Reaction products of diazotised 4,4-diaminodiphenylamine-2-sulfonic acid, subsequently coupled with 6-amino-4-hydroxynaphthalene-2-sulfonic acid, further diazotised and coupled with metaphenylendiamine, sodium salts

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

other: experimental study on similar substance

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 4-amino-3,6-bis[[4-[(2,4-diaminophenyl)azo]phenyl]azo]-5-hydroxynaphthalene-2,7-disulphonate

EC Number:

229-208-1

EC Name:

Disodium 4-amino-3,6-bis[[4-[(2,4-diaminophenyl)azo]phenyl]azo]-5-hydroxynaphthalene-2,7-disulphonate

Cas Number:

6428-31-5

Molecular formula:

C34H29N13O7S2.2Na

IUPAC Name:

disodium 4-amino-3,6-bis({4-[(2,4-diaminophenyl)diazenyl]phenyl}diazenyl)-5-hydroxynaphthalene-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:

TEST ANIMALS
- Animal arrival: 14.03.2012
- Selection: 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
- Age of animals: males, females - sexually adult; 10 weeks on arrival
- Number of animals: 12 females and 12 males per group, 6 males and 6 females per satellite group
- Housing conditions: the study will proceed in SPF conditions according to SOP No.12.
pre-mating period 2 rats of the same sex in one cage
during mating period one male and one female in one cage
pregnant females individually; offspring with mother
satellite animals 2 rats of the same sex in one cage
- Bedding: sterilized shavings of soft wood
- Food: complete pelleted diet for rats and mice in SPF breeding
- Water: drinking water ad libitum, quality standard ČSN 757 111
- Acclimatization: at least 5 days. During the acclimatisation period the health condition of all animals was controlled daily.

ENVIRONMENTAL CONDITIONS
- Light cycle: 12 hour light / 12 hour dark
- Temperature: 22 ± 3°C
- Humidity: relative humidity 30-70%

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

aqua pro injection

Details on oral exposure:

ADMINISTRATION
The test substance was administered to the stomach by gavage as the solution in aqua pro injection.
Oral way of administration was chosen according to the guideline and it was approved by sponsor.

PREPARATION OF DOSING SOLUTIONS
The concentrations of solutions at all dose levels were adjusted to ensure the administration of 1 mL per 100 g of body weight.
The application form (test substance solution in aqua pro injection) 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:
20 mg/kg/day
Basis:
actual ingested

Doses / concentrations 2

Remarks:

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

Doses / concentrations 3

Remarks:

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

No. of animals per sex per dose:

12 males + 12 females for all tested and control group.
6 males + 6 females in satellite groups

Control animals:

yes

Details on study design:

ANIMAL
- Selection of test animals: animals were randomly divided into the control and test groups and they were marked individually.
- Identification: 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

DOSE SELECTION
1) Dose Range findings
The dose-range finding experiment with 14-day application period was performed with 4 groups of treated animals without control group.
The dose levels for the dose-range finding experiment have been chosen according to the request of sponsor and with respect to the information in Material Safety Data Sheet obtained from sponsor (LD50, oral rat - 4900 mg/kg).
Appropriate dose levels of the test substance for the 14-day dose-range finding experiment was chosen as follows:
Dose levels: 125, 250, 500 and 1000 mg/kg/day
In the dose-range finding experiment with test substance two females and one male died at the dose level 1000 mg/kg/day during the 1st week of application and two males and one female died at the dose level 500 mg/kg/day during the 2nd week of application. Four males and three females at the dose level 1000 mg/kg/day were humanely killed at the end of the first week of application and three males and four females were killed at the dose level 500 mg/kg/day during the 2nd week of application by the reason of bad health condition and prevention suffering of animals.
Marked fall of weight was recorded in males and females of the dose level 1000 mg/kg/day. The body weights and body weight increments of animals at the dose levels 500 mg/kg/day were decreased.
Clinical observation detected the impact of the test substance on the health condition of animals at the dose levels 500 and 1000 mg/kg/day.
Results of haematology examination showed that the test substance had influence on red and white blood component in both sexes mainly at the dose level 500 mg/kg/day.
Pathological examination revealed change of colour of skin, subcutis, muscle and peritoneum at the dose level 500 and 1000 mg/kg/day. Other macroscopic changes were found mainly in digestive system, in lungs and in spleen of animals at the dose levels 500 and1000 mg/kg/day.
On the basis of the results obtained the following dose levels 20, 80 and 320 mg/kg/day were chosen for the main Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test.

MATING PROCEDUIRE
Animals were mated from the 15th day of study. Mating 1:1 (one male to one female) was used in this study. Each morning the females were examined for presence of spermatozoa in vaginal smears Day 0 of pregnancy was defined as the day the sperms were found.

