipper tubes and to pelleted SDS Rat and Mouse No. 1 modified Maintenance Diet, except as noted under laboratory investigations.

An acclimatisation period of 6 days allowed between arrival/ allocation and the start of treatment.

The remaining spare rats not allocated to study groups were retained during this period then discarded from the study on day 29 without further investigation.

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The rats were dosed orally as this is a possible route of human exposure to the test substance, which may be ingested accidentally.

Vehicle:

other: 1% aqueous methyl celluloe + 0.5 % Tween 80.

Details on oral exposure:

Preparation of formulations
The test substance,ST463,was administered as a suspension in 1 % aqueous methyl Cellulose + 0.5% Tween 80, A series of suspensions were prepared by direct diludon of the test substance into the vehicle 1 % aqueous mehyl cellulose + 0.5%Tween 80.The test substance was used as supplied and administered in the vehicle(in 1 % aqueous methyl cellulose + 0.5%Tween 80) at concentrations of 1.5,15 and 100 mg/ml.
All suspensions were prepared daily on the day before dosing. Control animals received the vehicle alone at the same dosage volume.

Administration of formulations.
The test substance, ST463,was administered as a suspension in 1 % aqucous mettyl cellulose + 0.5 % Tween 80. Control animals (Group l) received the vehicle alone. The anlmals were dosed at
approximately the same time each day, where possible, using a suitably graduated synnge and a rubber catheter inserted via the mouth into the stomach.The dosage volume administered
to each animal was calculated according to he most recent recorded bodywelght,with individual dose volumes adjusted to the nearest 0.1 ml. A constant dosage volume of 10 ml/kg bodyweight was used.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

FORMULATION SAMPLING AND ANALYSIS
Before commencement of treatment, the suitability of the mixing procedures was determined and specimen formulations at concentrations of 1 and 100 mg/ml were analysed to assess the homogeneity and stability of the test formulation. In addition,samples of formulations prepared for use on Day 1
and Day 9 were analysed to asess achieved concentration.

The concentration of ST463 in the final solution was quantiied by high performance liquid chromatography using ultra-violet detection with reference to dupticate extracted calibration standards prepared at a nominal concentration of 40 μg/ml.

Conclusion:
The mean concentration of ST463 in test formulation analysed during the study were within -19.5 % of the nominal concentrations confirming the accuracy of formulation.

The homogeneity and stability of ST463 in the 1% aqueous methyl cellulose + 0.5% Tween 80 formulations were confirmed at nominal concentration of 1 mg/ml and 100 mg/ml during storage at ambient temperature for 2 days and refrigeration for 2 days. The storage period represented the maximum time from preparation to completion of use.

The analytical procedure was validated for ST463 in aqueous 0.5 % Tween 80 / 1% MC with respect to the specificity of the chromatographic analysis, linearity of detector response, precision of injection, limit of detection, method of accuracy and precision.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control (group 1)

Doses / concentrations 2

Dose / conc.:

15 mg/kg bw/day (nominal)

Remarks:

group 2

Doses / concentrations 3

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

group 3

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

group 4

No. of animals per sex per dose:

5 males and 5 females at 0 mg/kg
5 males and 5 females at 15 mg/kg
5 males and 5 females at 150 mg/kg
5 males and 5 females at 1000 mg/kg

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The high dosage was selected on the basis of a seven day preliminary oral toxicity study. In that study, rats were dosed at levels of 0, 250, 500 or 1000 mg/kg/day. Treatment was well tolerated at 1000 mg/kg/day for seven consecutive days, thus, a dose regimen of 0, 15, 150 and 1000 mg/kg/day was proposed for the main study.

Positive control:

None.

Examinations

Observations and examinations performed and frequency:

The following observations and measurements were made during the course of the study:

Clinical signs and mortality:
All animals were observed at least once each day from arrival to termination. During the treatment period all anilnals were observed at intervals after dosing each day to monitor any signs of ill health, behavioural changes or reacion to treatment. Rccords of these post dose checks were made daily throughout out the treatment peiod.

A detailed physical examinaion was performed weekly during pre-treatment, weeks 1, 2, 3 and 4 at approximately the same time of day on each occasion. after removal from the home cage, animals were assessed for physical condition and behaviour during handling and after being placed in a standard arena. Any deviations from normal were recorded.

In addition all animals were checked early in each working day and again in the late afternoon to look for dead ormoribund animals.

Neurobehavioural screening:
During Week 4 of treatment, sensory reactivity, grip strength and motor activity assessments were perfomed. Animals were not all tested in one day, but the numbers of animals and the time of testing were balanced across the groups. These procedures were performed prior to any laboratory invesigations and before daily dosing.

Each animal was subjected to the procedures detailed below, on the specified occasions, by an observer who was unaware of the treatment group to which each animal belonged. Before the start of each set of observations, cage labels showing the treatment group were replaced by lables stating only the study, animal and cage number.

