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

Endpoint summary

Description of key information

NOAEL oral rat > 2000 mg/kg bw

NOAEL dermal rat > 2000 mg/kg bw

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 august 1995 - 14 september 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

other: Alpk: APfSD (Wistar)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals:
species: rat
strain: Alpk: APfSD (Wistar)
source: Barriered animal Breeding unit, Zeneca Pharmaceuticals, Alderley Park
Sex: 5 male and 5 female
Specification: Young Adults, Males weighed 279-294g and the females weighed 200-256g at the beginning of the study.

Accomodation and husbandry:
A maximum of 5 rats was housed per cage, sexes separately, in multiple rat sacks suitable for animals of this strain and the weight range expected during the course of the study.
The rats were fasted overnight immediately prior to dosing to ensure that the stomachs were void of food (the presence of food could affect the rate of absorption of the test substance)
The rats were transferred to clean cages and racks,as necessary, during the study.
The animal room was designed to give the environmental conditions shows as follows:
temperature: 21±2°C
Relative humidity: 40-70%
Air: approximately 25-30 changes/hour
Light cycle: artifical giving 12 hours light, 12 hours dark

Both temperature and relative humidity were monitored continuously using an automated system which triggers an alarm if values are outside specified ranges. In general, the recorded values were within the specified ranges and any deviations that were observed are considered not to have affected the integrity of the study.

Diet (PCD) supplied by Special Diet Services Limited, Witham, Essex, UK and mains water, supplied by an automatic system were available ad libitum.

Each batch of diet is routinely analysed for composition and for the presence of contaminants. Water is also periodically analysed for the presence of contaminants. No contaminants were found to be present in the diet or water at levels considered to be capable of interering with the puropose or outcome of the study.


Acclimatisation:
The animals were housed under the experimental conditions for at least 6 days, prior the start of the study.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

A standard volume of 10 ml/kg of the dosing preparation was administered by oral gavage using a plastic catheter attached to a sterile disposable plastic syringe of suitable size.

Doses:

The dose levels selected for this study were based on the results of a preliminary study (speculative oral dosing study) in the rat.
The animals were given a single dose.
As no deaths were noted in the speculaive study, a single, limit dose level of 2000 mg/kg was administered.
The volume of the dose was calculated for each animal according to its weight at the time of dosing.

No. of animals per sex per dose:

5 males and 5 females

Control animals:

not specified

Details on study design:

Five male and five female rats were allocated to the study. animals were individually identified with a number, unique within the study, by ear punching.
On the front of each cage of animals was a card identifying the contained animals by procedure code, test substance,date of administration, dose level, individual number, sex and study.
A card was also displayed on the cage to indicate the date of the pre-dose fasting period.

Prior to the start of the sudy, all rats were examined to ensure that they were physically normal and exhibited normal activity. The animlas were observed for signs of systemic toxicity once within 2 hours of dosing and again between 4 and 7 hours after dosing. Subsequent observations were made once daily up to day 15.

The animals were weighed on the day before dosing (day -1), immediately before dosing (day 1) and at days 1, 3, 6, 8, 15.

All rats were killed by exsanguination under terminal anaesthesia induced by halothane vapour.

All animals were subjected to an examination post mortem. This involved an external observation and a careful examination of all thoracic and abdominal viscera. All abnormalities were recorded but tissues were not submitted for histopathological examination.

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

no deaths

Clinical signs:

other: no significant signs of toxicity. Upward curvature of the spine and piloerection were noted in all animals for up to 2 days. All animals showed yellow staining of the fur throughout the study and two males and one female had coloured faeces on day 1.

Gross pathology:

Post mortem examination revealed mottled areas in the lung of one female and red areas in the thymus of another female. These are common spontaneus findings in rats of this age and strain and are considered not to be treatment-related. There were no macroscopic finding in the males.

Interpretation of results:

other: not classified

Conclusions:

The acute oral medina lethal dose of the tested substance is in excess of 2000 mg/kg to male and female rats.

Executive summary:

Groups of five male and five female Alpk:APfSD(Wistar-derived) rats received a single dose of 2000 mg/ks of the test sample.

