Reaction mass of disodium 4-amino-6-[[4-[(2,4-diaminophenyl)diazenyl]phenyl]]-3-((E)-[4-((E)-(2,4-diaminophenyl)diazenyl]phenyl )diazenyl)-5-hydroxynaphthalene-2,7-disulphonate and disodium 4-amino-6-((E)-(4-aminophenyl)diazenyl)-3-((E)-(4-((E)-(2,4-diaminophenyl)diazenyl)phenyl)diazenyl)-5-hydroxynaphthalene-2,7-disulfonate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

other: experimental result on similar substance

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The test has been performed following official guidelines, that completely assesses the end point, on a similar substance differing only by the counter ion potassium instead of sodium cation.

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:

other: 422/Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

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

Details on test animals or test system and environmental conditions:

For parental animals:
Age of animals: males, 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 cage, 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 exposure:

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. 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

Details on mating procedure:

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.

Duration of treatment / exposure:

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

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

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:
20 mg/kg bw /day
Basis:
actual ingested

Doses / concentrations 3

Remarks:

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

Doses / concentrations 4

Remarks:

Doses / Concentrations:
320 mg/kg bw /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

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.

Examinations

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of corpora lutea: Yes, average number of corpora lutea were decreased only at the highest dose level
- Number of implantations: Yes, average number of number of implantation were decreased only at the highest dose level
- Number of early resorptions: No data
- Number of late resorptions:No data
- Gravid uterus weight: No data

Fetal examinations:

- External examinations: No data
- Soft tissue examinations: No data
- Skeletal examinations: No data
- Head examinations: No data

Indices:

Calculated parameters see table reported in next section for indices

Results and discussion

Results: maternal animals

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

ca. 80 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no data

Effect levels (fetuses)

Dose descriptor:

NOAEL

Remarks on result:

not measured/tested

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Number and Sex Ratio of Pups

The statistical evaluation of the number of live born pups/per litter, number of corpora lutea and number of implantations was performed. No statistically significant intergroup differences were recorded.

The total number of live pups and mean number of pups per litter at the dose level 320 mg/kg/day was markedly decreased in comparison with the control.The total numbers of live pups and mean number of pups per litter at the dose levels 20 and 80 mg/kg/day were similar or higher than control. The number of stillborn pups was increased at the dose level 320 mg/kg/day. 

In sex ratio no significant differences were recorded in treated groups.

 

Number of pups and sex(total number and mean per litter)
Dose level	0		20		80		320
	Total number (mean number of live born pups)	Total number of males and females (live born)	Total number (mean number of live born pups)	Total number of males and females (live born)	Total number (mean number of live born pups)	Total number of males and females (live born)	Total number (mean number of live born pups)	Total number of males and females (live born)
0/1 day (first check of litter after parturition)*	124/124 (12.40)	59M 65F	128/126 (14.00)	54M 72F	142/139 (13.90)	68M 71F	99/86 (10.75)	44M 42F
	Total number (mean number of live pups)	Total number of males and females	Total number (mean number of live pups)	Total number of males and females	Total number (mean number of live pups)	Total number of males and females	Total number (mean number of live pups)	Total number of males and females
4^thday of lactation	124 (12.40)	59M 65F	124 (13.78)	53M 71F	137 (13.70)	67M 70F	86 (10.75)	44M 42F

Note: * - total number of pups/total number of live born pups, M - male pups, F – female pups 

Body Weight

The statistical evaluation of mean weight of pup on the 0./1^stday and mean weight of pup on the 4^thday was performed. No statistically significant intergroup differences were recorded.

Mean body weights of litters at the dose levels 20 and 80 mg/kg/day were increased compared to control. Mean weight of the litter at the dose level 320 mg/kg/day was markedly decreased against control. Mean weights of pup recorded at the 1^stcheck of litter after parturition in treated and control groups were similar. Mean body weight increment of pup (from the 1^stcheck of litter after parturition to the 4^thday of lactation) was slightly decreased at the highest dose level.  