Examinations

Observations and examinations performed and frequency:

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

HEALTH CONDITIONS
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
Adult animals
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 (11.00 a.m. - 13 p.m.) – at the time of expectation of maximal effect of the test substance. Animals were observed in natural conditions in their cages.

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.

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, daily in mating period
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.

BODY WEIGHT
Males weekly and females weekly in premating and mating period; during pregnancy at 0, 7th, 14th, 20th day; during lactation at 0 or 1st and 4th day.
Pups (litters) at 0 or 1st and 4th day

FOOD CONSUMPTION
Males weekly (except the mating period) and females weekly during premating period and after mating period; during pregnancy and lactation on the same days as body weight.

URINALYSIS: only males 42nd and 56th day of study
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 parameters were determined by analyser PocketChem PU-4210 (Arkray, Inc., Japan).

HAEMATOLOGICAL EXAMINATION
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
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.

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 test 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 mortality
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 males and females, from each group + satellite group);
testes or ovaries, epididymides or uterus, prostate gland, pituitary gland (reproduction part of study – all animals). 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
The following list contains all the examined organs which were collected at necropsy and fixed:

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 (Analysis of Variance)

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No mortality at any dose both for males and females

Mortality:

no mortality observed

Description (incidence):

No mortality at any dose both for males and females

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males: body weights were markedly decreased at the highest dose from the 2nd week of application. Females: average body weight increment was decreased at the highest dose level. No effects during pregnancy and lactation.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

During pregnancy the food consumptions was decreased compared to control group. Pregnant females food consumption was similar to the control during pregnancy.

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 increased for males and females after the second week at the highest dose

Ophthalmological findings:

effects observed, treatment-related

Description (incidence and severity):

Natural reaction for each dose

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Males and females: statistically significant differences were not detected in treated animals. Parameters of red and white blood cell were similar in control and treated males.

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 no effect for each dose level

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Males and females: Statistically significant differences were detected at the middle and highest dose level for liver, decrease of absolute weight

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Change of colour in some tissues and organs at the highest dose level

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Partly irreversible and reversible occurrence of the dye in some tissues and organs at the middle and highest dose levels

Details on results:

REPEATED DOSE TOXICITY
Repeated oral administration of test item to rats by gavage at the dose levels 20, 80 and 320 mg/kg/day did not cause any mortality. No negative treatment-related effects were detected during functional observation of animals.

20 mg/kg/day
Body weight, food consumption, water consumption, clinical status of animals, urinalysis, weight of organs, macroscopical and microscopical structure of organs were not seriously affected. Slight decrease of food conversion in males, change of one haematological parameter prolonged tromboplastin time in females (above historical control) and statistically significant decrease of ALP activity in males were recorded at the dose level.

80 mg/kg/day
Food consumption, water consumption, clinical status of animals, relative weight of organs and macroscopical structure of organs were not seriously affected.
Decreased body weight increments at the end of application period in males, decreased food conversion in males, changes of some haematological parameters (increase of total leukocyte count – above historical control in females), differences in biochemical blood parameters (statistically significant decrease of urea concentration, decrease ALP and concentration of inorganic phosphorus in males, statistically increase of urea concentration in females), changes of absolute weight of liver (statistically significant decrease of absolute weight of liver in males and statistically significant increase of absolute weight of liver in females), increased occurrence of microscopical changes of brain, cerebellum, spleen and thyroid gland in males (deposits of pigment), of kidneys, spleen and thyroid gland in females (deposits of pigment) and of heart (sporadically regressive changes in myocardium) were detected.

320 mg/kg/day
Growth of animals was influenced by the test substance treatment (decrease of body weight increments or fall of body weight, food consumption and food conversion). Clinical status of animals after application was also influenced by the test substance treatment (red-coloured bedding, test-substance coloured excrements, reversible change of colour of skin and irreversible change of colour of visible mucous membranes). Haematological examination (delayed significant increase of platelets count - above historical control and change of differential leukocyte count in males; reversible increase of total leukocyte count and delayed significant increase of PT value, delayed increase of APTT value in females), blood biochemical examination (reversible statistically significant and dose dependent decrease of ALP activity and urea concentration in males; delayed statistically significant increase of urea, albumin, inorganic phosphorus and potassium ions concentration in males; irreversible significant increase of urea concentration – above historical control and decrease of total protein concentration, reversible increase of AST activity – above historical control in females, delayed statistically significant increase of ALP activity and inorganic phosphorus in females), urinalysis (change of colour of urine in males), biometry of organs (irreversible increase of relative weight of testes, reversible statistically significant and dose dependent decrease of absolute and relative weight of liver, delayed statistically significant increase of absolute and relative weight of adrenal glands and relative weight of spleen, reversible decrease of absolute weight of heart in males; irreversible statistically significant and dose dependent increase of absolute and relative weight of liver, reversible and dose dependent decrease of absolute and relative weight of thymus),gross examination of organs and tissues(irreversible change of colour of mucous membranes, muscles, brain, spleen, thyroid gland, reversible change of colour of skin, subcutis and peritoneum in males; irreversible change of colour of mucous membranes, muscles, spleen, thyroid gland, reversible change of colour of skin, subcutis, brain, organs of thoracic and abdominal cavity and peritoneum, reduction of thymus in females) and histological examination of organs and tissues (irreversible regressive changes in myocardium accompanied by reparative fibrosis and accumulation of pigment phages in males and females, irreversible occurrence of pigment in brain, cerebellum, heart, spinal cord, kidneys, spleen and thyroid gland in males and females, focal atrophic or necrotic changes in skeletal muscle of females; reversible dystelectasis in lungs and activation of white pulp in spleen of males; reversible inflammation or necrosis of muscle fibres in larynx, atrophy of cortex in thymus in females) of animals at the dose level revealed significant changes attributable to the test substance administration.