The animals were assessed daily for the following 15 days for any signs of systemic toxicity and their bodyweights were recorded at intervals throughout the study. At the end of the study all the animals were killed and subjected to a macroscopic examination post mortem. Following a single oral dose of 2000mg/kg, none of the animals died and there were no significant signs of toxicity.

The acute oral median lethal dose of the tested substance is in excess of 2000mg/kg to male and female rats.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Acute toxicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Acute toxicity: via dermal route

Link to relevant study records

Reference

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 October 1995-26 October 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

fixed dose procedure

Limit test:

yes

Species:

rat

Strain:

other: Alpk: APfSD (Wistar)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species: Rat
strain: Alpk: APfSD (Wistar)
source: Barriered Animal Breeding Unit, zeneca Pharmaceuticals, Alderley Park
Sex: female and male
specification: young adults. Males weighed 247-273g and the females weighed 178-210g at the beginning of the study

The rats were housed individually, in multiple rat racks suitable for animlas of this strain and the weight range expected during the course of the study.
The rats were transferred to clean cages and racks, as necessary, during the study.
The animal room was designed to give the environmental conditions shown below:
temperature: 21±2°C
relative humidity: 40-70%
Air: approximately 25-30 changes/hour
light cycle: artificial giving 12 hours light, 12 hours dark.

Both temperature and relative humidity were monitored continuosly using an automated system which triggers an alarm if values are outside specified ranges. In general, the recorded values were within the specified ranges and any deviations that were observed are considered not to have affected the integrity of the study.
Diet (PCD) supplied by Special diet Services Limited, Witham, Essex, UK and mains water, supplied by an automatic system were available ad libitum.
Each batch of diet is routinely analysed for composition and for presence of contaminants. Water is also periodically analysed for the presence of contaminants. No contaminants were found to be present in the diet or water at levels considered to be capable of interfering with the purpose or outcome of the study.

The animals were housed under the experimental conditions for at least 6 days, prior to the start of the study.

Five male and five female rats were allocated to the study. Animals were individually identified with a number, unique within the study, by ear punching.
On the front of each cage of animals was a card identifying the contained animals by procedure code, test substance, date of dministration, dose level, individual number, sex and study.

Type of coverage:

occlusive

Vehicle:

other: deionised water

Duration of exposure:

24 h

Doses:

single limit dose level of 2000 mg/kg

No. of animals per sex per dose:

5 males and 5 females

Control animals:

no

Details on study design:

As no deaths were noted in the speculative study, a single, limit dose level of 2000 mg/kg was administered.
The appropriate amount of the test sample was weighed onto a plastic weighing boat and made into a paste by adding a small amount (0.5-0-6ml) of deionised water. The amount applied was calculated for each animal according to its weight at the time of dosing. The test sample covered approximately two-thirds of the application site and the estimated amount applied per unit area of skin was 14.8-16.4mg/cm2 for males and 10.7-12-6mg/cm2 for females. The paste, covered by a 4 ply gauze patch (approximately 7cmx7cm) was applied to the shorn back of each animals and was kept in contact for 24 hours using an occlusive dressing. The gauze patch was covered by a patch of plastic film (7cmx7cm) and was held in position using adhesive bandage (25cmx7.5cm) which was secured by two pieced of PVC tape (approximately 2.5cmx20cm).

At the end of the 24-hour contact period, the dressing were carefully cut, using blunt tipped scissors, removed and discarded. the skin, at the site of application, was cleansed free of any residual test sample using clean swabs of absorbent cotton wool soaked in clean warm water and was then dried with clean tissue paper.

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

none of the animals died

Clinical signs:

other: no sign of toxicity. The skin of all the animlas was stained black or purple by the test sample and in some animals the application site was obscured, preventing the full assessment of irritation. there were pratically no signs of skin irritation (one mal

Other findings:

Apart from staining of the hair/skin by the test sample, there were no abnormalities in any animal.

Interpretation of results:

other: not irritant

Conclusions:

LD50 > 2000 mg/kg to male and female rats.

Executive summary:

A group of five male and five female Alpk: APfSD (Wistar) rats received a single dermal application of substance.

The animals were assessed daily for the following 14 days for any signs of systemic toxicity and their bodyweights were recorded at intervals throughout the study. At the end of the study all the animals were killed and subjected to a macroscopic examination post mortem.