 

Mean body weight(grams)
Dose level	0		20		80		320
Day of study	Litter	Pup	Litter	Pup	Litter	Pup	Litter	Pup
0/1 day first check of litter after parturition	84.02	6.87	91.69	6.55	87.09	6.16	76.01	6.20
3^rdday of lactation	117.07	9.71	123.86	9.13	122.00	8.99	102.54	8.42
4^thday of lactation	129.09	10.77	138.40	10.20	142.33	10.47	113.77	9.36

 

Development of Pups

Increased number of stillborn pups was recorded at the highest dose level. At the lowest and the middle dose level the number of stillborn pupswas negligible[fd1] .

Mortality of pups in lactation period was sporadically detected only at the lowest and the middle dose level. 

No differences in development of pups were observed at the control and treated groups.

 

Pathological Examination

  The macroscopic examination was performed in all pups. Pathological findings were observed only in stillborn pups: lungs without air, empty stomach. In other examined pups of control and treated groups no pathological findings were recorded

Applicant's summary and conclusion

Conclusions:

The values of NOAEL for developmental of pups was established to be 80 mg/kg bw/day

Executive summary:

Introduction

The test substance,Direct Black 19,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 20, 80, 320 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 (320 mg/kg/day).The dose levels for study were determined on the basis of results of adose-range finding experiment (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,

non pregnant 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. The functional observation was performed at the end of application and observation period. 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.

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 ofDirect Black 19to rats by gavage at the dose levels20, 80 and 320 mg/kg/daydid not cause any mortality. No negative treatment-related effects were detected during functional observation of animals.

Body weight, food consumption, water consumption, clinical status of animals, urinalysis, weight of organs, macroscopical and microscopical structure of organs were not seriously affected by treatment of the test substance at the dose level20mg/kg/day.Slightdecrease of food conversionin males, change of one haematological parameter –prolonged tromboplastin timein females (above historical control),statistically significant decrease of ALP activityin males were recorded at the dose level20 mg/kg/day.

 

Food consumption, water consumption, clinical status of animals, relative weight of organs and macroscopical structure of organs were not seriously affected by treatment of the test substance at the dose level80mg/kg/day. Decreased body weight incrementsat the end of application period in males,decreased food conversionin males,changes of some haematological parameters(increase of total leucocyte 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 ofmicroscopical changes of brain, cerebellum, spleen and thyroid gland in males (deposits of pigment), ofkidneys, spleen and thyroid glandin females (deposits of pigment) and ofheart(sporadical regressive changes in myocardium) were detected at the dose level80 mg/kg/day.

 

Growth of animalsat the dose level320mg/kg/daywas influenced by the test substance treatment (decrease of body weight increments or fall of body weight, food consumption and food conversion).Clinical status of animalsafter 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 leucocyte count in males; reversible increase of total leucocyte 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, reducement of thymus in females) andhistological examination of organs and tissues(irreversible regressive changes in myocardium accompanied by reparative fibrosis and accumulation of pigmentophages 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 level320mg/kg/dayrevealed significant changes attributable to the test substance administration.    

 

Reproduction part of study:

The course of mating, pregnancy and lactation of parental animals, number of femalesachieving pregnancy and bearing live pups, weights of reprodutive organs and pituitary gland, spermiogenesis 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 by the test substance treatment at the dose levels20 and 80 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, spermiogenesis 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 by the test substance treatment at the dose level320 mg/kg/da

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 of the dose level 320 mg/kg/day revealed adverse effects attributable to test substance.

 

Conclusion

Administration of the test substance,Direct Black 19, 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-relatedreduction 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. Somebiochemical 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

Theheartappeared to be atarget organfollowing repeated oral exposure of the test substance,Direct Black 19, 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 fibers of larynx andskeletal muscle. The above mentioned 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 pigmentin 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 thenumber 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 fertilise eggs and female ability to impregnate was not significantly changed.

 

The value of NOAEL (No Observed Adverse Effect Level)for theREPRODUCTIONand for DEVELOPMENT OF PUPS was established as 80mg/kg body weight/day.