Pentasodium bis[5-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-4-hydroxy-3-[(2-hydroxy-5-nitrophenyl)azo]naphthalene-2,7-disulphonato(4-)]chromate(5-)

Endpoint summary

Description of key information

The NOAEL for systemic toxicity via oral route was found to be 500 mg/kg/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

Principles of method if other than guideline:

None

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SPF breeding VELAZ Černý Vůl
- Age at study initiation: 6-8 weeks
- Housing: 2-3 rats of the same sex in one plastic cage containing dry and sterilized clean shavings of soft wood.
- Diet (e.g. ad libitum): standard complete pelleted diet ST1 (supplier Bergman, Jesenice u Prahy)
- Water (e.g. ad libitum): free access to drinking water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 23 ±-3 °C
- Humidity: 40-60 %
- Photoperiod: 12-hour light/12-hour dark cycle

Route of administration:

oral: gavage

Vehicle:

other: water with addition of 0.5 % methylcellulose

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: daily just before administration. The concentrations of suspensions at each dose levels were adjusted, so that the administration would ensure the equals 1 mL per 100 g of body weight of the test animal and the volume was constant for each dose levels. During the application, the suspension was permanent mixed by electric stirrer.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

None

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

5 males and 5 females per dose

Control animals:

yes, concurrent no treatment

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on the results of the dose-range finding experiment
- Post-exposure recovery period in satellite groups: 14 days

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: in 7-days intervals

BODY WEIGHT: Yes
- Time schedule for examinations: in 7-days intervals

FOOD CONSUMPTION:
- Food consumption for each animal determined: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time x 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Before the start of the study and than in the 28th and the 42nd day of the study. In satellite groups was the examination performed in the 42nd day.
- Anaesthetic used for blood collection: Yes - ether
- Animals fasted: Yes (18 h)
- How many animals: all animals
- Parameters examined: Haemoglobin, Total count of Erythrocytes, Haematokrit , MCV (the mean corpuscular volume), Total count of Leucocytes, Thrombocytes, Differential leukocyte count

BIOCHEMICAL EXAMINATION

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in the 28th and the 42nd day of the study
- Animals fasted: Yes (18 h)
- How many animals: all animals
- Parameters examined: Total protein, Glucose, Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Total bilirubin, Creatinine, Urea, Albumin, Alkaline phosphatase (ALP), Na+, K+, Ca2+, Cl-

URINALYSIS: Yes
- Time schedule for collection of urine: in the 28th and the 42nd day of the study
- Metabolism cages used for collection of urine: Yes
- Parameters examined: quantity of erythrocytes, haemoglobin, ketones, bilirubin, urobilinogen, glucose, protein and pH.

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- macroscopic revision of thoracic, abdominal and cranial cavity
- selected organs were collected for weighing and further histological examination
- the absolute weights of liver, adrenal glands, kidneys, brain, and in males also of testes were recorded
- somatic indexes were calculated

HISTOPATHOLOGY: Yes
- brain, lungs, heart, liver, spleen, pancreas, adrenal glands, kidneys, esophagus, stomach, small and large intestine, testes, epididymides, uterus and ovary

Other examinations:

None

Statistics:

The non-parametric Mann-Whitney test was used for statistical analysis of results.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY
Changes in the stool consistence were observed in some animals during the clinical observation. An increased restlessness and different coloration of the auricles, nostrils and fingers were found in animals at the highest dose level.

HAEMATOLOGY
The dose level - 100 mg/kg: The values of total leucocytes count in treated animals were on the upper limit of physiological norms. Other haematological values were in the physiological range.
The dose level - 500 mg/kg: All of haematological values were in the physiological range.
The dose level - 1000 mg/kg: The haematological examination of animals showed changes in the differential leukocyte count with occurrence of young developmental forms especially myelocytes and metamyelocytes. In most of animals at the highest dose level occurrence of stomatocytes were observed in the microscopic examination of blood smears. Increasing of total number of leukocytes above limit of physiological norms was observed in males. In females, the value of MCV (mean corpuscular volume) was in the physiological range and haemoglobin was at the lower limit.