Following a single dermal application of  2000 mg/kg, none of the animals died. There were no signs of  toxicity and practically no signs of skin irritation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Additional information

ACUTE TOXICITY - ORAL ROUTE

The acute toxicological characterization of the substance was determined by Acute toxicity test, according to the OECD guideline 401 (Stahl, 1996).

Groups of five male and five female Alpk:APfSD(Wistar-derived) rats received a single dose of 2000 mg/ks of the test sample.

The animals were assessed daily for the following 15 days for any signs of systemic toxicity and their bodyweights were recorded at intervals throughout the study. At the end of the study all the animals were killed and subjected to a macroscopic examination post mortem. Following a single oral dose of 2000mg/kg, none of the animals died and there were no significant signs of toxicity.

The acute oral median lethal dose of the tested substance is in excess of 2000mg/kg to male and female rats.

Moreover, another test is available on Acid Black 210, potassium salt (Clariant, 1995).

The acute toxicity of the substance was assessed folllowing oral administration in Sprague Dawley rats, by the fixed dose method. The substance was administered at the dose of 2000 mg/kg. None of the animals tretaed at the dose of 2000 mg/kg died in the course of the study.

The only observation made on the day of treatment was the execretion of deeply yellow-coloured urine by all the animals. This observation was also seen on the following day poste-treatment, accompained by black-coloured faeces.

The evolution in the bodyweights of all the animals was normal. In the necropsies there were no alterations detected

 

ACUTE TOXICITY - INHALATION ROUTE

No acute toxicity studies by inhalation route are available on Acid Black 210.

Nevertheless, because of the physical state and the trade forms of the substance inhalation is not an appropriate route of exposure. Due to the very low vapour pressure and the production procedures where antidusting additives are added to the substance, the inhalatory route has been considered as secondary with respect to the dermal one. The chosen route has been then dermal, for which a study has been performed.

Moreover, Particle size distribution (Acid Black 210 Consortium, 2011) showed that ABk210 is characterized by particles that are expected to remain in the upper respiratory tract, which is characterized by efficacious defence mechanisms able to remove them. The laser diffraction analysis recorded that the 90 and 50 percent of particles have a diameter higher than 50 µm, and the 10 percent have a diameter higher than 10 µm.

From this point of view, inhalation route is expected to be an unlikely route of absorption of the substance.

 

ACUTE TOXICITY - DERMAL ROUTE

Regarding the acute toxicity by dermal route, the substance was assayed in a toxicity test, according to the OECD guideline 402 (Stahl, 1996).

A group of five male and five female Alpk: APfSD (Wistar) rats received a single dermal application of substance.

The animals were assessed daily for the following 14 days for any signs of systemic toxicity and their bodyweights were recorded at intervals throughout the study. At the end of the study all the animals were killed and subjected to a macroscopic examination post mortem.

Following a single dermal application of  2000 mg/kg, none of the animals died. There were no signs of  toxicity and practically no signs of skin irritation.

 

 

 

Justification for selection of acute toxicity – oral endpoint

Study conducted according to internationally accepted testing guideline.

 

Justification for selection of acute toxicity – dermal endpoint

Study conducted according to internationally accepted testing guideline.

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), 3.1 Acute toxicity section, substances can be allocated to one of four toxicity categories based on acute toxicity by the oral, dermal or inhalation route according to the numeric criteria. Acute toxicity values are expressed as (approximate) LD50 (oral, dermal) or LC50 (inhalation) values or as acute toxicity estimates (ATE).

 

The oral LD50 value was established to be 2000 mg/kg body weight, therefore the test substance is out of any classification limit for acute oral toxicity (oral acute toxicity category 4: 300 < ATE ≤ 2000 mg/kg bw).

 

No data about acute toxicity by inhalation route is available. Nevertheless, based on the physical state and the trade forms of the substance, there are no reasons of concern and the inhalation can be considered as a non appropriate route of exposure.

 

The dermal LD50 value was established to be 2000 mg/kg body weight, therefore the test substance is out of any classification limit for acute dermal toxicity (dermal acute toxicity category 4: 1000 < ATE ≤ 2000 mg/kg bw).

 

In conclusion, the test substance is non classified for oral/dermal acute toxicity, according to the CLP Regulation (EC 1272/2008).