REPRODUCTIVE TOXICITY
20 and 80 mg/kg/day
The course of mating, pregnancy and lactation of parental animals, number of females achieving pregnancy and bearing live pups, weights of reproductive organs and pituitary gland, spermatogenesis and sperm parameters, macroscopical and microscopical structure of reproductive organs and pituitary gland of parental animals, number of pre-implantation, post-implantation and post-natal losses of mothers and number, weight, sex ratio and development of pups were not adversely affected.

320 mg/kg/day
The course of mating, pregnancy and lactation of parental animals, number of females achieving pregnancy, absolute weight of reproductive organs and pituitary gland and relative weight of pituitary gland, spermatogenesis and sperm parameters, macroscopical and microscopical structure of reproductive organs and pituitary gland of parental animals, number of post-implantation and post-natal losses of mothers, sex ratio and development of pups were not adversely influenced.
Evaluation of relative weights of reproductive organs (increase of relative weight of testes and epididymis), examination of number of pups(decrease of the total number of live pups and mean number of pups per litter), calculation of reproduction parameters (decreased number of females bearing live pups, increased number of stillborn pups), calculation of pre-implantation losses (decreased number of corpora lutea and uterus implantations) and evaluation of pup weights(decrease of mean litter weight, decrease of mean pup weight on day 4 after parturition) in animals revealed adverse effects attributable to test substance.

Effect levels

Dose descriptor:

NOAEL

Effect level:

ca. 80 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

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 80 mg/kg bw/day both for males and females.

Executive summary:

The test substance 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.

Conclusion

Administration of the test substance, had not adverse effect on mortality, parameters of functional observation and on some reproduction parameters - course of mating, pregnancy and lactation, weights of reproductive organs and pituitary gland, spermiogenesis, macroscopical and microscopical structure of reproductive organs and pituitary gland of parental animals, number of post-implantation and post-natal losses of mothers, sex ratio and development of pups.  

Test-related reduction in body weight gains (reduced body weight increments in both sexes, fall of body weight in females, decreased food consumption and conversion in both sexes) were noted at the dose of 320 mg/kg/day. Some biochemical parameters(changes of enzymes activity, urea, inorganic phosphorus, total protein and albumin concentration) and biometry of organs (changes of thymus, heart and spleen weight) were significantly changed especially in animals of the highest dose level.

The heart appeared to be a target organ following repeated oral exposure of the test substance at the highest dose level. Serious irreversible regressive lesions accompanied by accumulation of pigment were diagnosed in myocardium. In addition, similar changes were observed in muscle fibres of larynx and skeletal muscle. This findings tended to be more serious in females than in males. The test had influence on macroscopical and microscopical structure of some other organs and tissues. Irreversible accumulation of pigment in brain, cerebellum, spinal cord, spleen, thyroid gland and kidneys was detected in animals of the middle and the highest dose level. Atrophic changes occurred in thymus of females at the highest dose level.

Daily oral administration of the test substance at the dose level 320 mg/kg/day also affected the number of live pups (decrease of the number of females bearing live pups, decrease of total number of live pups and mean number of pups per litter), early prenatal development of organism in uterus (increased pre-implantation losses), growth of pups(decreased mean litter weight, decreased mean pup weight on day 4 after parturition). However, male ability to produce sperm that can fertilize eggs and female ability to impregnate was not significantly changed.

     

The value of NOAEL for REPEATED DOSE TOXICITY was established as 80 mg/kg body weight/day both for males and females.

The value of NOAEL for the REPRODUCTIVE and DEVELOPMENTAL TOXICITY was established as 80 mg/kg body weight/day