CLINICAL CHEMISTRY
Values of urea concentration, potassium and chloride ions were statistically significantly increased in males at the lowest dose level (100 mg/kg), but the values of concentration of sodium and calcium ions were decreased. In females at the highest dose level, increased values of cholesterol and concentration of potassium were recorded. Although the parameters were statistically significant, all of them were within physiological range.
Values of activity of AST were recorded statistically significantly increased and concentrations of creatinine, sodium and calcium ions were statistically significantly decreased in males at middle dose level (500 mg/kg). In females, increased values of concentration of potassium ion and activity of ALT, AST, ALP were recorded. Albumin and sodium ions concentration were decreased. Nevertheless all of biochemical parameters were within the physiological range.
Statistically significantly decreased concentrations of creatinine and sodium ions were recorded in animals at the highest dose level (1000 mg/kg) Decreased activity of ALP and concentration of sodium ions and increased concentration of total bilirubin and concentration of potassium ions were recorded in females. Although the parameters were statistically significant, all of monitored values were within physiological range. Increased concentration of chloride ions above physiological range was recorded in both sexes at the highest dose level. Concentration of sodium ions decreased below the limit of physiological range.
In males from the satellite group, significantly statistically increasing of concentrations of total protein and urea were recorded. The albumin and calcium concentration was decreased. In females were recorded decreased values of urea, AST, total bilirubin, sodium and calcium ions. Although the parameters were statistically significant, all of monitored values were within the physiological range.

GROSS PATHOLOGY
Bluish coloration of tail skin was observed in males at middle dose level (500 mg/kg), who were sacrificed at the end of application period. Two males and one female at the highest dose level (1000 mg/kg) showed also stomach mucous membrane coloration. Enlargement of spleen was observed in two males and four females at the highest dose level and testes reducing was observed in one male.

HISTOPATHOLOGY: NON-NEOPLASTIC
The significant histopathological changes - macroscopically enlarged spleen, were found at the highest dose level (1000 mg/kg). In the histological examination was evident enlargement of a red pulp with dilatation of the sinuses with erythrocytes and numerous of scavenger cells with a rusty pigment. Particles of pigment were also situated freely. The affect with this character was in two males and one female. The kidney cortex with a round-cell infiltration was observed in three males. There were observed advanced degree of dystrophy of the germinal epithelium of testes with a total disappearance of all layers of this epithelium (maintenance of the Sertoli cell only) in one male. The dark-blue pigment residues in the stomach mucous membrane were recorded in two females at the highest dose level. The fatty droplets were found only in the interstitium of the renal papilla in one male.

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The changes of the haematological parameters and increased erythrocyte haemolysis, which caused damage of spleen tissue and reversible changes of the biochemical parameters.

Critical effects observed:

not specified

DISCUSSION

Changes in the stool consistence were observed in some animals during the clinical observation. An increased restlessness and different coloration of the auricles, nostrils and fingers were found in animals at the highest dose level. The body weight increment of treated animals was not significantly different in comparison with the animals from the control group. Although the results of biochemical parameters were statistically significant, most of monitored values were within the physiological range. Only the concentration of chloride ions was on the upper limit of physiological norms and oppositely, the concentration of sodium ions was on the lower limit. The concentrations of ions returned on physiological range in satellite groups. The spleen oedema was found during the histopathological examination in seven animals (in two males and five females) at the highest dose level and in three females at satellite group administered by the highest dose level of the test substance. It was apparent, that there was no inflammatory – septic splenomegaly. The occurrence of the russet pigment in the scavenger cells or freely placed as a particles in the red pulp, is probably associated with occurrence of the stomatocytes in blood smears, when the inverted shape of erythrocytes were caused by poor membrane. So the occurrence of haemolysis of erythrocytes was increased. Other haematological parameters correspond to this pathological status, especially decreased concentration of haemoglobin and increased volume of erythrocytes. The reaction of the tissue was not in any case caused by macroscopically and microscopically detectable residues of dark-blue pigment on the mucous membrane of stomach. Also the small nodular clusters of the mononuclear cells in liver were also occurred in limited number of animals at all dose levels. Their character did not demonstrate the toxic effect of the test substance, because the occurrence of alteration of liver cells is missing, nor the results of biochemical examination did not indicate the toxic effect. With increasing dose level, any increased frequency or intensity of mentioned affections were recorded. The consequence of the quantitative differential in the quantity of neutral lipids in the plasma of hepatocytes is given by individual difference of lipids metabolism. In all cases it was finely droplet or irregular steatosis, not dystrophic steatosis with character of dying liver cells. The other pathomorphological affection has not integral character (spermatic granuloma in epididymides, advanced dystrophy of testes and occurrence of non-septic nephritis of interstitium) in animals at the highest dose level. It may be classified like a spontaneous and occasional. After the examinations may be stated, the test substance had a negative effect in dependence of sexes and doses, because it caused a dose dependent hard harm in females at the highest dose level (1000 mg/kg).