Sensory reactivity:
Approach response:
A blunt probe was brought towards the animal’s head until it was close to the animal’s nose (but not touching the vibrissae). The animal’s reaction was recorded as:
1: No reaction or ignores probe
2: Normal awareness and reaction e.g. approaches and/or sniffs probe
3: Abnormally fearful or aggressive reaction

Touch response:
The animal’ s flank was stroked gently with a blunt probe and the reaction recorded as:
1: No reaction or ignores probe
2 : Normal awareness and reaction e.g . turns towards or moves away
3 : Abnormally fearful or aggressive reaction

Auditory startle response
The animal’s response to a sudden loud noise was assessed and scored as:
1 : No response
2: Weak response e.g. ear twitch only
3: Normal response e.g. obvious flinch or startle
4: Exaggerated response e.g. all feet off floor

Tail pinch response:
The animal’s tail was pinched sharply with forceps approximately one third from the tip and the response graded as:
1: No response
2: Weak response e.g. turns around slowly or weak vocalisation without moving away
3: Normal response e.g. jumps forward or turns around sharply, usually with vocalisation
4: Exaggerated response e.g. excessive vocalisation, body movement or aggression

Grip strength:
Forelimb and hindlimb grip strength was measured using strain gauges. Two trials were performed.

Motor activity:
Motor activity was recorded using a Coulboum Infra-Red Activity Monitoring System.
This system uses infra-red detectors to monitor activity. The following categories of activity are recorded: the time spent in locomotor activity, non-locomotor activity and in no movement. The
number of occurrences (events) of each category is also recorded . For reporting purposes, only the time spent in locomotor activity is presented routinely.
For testing, designated animals were placed singly into observation cages. The test session for each animal was 1 hour , with data being collected every 2 minutes.

Bodyweight:
All rats were weighed when allocated to treatment groups on arrival (Day - 6), prior to dosing on Day 0 and on Days 7, 14, 21 and 27 (prior to overnight starvation for clinical pathology). In addition, the bodyweights of all animals were recorded prior to necropsy (Day 28).

Food consumption:
The quantity of food consumed by each cage of rats was measured on a weekly basis (from week 1).
Food intake per rat (g/rat/week ) was calculated using the total amount of food given to and left by each cage in each group and the number of rats in each cage .

Food conversion efficiency:
Food efficiency was calculated over the treatment period Weeks 1 to 4 from bodyweight and food consumption data as the bodyweight gain per unit food consumption expressed as a percentage . The following formula was used:
Food efficiency +(bodyweight gain (g)/ Food consumed (g)) x 100

The food consumed was calculated as indicated in the Food consumption section. The bodyweight gain was calculated from the gain of each animal and used the mean gain in the formula.

Water consumption:
Daily monitoring by visual appraisal was maintained throughout the treatment period. No formal measurements were made.

LABORATORY INVESTIGATIONS
Samples of whole blood were withdrawn from the retro-orbital sinus of all rats under light anaesthesia, using isoflurane, on Day 29. Food was removed overnight from animals prior to blood sampling. The blood samples were collected and divided into tubes as follows:
- 0.5 ml into EDTA anticoagulant for haematological investigations
- 0.5 ml into citrate anticoagulant for coagulation tests
- 0.7 ml into lithium heparin anticoagulant for blood chemistry investigations

Haematology:
The following estimations were performed:
Haematocrit (Hct) L/L
Haemoglobin concentration (Hb) g/dL
Erythrocyte count (RBC ) X 10E12/L
Mean cell haemoglobin (MCH) pg
Mean cell haemoglobin concentration (MCHC) g/dL
Mean cell volume (MCV) fL
Total leucocyte count (WBC) X 10E9/L
Differential leucocyte counts:
Neutrophils (N) X 10E9 /L
Lymphocytes (L) X 10E9 /L
Eosinophils (E) X 10E9 /L
Basophils (B) X 10E9 /L
Monocytes (M) X 10E9 /L
Large unstained cells (LUC)
Cell morphology: The most common morphological changes
(anisocytosis [Aniso-cytosis] , micro/ macrocytosis [Micro-cytosis/Macro-cytosis],
hypo/hyperchromasia [Hypo-chromasia/Hyper-chromasia]), were recorded
as follows:
no abnormalities detected
Platelet count (Pit) X 10E9 /L
The following were performed using the appropriate methodology
as described below:
Prothrombin time (PT) - Method of Quick, A.J.
(The haemorrhagic disease and the physiology of Haemostasis, 1942), using
an ACL 3000 Plus analyser and IL PT-fibrinogen reagent. (sec)
Activated Partial Thromboplastin Time (APTT) - Method of Proctor,
R.R. & Rapaport, S.I (1961), using an ACL 3000 plus analyser and
IL APTT reagent. (sec)

