disodium 4-amino-6-((4-((4-(2,4-diaminophenyl)azo)phenylsulfamoyl)phenyl)azo)-5-hydroxy-3-((4-nitrophenyl)azo)naphthalene-2,7-disulfonate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening - extended , considered as a subchronic toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 1,2010-January 20, 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 422

GLP compliance:

yes

Limit test:

no

Test material

Test material information

1

Reference substance name:

-

EC Number:

421-880-6

EC Name:

-

Cas Number:

201792-73-6

Molecular formula:

C34H25N11Na2O11S3

IUPAC Name:

disodium 4-amino-6-{2-[4-({4-[2-(2,4-diaminophenyl)diazen-1-yl]phenyl}sulfamoyl)phenyl]diazen-1-yl}-5-hydroxy-3-[2-(4-nitrophenyl)diazen-1-yl]naphthalene-2,7-disulfonate

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Age of animals: male, females – sexually adult (10 weeks – on arrival)
Selection of animal species: laboratory rat has been chosen because our testing laboratory has long experience with this species
Acclimatization: at least 5 days
Number of animals: 12 females and 12 males per group, 6 males and 6 females per satellite group
Housing conditions: SPF – 2 rats of the same sex in one cage in pre-mating period, during mating period – one male and one female in one c age, pregnant females – individually, offspring – with mother, satellite animals - 2 rats of the same sex in one cage
- Food: complete pelleted diet for rats and mice in SPF breeding
- Water: drinking water ad libitum, quality standard ČSN 757 111
- Light cycle: 12 hour light / 12 hour dark

- Microclimate: 22  3°C, relative humidity 30-70%
- Bedding: sterilized shavings of soft wood
Selection of animals: random selection according to the internal rule – at the beginning of the study the weight variation of animals in groups of each sex should not exceed + 20% of the mean weight
Identification of animals: the animals will be identified by the colour marks on their fur, each cage will be marked with the number of animals, sex, number of cage, name and dose level of the test substance
Animal housing: The study will proceed in SPF conditions according to SOP No.12.

During the acclimatisation period the health condition of all animals was controlled daily.

Then the animals were randomly divided into the control and test groups and they were
marked individually.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

The test substance was administered to the stomach by gavage as the solution in aqua pro injectione. Oral way of administration was chosen according to the guideline and it was approved by sponsor. The animals were treated 7 days per week at the same time (8.00 – 10.00 am).
The concentrations of solutions at all dose levels were adjusted to ensure the administration of 1 mL per 100 g of body weight. The vehicle control group was administered by aqua pro injectione in the same volume. The application form (test substance solution in aqua pro injectione) was prepared daily just before administration.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

Parental males:
1st day – 14th day (pre-mating) → 28th day (mating) → 42nd day of study

Satellite males:
1st day → 42nd day (administration) → 56th day (observation)

Parental females:
1st day – 14th day (pre-mating) → 28th day (mating) → gestation → lactation → day 4 post partum

Satellite females:
1st day → 42nd day (administration) → 56th day (observation)


Non-pregnant females (without evidence of copulation):
1st day – 14th day (pre-mating) → 28th day (mating) → 54th day of study

Non-pregnant females (with evidence of copulation):
1st day – 14th day (pre-mating) → 28th day (mating) → 25th day after confirmed mating (max. 54th day of study)

Frequency of treatment:

The animals were treated 7 days per week at the same time (8.00-10.00 am)

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0 mg/kg/day
Basis:

Doses / concentrations 2

Remarks:

Doses / Concentrations:
50 mg/kg/day
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
150 mg/kg/day
Basis:
actual ingested

Doses / concentrations 4

Remarks:

Doses / Concentrations:
450 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

Basic groups:
1. Control 0 12 males + 12 females
2. Low dose 50 mg/kg/day 12 males + 12 females
3. Intermediate dose 150 mg/kg/day 12 males + 12 females
4. High dose 450 mg/kg/day 12 males + 12 females

Satellite groups:
5. Control – vehicle – satellite: 0 6 males + 6 females
6. High dose – satellite: 450 mg/kg/day 6 males + 6 females

Control animals:

yes

Details on study design:

Study Time Schedule
Main Study:

Animal arrival: 01. 09. 2010
Acclimatisation: at least 5 days
Administration: 08. 09. – 22. 10. 2010

Urinalysis: only males – 42nd and 56th day of study

Haematology and necropsies: parental males – 43th day of study
satellite males – 57th day of study
parental females – 4th day of lactation
satellite females – 57th day of study
non-pregnant females – 55th day of study or 26th day after confirmed mating

-Preparation of Experimental Animals
During the acclimatisation period the health condition of all animals was controlled daily.
Then the animals were randomly divided into the control and test groups and they were
marked individually.

