Disodium; 4-Amino-3-{4-[4-(2,4-diamino-phenylazo)-benzenesulfonylamino]-phenylazo}-5-hydroxy-6-phenylazo-naphthalene-2,7-disulfonate

Endpoint summary

Description of key information

NOAEL oral rat > 2000 mg/kg

NOAEL dermal rat > 2000 mg/kg

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:

2020

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 420 (Acute Oral Toxicity - Fixed Dose Method)

GLP compliance:

yes (incl. QA statement)

Test type:

fixed dose procedure

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

Species: laboratory albino rat (it is the preferred rodent species according to the guideline), females, nulliparous and non-pregnant
Strain: Wistar Han, monitored quality
Source: breeding farm VELAZ s.r.o., Koleč u Kladna, Czech Republic, RČH CZ 21760118
Sex: females
Age: 6-7 weeks at the time application
Acclimatisation: 6 days
Total number: 5 females
Housing: animal room with monitoring conditions, plastic breeding cages Velaz T4, sighting study 1 animal/cage, main study - 4 animals/cage
Diet: Altromin for rats, Manufacturer: Altromin Spezialfutter GmbH & Co. KG, Germany supplied via VELAZ
Water: drinking tap water ad libitum (quality corresponding to Regulation No. 252/2004 Czech Coll. of Law)
Microclimatic conditions: room temperature 22 + 3°C, permanently monitored
relative humidity 30 – 70 %, permanently monitored
light period 12-hour light/12 hour dark
Bedding: sterilized shavings of soft wood
Randomisation: according to the internal rule, at the start of the study the weight variation of animals was minimal
Identification of animals: colour marks on tail of animals, each cage was marked with the number of study, sex and dose of the test item
Health condition: certificate of good health condition – from breeding farm; no signs of diseases were observed at clinical check-in, during the acclimatisation period and before the start of study

About twenty hours before oral administration the animals were not fed, water was given ad libitum. Immediately before application the animals were weighed. The feed was given to animals 3 hours after application of the test item.

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

Immediately before application the test item was weighed and mixed in vehicle (Aqua pro iniectione) and resulting suspension was administered to the stomach by tube. The single volume of administered suspension was 1 ml/100 g of animal body weight

Doses:

2000 mg/kg

No. of animals per sex per dose:

5 females

Control animals:

no

Details on study design:

Sighting study - the dose level of 2000 mg/kg was used as the starting dose - no death of animal was recorded.
Main study - on the basis of result of sighting study dose level 2000 mg/kg with four animals was used. No death of animals was detected.
Final number of animals for dose level 2000 mg/kg – 5 animals (1 animal – sighting study and 4 animals - main study).

Animals were weighed before application, at the 8th day of study and at the 15th day, before euthanasia of animals. Average body weight in a group was calculated from individual body weights. Body weight increments were calculated from body weight at the start of the study, the first week and at the end of the study.

After application the animals were observed individually:
- the first day: twice (30 minutes and 3 hours after application)
- the second day: twice (in the morning and in the afternoon) and daily thereafter for 14 days.
Observations included changes in skin and fur, eyes, visible mucous membranes, behaviour of animals, somatomotoric activity, reactions to stimuli, and presence of lacrimation, salivation and discharge from nostrils, function of respiratory, digestive and urogenital system.
The results of the observations were recorded on special data sheets.

All test animals survived to the end of study were sacrificed on the 15th day and gross necropsy was carried out. Nutritious status, body surface, body foramina, thoracic, abdominal and cranial cavity were evaluated. All gross macroscopic changes of organs and tissues were recorded on special data sheets

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No death recorded

Clinical signs:

other: No clinical sings of intoxication were observed during the study

Gross pathology:

No pathologic macroscopic changes were diagnosed during pathological examination.

Interpretation of results:

other: not classified for acute oral toxicity

Conclusions:

The test item toxicity was evaluated on the basis of mortality, clinical signs of intoxication, body weight increments during the observation period and necropsy findings at the end of study.
The test item administered at the dose of 2000 mg/kg caused no death of animals. No clinical sings of intoxication were observed.
Body weight and weight increments were adequate to species, sex and age of animals in experiment. No pathologic macroscopic changes were diagnosed during pathological examination.
According to the study results the value of LD50 of the test item, Acid Black 234, for female rats is higher than 2000 mg/kg of body weight

Executive summary:

The aim of the study was to investigate acute toxic effects of the test item Acid Black 234, after a single oral administration to Wistar rats.

The testing was performed according to the OECD Test Guideline No. 420: Acute Oral Toxicity – Fixed Dose Procedure, adopted 17th December 2001.

With respect to information available indicating that the test item is likely to be nontoxic, the study was performed as limit test.

First the sighting study was performed, using the starting dose of 2000 mg/kg of body weight with one female, then followed main study with group of four females, dosed by the same dose of 2000 mg/kg of body weight.

The test item was administered in a single dose by gavage using a stomach tube to female Wistar CRL rats. After dosing the animals were observed individually for a total of 14 day. At the end of the test surviving animals were killed. Necropsy of all animals were carried out, and all gross pathological changes were recorded.

The test item administered at the dose level of 2000 mg/kg did not cause the death of animals. No clinical signs of intoxication were recorded. No pathologic macroscopic changes were diagnosed during pathological examination.

According to the study results the value of LD50 of the test item, Acid Black 234, for female rats is higher than 2000 mg/kg of body weight.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Acute toxicity: via dermal route

Link to relevant study records

Reference

Endpoint:

acute toxicity: dermal

Type of information:

other: read across from supporting substance

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 420 (ABK210 Consortium, 2020).

Groups of five female Wistar rats received a single dose of 2000 mg/ks of the test sample.

First the sighting study was performed, using the starting dose of 2000 mg/kg of body weight with one female, then followed main study with group of four females, dosed by the same dose of 2000 mg/kg of body weight.

The test item was administered in a single dose by gavage using a stomach tube to female Wistar CRL rats. After dosing the animals were observed individually for a total of 14 day. At the end of the test surviving animals were killed. Necropsy of all animals were carried out, and all gross pathological changes were recorded.

The test item administered at the dose level of 2000 mg/kg did not cause the death of animals. No clinical signs of intoxication were recorded. No pathologic macroscopic changes were diagnosed during pathological examination.

Moreover, other two tests are available on similar substance 1, both confirming the non toxicity of the substance, with a LD50 > 2000  mg/kg.

ACUTE TOXICITY - INHALATION ROUTE

No acute toxicity studies by inhalation route are available on Acid Black 234.

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 234 Consortium, 2011) showed that ABk234 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 20 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 similar substance 1 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).