Blood chemistry :
The following parameters were analysed:
Alanine aminotransferase (ALT) U/L
Aspartate aminotransferase (AST) U/L
Urea (Urea) mmol/L
Creatinine (Creat) pmol/L
Glucose (Glue) - Hexokinase mediated assay mmol/L
Cholesterol-Total (Choi) mmol/L
Sodium (Na) mmol/L
Potassium (K) mmol/L
Total protein (Total Prot) g/L
Albumin by chemical assay (Alb) g/L
A/G ratio (Albumin/globulin ratio) Calculated from Total protein and Albumin concentrations

Sacrifice and pathology:

Necropsy:
On completion of the 4-week treatment period (Day 29), all animals were humanely killed by carbon dioxide asphyxiation and subjected to the following detailed necropsy procedure:

All superficial tissues were examined visually and by palpation and the cranial roof removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral mid-line incision and skin reflection, all subcutaneous tissues were examined. The condition of the thoracic viscera was noted, with due attention to the thymus, lymph nodes and heart.

The abdominal viscera were examined before and after removal; the urinary bladder was examined externally and by palpation. The gastrointestinal tract was examined as a whole and
the stomach and caecum were incised and examined. The lungs were removed and all pleural surfaces examined under suitable illumination. The liver was sectioned at intervals of a few
millimetres; the kidneys were incised and examined. Any abnormalities in the appearance and size of the gonads, adrenals, uterus, intra-abdominal lymph nodes and accessory reproductive
organs were recorded.

Organ weight:
The following organs from each animal were dissected free of fat and weighed:
adrenals
brain
epididymides
heart
kidneys
liver
spleen
testes
thymus
Bilateral organs were weighed together.

Preservation of tissues:
Samples of all the tissues, listed on the following page, from all animals were preserved in 10% Neutral Buffered Formalin (except testes/epididymides which were fixed in Bourn’s solution and then
transferred to 70% IMS (Industrial Methylated Spirits)).

In addition, samples of any macroscopically abnormal tissues were routinely preserved, along with samples of adjacent tissue where appropriate.
adrenals
brain (cerebellum, cerebrum and midbrain sections)
caecum
colon
duodenum
epididymides
femur (with joint)
head#
heart
ileum (including peyer’s patches)
jejunum
kidneys
spinal cord (transverse and longitudinal sections at the cervical, thoracic and lumbar levels)
liver (section from all main lobes)
lungs (including bronchi)
lymph nodes (mandibular and mesenteric)
oesophagus#
spleen
sternum#
stomach
testes
thymus
thyroid (with parathyroid)
trachea
urinary bladder
uterus (with cervix)
other macroscopically abnormal tissue
ovaries
pancreas#
prostate
rectum
sciatic nerves (only one examined)
seminal vesicles
# Tissues not examined.
This extensive list of preserved tissues is intended to satisfy any possible future requirements for
further examination of tissues.

Histopathological examination:
Tissues required for microscopic examination in this study are indicated in the previous list. For testes, tissues were embedded in paraffin wax and sections were stained using a standard PAS
(Periodic Acid Schiff ) method. Other tissues were embedded in paraffin wax and sections cut at 4 - 5 micrometres were stained with haematoxylin and eosin .

For bilateral organs, sections of both the left and right organs were examined. Single sections were prepared from each of the remaining tissues required for microscopic examination.

Microscopic examination of prepared slides (from tissues indicated under Preservation of Tissues) was carried out for all main group animals of Groups 1 and 4. Abnormalities were processed and
examined for all animals on the study.

Statistics:

Yes - all statistical analyses were carried out separately for males and females.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical findings observed during the study that were considered to be indicative of toxicity, however in animals dosed at 1000 mg/kg/day red staining of the dorsal body surface was seen in two males, (for one animal sign was seen on Day 6 only, the other male showed the sign from Day 6 to termination). Red staining of the tail was seen in one female (Day 27 to termination).

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Overall mean bodyweight gains for treated groups was comparable or superior to controls. Thus there was no adverse effect of treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no effect of treatment on food consumption. Food intake for treated groups was comparable with controls

Food efficiency:

no effects observed

Description (incidence and severity):

All treated groups generally showed similar efficiencies of food utilisation, in comparison with controls.

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Females receiving 1000 mg/kg/day showed statistically significantly lower mean APTT values compared with controls. Mean PT values for these females and mean PT and APTT values for treated males were comparable with controls.

There were no other differences from controls which were considered to be possibly attributable to treatment.