-Experimental Data Collection

Health condition control: daily - during the acclimatization and the experimental part
Body weight: males - weekly
females - weekly in premating and mating period,
during pregnancy: 0., 7th, 14th, 20th day,
during lactation: 0. or 1st and 4th day;
pups (litters) – 0. or 1st and 4th day;
Food consumption: males - weekly (except the mating period)
females - weekly during premating period and after mating period
during pregnancy and lactation – on the same days as
body weight
Clinical observations: males and females - daily during the administration period
pups - as soon as possible after delivery and then daily

Mortality control: daily
Laboratory examinations:
- vaginal smears: daily in mating period
- pathological examination: males and nonpregnant females - after the end of administration period
parental females and pups - on the 4th day of lactation
- weight of organs: during necropsy
- sperm observation: all males after necropsy
- histopathological examination: all males and females after necropsy

Examinations

Observations and examinations performed and frequency:

Mortality Control
All rats during the treatment periods were examined for vitality or mortality changes daily.


Health Condition Control
All rats were observed pre-experimentally to ensure that only the animals exhibiting normal behavioural activity would be entered into the study. During the administration period they were examined for behaviour changes each day before and during application.


Clinical Observations of Males and Females
All rats were observed daily during the administration period.
This observation was made in order to record possible clinical effects after application and all changes in behaviour of animals. So it was done after application at the same time every day (12.00 - 14 p.m.) – at the time of expectation of maximal effect of the test substance. Animals were observed in natural conditions in their cages.


Clinical Observation of Pups
All pups were observed in natural conditions in their cages daily during the lactation. Changes in behavioural abnormalities were recorded. Detailed examination of each litter was performed as soon as possible after delivery (day 0 or 1 post-partum) and the 4th day of lactation. The number and sex of pups, stillbirths, live births and presence of gross anomalies were recorded.


Detailed Clinical Observation
This observation was carried out before the first application and then weekly. At the first part of observation the behaviour of animals in the cage was monitored: piloerection, posture, position of eyelids, breathing, tonic or clonic movements, stereotypes or bizarre behaviour.
The second part was the observation during the removal from cage: reaction to handling, elasticity of skin, colour of mucous membranes, salivation, lacrimation, cleanliness of fur around foramina.


Functional Observation
This observation was done at the end of administration period and recovery period.
During functional examination, the sensory reactivity on auditory, visual, proprioceptive stimuli and pupillary reflex were evaluated and motor activity assessment was conducted. Moreover the individual observations of grip strength were performed using grip strength meter. Measurements were made on: 1) pectoral legs, 2) pelvis legs. Grip power was expressed in Newtons.

Laboratory examination
Examination of Vaginal Smears
The pregnancy was determined by the presence of spermatozoa in vaginal smear. The vaginal smears were carried out daily in the morning during mating period. The smears were stained
and the presence of sperm was evaluated. Day 0 of pregnancy was defined as the day when sperms were found.

Urinalysis
The rats were kept in the metabolic cages for the collection of urine for two hours. Immediately before entering metabolic cages the animals were administered 2 mL of drinking water for 100 g of body weight by gavage to the stomach.
The following parameters were determined by analyser PocketChem PU-4210 (Arkray, Inc., Japan).

Haematological Examination
The blood samples were collected from the orbital plexus by glass micropipette under the light ether narcosis into the PVC test tubes containing anticoagulation systems.
Haematology analysers Coulter  AC.T diffTM, Celltac alfa and Coagulometer ACL 200 were used for examination and the following parameters were determined.

Biochemical Examination
The animals starved approximately for 18 hours before blood collection but they were supplied by drinking water ad libitum.
The blood samples were collected from the orbital plexus under the light ether narcosis. Biochemical parameters were measured in serum.
The following parameters were determined by automatic biochemical analysers SPOTCHEMTM EZ SP-4430 and SPOTCHEMTM EL SE-1520 (Arkray, Inc., Japan).


Pathological Examination
During the necropsy a revision of the external surface of the body, of all orifices and the cranial, thoracic and abdominal cavities were carried out. Organs for consequent histopathological examination were taken out and stored in containers with fixative (buffer 4% formaldehyde). Testes and epididymides were fixed in modified Davidson’s fixative.

Observation of Sperm
In all males of all groups surviving to scheduled necropsy the sperm parameters were examined: sperm motility and sperm morphology. Sperm specimens were prepared and examined according to the SOP No. M/45.

Sperm motility
Sperm samples were taken from one epididymis and sperm motility was assessed from these samples. The motility of sperm was determined by microscopic examination of the prepared sperm suspension. The result of observation was evaluated subjectively according to following grades: 1 - fast progressive motility, 2 - slow progressive motility, 3 - no progressive motility, 4 - non-motile sperm.