Conclusions:

The mid dose of 500 mg/kg may be considered as NOEL (no-observed-effect level).

Executive summary:

The test substance Ostazin Black H-N was tested in the 28-days study according to the guideline OECD No. 407 (OECD, 1981). The test substance was administered to Wistar rats as a suspension in water using a stomach tube. The concentrations of suspensions were 100, 500 and 1000 mg/kg of the body weight. During the study, checking of the health status was performed daily. The detailed clinical observation, food consumption and record of the body weight were performed weekly. At the same time interval (weekly), test substance volume was adjusted according to the body weight of the animals. Haematological and biochemical analysis and the urinalysis were performed of the 28th and 42nd day of study. At the end of application period of the test substance or the observation period, gross necropsy of animals with the macroscopic revision of the thoracic, abdominal and cranial cavity was performed. The selected organs for histopathology examinations were removed. The test substance at the lowest (100 mg/kg) dose level and at the middle dose level (500 mg/kg) did not affect a behaviour of the test animals and body weight increment and did not cause changes in haematological and biochemical parameters with a deviation from physiological norms. Also the results of histopathological examinations did not demonstrate changes depending on the test substance either. Therefore the middle dose level (500 mg/kg) may be considered as NOEL (no-observed-effect level). The test substance at the highest dose level (1000 mg/kg) caused changes in behaviour of the test animals and stool consistency. Changes in the haematological parameters were detected. There was a change in the differential leukocyte count with occurrence of young developmental forms, especially myelocytes and metamyelocytes. An occurrence of the stomatocytes was observed in a microscopic examination of blood smears. Increased total number of leukocytes above the limit of physiological norms was observed in males. In females, the value of MCV (the mean corpuscular volume) was increased above the limit of physiological ranges and haemoglobin was at a lower limit. Biochemical parameters showed increasing concentration of Cl^- ions above the limit of physiological norms and the concentration of Na⁺ions decreased below the limit of physiological norms. The histopathological examination of the animals at the highest dose level revealed enlargement of the spleen with russet pigment and numerous scavenger cells in a red pulp. In the satellite groups the haematological and biochemical values returned to the physiological range, however increased total number of leukocytes were still seen in males only. Histopathological changes at the highest dose level (1000 mg/kg bw/day) persisted in both sexes, especially in females. Based on the above findings, it can be said that this substance (or metabolites) caused changes dependent on dose levels and sex of the animals. The changes of the haematological parameters and increased erythrocyte haemolysis, which caused damage of spleen tissue, were recorded. The test substance also caused reversible changes of the biochemical parameters. With respect to the results of the study, the middle dose level 500 mg/kg may be considered as NOEL (no-observed-effect level).       

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

500 mg/kg bw/day

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated dose toxicity: oral

The test substance Ostazin Black H-N was tested in a 28-day study according to the guideline OECD No. 407 (OECD, 1981). The test substance was administered to Wistar rats as a suspension in water using a stomach tube. The concentrations of suspensions were 100, 500 and 1000 mg/kg of the body weight. During the study, checking of the health status was performed daily. The detailed clinical observation, food consumption and record of the body weight were performed weekly. At the same time interval (weekly), test substance volume was adjusted according to the body weight of the animals. Haematological and biochemical analysis and the urinalysis were performed of the 28th and 42nd day of study. At the end of application period of the test substance or the observation period, gross necropsy of animals with the macroscopic revision of the thoracic, abdominal and cranial cavity was performed. The selected organs for histopathology examinations were removed. The test substance at the lowest (100 mg/kg) dose level and at the middle dose level (500 mg/kg) did not affect a behaviour of the test animals and body weight increment and did not cause changes in haematological and biochemical parameters with a deviation from physiological norms. Also the results of histopathological examinations did not demonstrate changes depending on the test substance either. Therefore, the middle dose level (500 mg/kg) may be considered as NOEL (no-observed-effect level).