Males at 1000 mg/kg/day showed a statistically significant higher mean MCHC value, whilst females at this level showed a lower mean MCHC value, in comparison with controls. However Hb or Hct values for these groups were comparable with controls and the differences from controls were opposite between the sexes. Females at 1000 mg/kg/day also showed a statistically higher mean neutrophil value compared with controls. The high mean value was mainly attributable to a single animal (No.26) which showed a higher individual value than the other animals in the group and hence unduly affected the group mean .

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Females receiving 1000 mg/kg/day showed a statistically significantly lower potassium value compared with controls. Potassium values for individual treated males were generally comparable with controls.

There were no other differences from controls which were considered to be possibly attributable to treatment.

All treated groups of males showed a statistically significant higher mean sodium values and males receiving 150 or 1000 mg/kg/day showed higher mean albumen values compared with controls. However there was no dosage-relationships and the magnitude of the differences from controls was marginal and similar differences from controls were not noted for treated females.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

An assessment of the detailed physical examination and arena observations carried out during the treatment period did not reveal any clinical signs or changes in behaviour there were considered indicative of neurotoxicity. Motor activity was similar between controls and all treated groups of both sexes.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no differences from controls that were considered to be attributable to treatment.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Pink contents of all or parts of the gastrointestinal tract were seen in all rats treated with 150 or 1000 mg/kg/day, compared with 0/5 male and female control rats. Pink staining on the tail was observed in 5/5 female rats treated with 1000 mg/kg/day compared with 0/5 female control rats.

The incidence and distribution of all other findings were considered to fall within the expected background range of macroscopic findings. These findings were not considered to be of toxicological importance.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Treatment-related findings:
No adverse treatment related changes were detected.

Other findings:
An intense red staining material was seen in the large intestine of some animals receiving 1000 mg/kg/day. This was considered to be due to the red colour of the test substance and coincided with the gross finding of pink contents from the gastrointestinal tract of animals in this treatment group.

Incidental findings:
Only minor changes were reported which were of a type and severity commonly seen in CD rats of this age at this laboratory and considered to be incidental and of no toxicological importance.

Histopathological findings: neoplastic:

not examined

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

haematology

other: In the absence of any clear evidence of toxicity in this study 1000 mg/kg/day can be classed as the No Observed Adverse Effect Level (NOAEL).

2

Key result

Dose descriptor:

NOEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Treatment with ST463 at either 15 mg/kg/day or 150 mg/kg/day for 28 -days was well tolerated and did not result in any treatment related findings.

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

In conclusion, 150 mg/kg/day represents the highest NOEL and 1000 mg/kg/day was the NOAEL on this study.

Executive summary:

This study was designed to assess the systemic toxicity of ST463 to the rat when repeatedly administered orally for a period of 28 consecutive days repeated once daily oral administration.

ST463 was administered by oral gavage, once daily, to three groups of five male and five female rats for twenty-eight consecutive days at dosage levels of 15, 150 or 1000 mg/kg/day. The test substance was used as supplied and administered as a suspension in 1% aqueous methyl cellulose + 0.5% Tween

80 at concentrations of 1.5, 15 and 100 mg/ml . In addition, five male and five female rats were held as a concurrent control receiving the vehicle (1% aqueous methyl cellulose + 0.5% Tween 80) alone at the same dose volume (10 ml/kg/ day) .

Bodyweights, food consumption and clinical observations were recorded during the study for all animals. Sensory reactivity, grip strength and motor activity were assessed in Week 4 of the study. Blood samples for clinical investigations were taken prior to termination and all animals were killed and examined macroscopically on Day 29. At the scheduled necropsy selected organ weights were recorded and a wide range of tissues were preserved. Histopathological examination of specified

tissues was then undertaken.

The following comments in relation to principal findings during the study are made in summary:

There were no unscheduled deaths and no clinical signs indicative of toxicity (including neurotoxicity) throughout the study.

Overall mean bodyweight gains for treated groups were comparable with controls for females at 1000 mg/kg/day.

Food intake for treated groups was generally comparable with controls.

All treated groups generally showed similar efficiencies of food utilisation, in comparison with controls.

Females receiving 1000 mg/kg/day showed statistically significantly lower mean APTT values compared with controls. Mean PT values for these females and mean PT and APTT values for treated males were comparable with controls. There were no other differences from controls which were considered to be possibly attributable to treatment.

There were no differences in organ weights compared with control that were considered to be attributable to treatment.

Macroscopic examination revealed pink contents of all or parts of the gastrointestinal tract in all rats treated with 1000 mg/kg/day, compared with 0/5 male and female control rats. Pink staining on the tail was observed in 1/5 female rats treated with 1000 mg/kg/day, compared with 0/5 female control rats.

An intense red staining material was seen in the large intestine of some animals receiving 1000 mg/kg/day of the compound. No adverse treatment related changes were detected.

In conclusion, 150 mg/kg/day represents the highest NOEL and 1000 mg/kg/day was the NOAEL on this study.