Sperm morphology
Sperm samples were taken from one epididymis and sperm morphology was assessed from these samples. A smear from the sperm suspension was prepared and stained (Giemsa staining). The morphology of sperm was determined by microscopic examination.
All deviations – e.g. broken tail, abnormal form of tail, double head, amorphous head, abnormal form of neck ¬– were recorded.

Biometry of Organs
At the end of study the experimental animals were narcotised and sacrificed by cutting the neck spine and medulla. After the gross necropsy of the cranial, thoracic and abdominal cavities the organs for weighing and further histological examination were collected. The absolute weights of liver, kidneys, adrenals, testes or ovaries, epididymides or uterus, prostate gland, thymus, spleen, brain, pituitary gland and heart were recorded (repeated dose toxicity part of study – 6 animals from group + satellite group); testes or ovaries, epididymides or uterus, prostate gland, pituitary gland (reproduction part of study – 12 animals from group). Afterwards the somatic indexes - SI (= relative weight of organ) were computed according to the following formula: SI = weight of organ x 100/ body weight.

Histopathological Examination
Samples of the following tissues and organs were collected at necropsy and fixed:

Table 4: Organs for histopathological examination
Reproduction part of study (12 males and 12 females from each main group: pituitary gland, ovaries, uterus, cervix of uterus, vagina, epididymis, prostate gland, testes, all gross lesions

Repeated dose toxicity part of study (6 males and 6 females in each main group + 6 males and 6 females in each satellite group)
Adrenal glands
Aorta
Brain (incl. cerebellum and med. oblongata)
Caecum
Coagulating gland
Colon
Duodenum
Pancreas
Rectum
Salivary glands
Sciatic nerve
Seminal vesicle
Skeletal muscle
Skin
Spinal cord – thoracic
Spleen
Stomach
Thymus
Thyroid gland incl. parathyroid
Trachea
Urinary bladder
Female mammary gland area
Femur
Heart
Ileum (incl. Peyer´s patches)
Jejunum (incl. Peyer´s patches)
Kidneys
Liver
Lungs
Lymph nodes – mesenteric, paraaortal
Oesophagus
All gross lesions

The mentioned tissue and organs were collected from all killed males and females at necropsy and fixed in neutral 4% formaldehyde solution (v/v) for further histopathological evaluation. For histopathological processing the routine histopathological paraffin technique with haematoxylin-eosin staining was used.
Detailed histological examination was performed on testes (with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure). Spermatogenesis and spermatogenic cycle were evaluated according to the publication: Hess, R.A.; Quantitative and qualitative Characteristics of the Stages and Transitions in the Cycle of the Rat Seminiferous Epithelium: Light Microscopic Observation of Perfusion-Fixed and Plastic-Embedded Testes (Biology of Reproduction 43, 525-542, 1990). Pathological changes were evaluated according to the publication: Creasy, D.M.; Evaluation of Testicular Toxicity in Safety Evaluation Studies: The Appropriate Use of Spermatogenic Staging (Toxicologic Pathology 25, 119-131, 1997) and Guidance Document for Histologic Evaluation of Endocrine and Reproductive Tests in Rodents, ENV/JM/MONO(2009)11.

Sacrifice and pathology:

At the end of study the experimental animals were narcotised and sacrificed by cutting the neck spine and medulla. During the necropsy a revision of the external surface of the body, of all orifices and the cranial, thoracic and abdominal cavities were carried out. Organs for consequent histopathological examination were taken out and stored in containers with fixative (buffer 4% formaldehyde). Testes and epididymides were fixed in modified Davidson’s fixative.

Statistics:

ANOVA

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

no mortality at any dose

Mortality:

no mortality observed

Description (incidence):

no mortality at any dose

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

weigth increments were lower in males of highest dose levels. Females: average body weight increment was decreased at the highest dose level in the 1st week of application. No effect during lactation

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

pregnant females food consumption was decreased also during lactation at the highest dose level

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

the observed effect on females on food consumption is confirmed

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

effects observed, treatment-related

Description (incidence and severity):

water consumption was incresead for males and females after the second week at the highest dose

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

see details for the highest dose

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

some effects on some parameters (cholesterol, glucose, AST, ALT, sodium, creatinine, chlorides) at the highest dose level for males and females

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

performed only on males with effect at the middle dose level:decrease of pH

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

slight increase of spleenweight (males and females) at the highest dose

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

change of colour in some tissues and organs and weight change in spleen at the highest dose level

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

partly irreversible and reversible occurence of the dye in some tissues and organs at the highest dose levels

Effect levels

Dose descriptor:

NOAEL

Effect level:

ca. 150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

food consumption and compound intake

food efficiency

gross pathology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

urinalysis

water consumption and compound intake

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The value of NOAEL for repeated dose toxicity was established at 150 mg/kg bw/day both for males and females.