At the highest dose level (1000 mg/kg) the test substance caused changes in behaviour of the test animals and stool consistency. Changes in the haematological parameters were detected. There was a change in the differential leukocyte count with occurrence of young developmental forms, especially myelocytes and metamyelocytes. An occurrence of the stomatocytes was observed in a microscopic examination of blood smears. Increased total number of leukocytes above the limit of physiological norms was observed in males. In females, the value of MCV (the mean corpuscular volume) was increased above the limit of physiological ranges and haemoglobin was at a lower limit. Biochemical parameters showed increasing concentration of Cl^- ions above the limit of physiological norms and the concentration of Na⁺ions decreased below the limit of physiological norms. The histopathological examination of the animals at the highest dose level revealed enlargement of the spleen with russet pigment and numerous scavenger cells in a red pulp. In the satellite groups the haematological and biochemical values returned to the physiological range, however increased total number of leukocytes were still seen in males only. Histopathological changes at the highest dose level (1000 mg/kg bw/day) persisted in both sexes, especially in females. Based on the above findings, it can be said that this substance (or metabolites) caused changes dependent on dose levels and sex of the animals. The changes of the haematological parameters and increased erythrocyte haemolysis, which caused damage of spleen tissue, were recorded. The test substance also caused reversible changes of the biochemical parameters. With respect to the results of the study, the middle dose level 500 mg/kg may be considered as NOEL (no-observed-effect level).       

Repeated dose toxicity: via oral route - systemic effects (target organ) cardiovascular / hematological: spleen

Repeated dose toxicity: inhalation

The test substance has very low vapour pressure (< 10E-05 hPa at 25 °C) so the potential for the generation of inhalable forms is low. It has a high water solubility of 443 g/L, indicating if vapours are produced will be trapped in the mucus, thereby limiting the absorption. The test item synthesis and spray drying is performed in a closed process; the final product consists of non-dusty granules. In addition, the use of this substance will not result in aerosols, particles or droplets of an inhalable size, so exposure to humans via the inhalation route will be unlikely to occur. Furthermore, the results of laboratory animal studies show low acute oral or dermal toxicity. The oral route proved to be the relevant route of exposure and due to the good water solubility, the oral route of exposure is the one deemed to prove the highest exposure to the test substance in repeat-dose animal studies to investigate possible adverse effects.Further results of exposure to test animals via the oral route from repeated dose toxicity study with the source substance are available, hence no elevated toxicity other than seen in this study, is expected via the inhalation route and safety for human health can be estimated via route to route extrapolation.Further studies for this endpoint are therefore not appropriate both on predictive toxicology and animal welfare grounds.

Repeated dose toxicity: dermal

Currently no study to assess repeated dose dermal toxicity of Reactive Black 8 is available. However, the molecular weight of the substance is1384.8g/mol, which indicates substance is too large for dermal absorption. Further, high water solubility (443 g/L) and low partition coefficient (<-6.29), indicate the substance may be too hydrophilic to cross the lipid rich environment of the stratum corneum. Hence, the dermal uptake for the substance is expected to be low.The oral route proved to be the relevant route of exposure and due to the good water solubility, the oral route of exposure is the one deemed to prove the highest exposure to the test substance in repeat-dose animal studies to investigate possible adverse effects.Further results of exposure to test animals via the oral route in a repeated dose toxicity study are available, hence no elevated toxicity other than seen in this study, is expected via the dermal route and safety for human health can be estimated via route to route extrapolation. Similarly, absence of systemic toxicity or mortality in skin irritation as well as sensitization studies with Reactive Black 8, further supports the conclusion that no additional adverse effects other than seen in repeated dose oral toxicity study are expected via dermal route. Further studies for this endpoint are therefore considered to be not appropriate both on predictive toxicology and animal welfare grounds.

Justification for classification or non-classification

Based on the above stated assessment of the repeated dose toxicity of Reactive Black 8, the substance does not have to be classified according to Council Directive 2001/59/EC and according to CLP (Regulation (EC) No 1272/2008 Of the European parliament and of the Council.