Executive summary:

Introduction

The test substance, Acid Black 210, was tested for reproduction and subacute toxicity using the OECD Test Guideline No. 422: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, Adopted by the Council on March 22^nd1996.

 

Methods

Wistar rats of SPF quality were used for testing. The test substance was administered in the form of solution in water for injection. Oral application by stomach tube was performed daily. The study includes four main groups and two satellite groups of animals. Each main group consisted of 12 males and 12 females; each satellite group consisted of 6 males and 6 females.Main groups contained 3 treated groups (doses 50, 150, 450 mg/kg of body weight /day) and one control group (vehicle only). The satellite groups contained one control group (vehicle only) and one treated group (450 mg/kg/day).The dose levels for study were determined on the basis of results of adose-range finding study phase (see the Annex 2).

The treated groups were administered daily for the following periods:

males and females – 2 weeks prior to the mating period and during the mating period,

pregnant females – during pregnancy and till the 3^rdday of lactation,

males – after mating period – totally for 42 days,

nonpregnant females (mated females without parturition) – for 25 days after the confirmed mating.

After the end of administration period the animals of main groups were sacrificed and satellite animals were observed for the next 14 days without treatment.

During the study clinical observation and health status control were performed daily. The body weight and food consumption were measured weekly or in the specified time intervals. Detailed clinical observation was carried out weekly. Water consumption was measured twice a week. Vaginal smears were prepared daily during the mating period (until the presence of spermatozoa). Reproduction parameters relevant to pups (number of pups, weight of litters, sex or vitality) were also recorded. Before the end of study the functional observation was accomplished.

The study was finished by urinalysis, haematological and biochemical analysis and gross necropsy of animals. In all males of main groups the sperm parameters, sperm motility and sperm morphology were examined. The selected organs from parental animals were removed for weighing and histopathological examination.

 

Results

Repeated dose toxicity part of study:

 

Repeated oral administration of Acid Black 210 to rats by gavage at the dose levels 50, 150 and 450 mg/kg/day did not cause any mortality. No negative treatment-related effects were detected during functional observation of animals.        

Body weight, food consumption and conversion, water consumption, clinical status of animals, haematological and biochemical blood parameters, biometry of organs, macroscopical and microscopical structure of organs were not seriously affected by treatment of the test substance at the dose level 50 mg/kg/day. Only some urine properties were changed in males of the dose level 50 mg/kg/day: glaringly yellow colour and presence of leucocytes.

 

Body weight, food consumption and conversion, water consumption, clinical status of animals, haematological and biochemical blood parameters and biometry of organs were not seriously affected by treatment of the test substance at the dose level 150 mg/kg/day. Increased occurrence of macroscopical changes of spleen in males and females (change of colour and enlargement), microscopical changes of spleen in males (mild complete extramedular haemopoiesis and mild enlargement of marginal zone of periarteriolar lymphoid sheats) and change of some urine properties in males (glaringly yellow colour and presence of leucocytes) were detected in animals of the dose level 150 mg/kg/day.

 

Growth of animals at the dose level 450 mg/kg/day was insignificantly influenced by the test substance treatment (slight decrease of body weight increments, food consumption and food conversion).Clinical status of animals after application was also influenced by the test substance treatment (red-coloured bedding, black colour of stool). Haematological examination (irreversible significant increase of MCV in males and females, reversible significant decrease of RBC and increase of WBC in males) and blood biochemical examination (reversible significant elevation of AST activity in males and females and activity of ALT in females, irreversible significant increase of creatinine in males, reversible changes: significant decrease of chloride concentration in males, increase of urea concentration in males and females),urinalysis (significant delayed increase of pH, glaringly yellow colour and presence of leucocytes), biometry of organs (significant absolute and relative weight elevation in spleen – partly irreversible, reversible decrease of absolute weight of brain in males), gross examination of organs and tissues (irreversible change of colour of brain, spleen, thyroid gland, epithelium of thoracic and abdominal cavity, mucosa, skeletal muscle and skin colour in males and females) and histological examination of organs and tissues (partly irreversible occurrence of pigment in kidneys, spleen, thyroid gland, liver and lymph nodes and ovaries, reversible increased occurrence of extramedular haemopoiesis and enlargement of marginal zone of periarteriolar lymphoid sheats in males) of animals at the dose level 450 mg/kg/day revealed significant changes attributable to the test substance administration.