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

not eye irritant for rabbits

not skin irritant for rabbits

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1996

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 404 (Acute Dermal Irritation / Corrosion)

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Species: Rabbit
Strain: New Zealand White albino
Source: Charles River UK Limited, Manston Road, Margate, Kent, UK
Sex:female
Specification: Young adults
The three animals weighed 2885g, 2210g and 2470g at the beginning ofthe study.

The rabbits were housed individually in aluminium sheet cages in racks suitable for animals of this strain and the weight range expected during the course ofthe study. The rabbits were transferred to clean cages and racks, as necessary, during the study.
The animai room was designed to give the environmental conditions shown as follows:

Temperature: 17±2°C
Relative humidity: 40-70%
Air:Approximately 25 changes/hour
Light cycle:Artificial 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 generai, 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 (ST ANRAB SQC) Standard Diet Services Limited, Stepfield, 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 interfering with the purpose or outcome of the study.

The animals were housed under the experimental conditions for at least 5 days prior to the start of the study.

Animals were individually identified with a number, unique within the study, which was written on the inner side of one ear, using a black, waterproof, marker-pen.
On the front of each cage was a card identifying the contained animal by procedure code, test substance, date of administration, dose level, individuai number, sex and study

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

water

Controls:

not required

Amount / concentration applied:

The test sample (approximately 500mg) was moistened with a small amount (0.5ml) of deionised water

Duration of treatment / exposure:

four hours of application

Observation period:

10 days

Number of animals:

3 females

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

ca. 0.33

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

ca. 0.33

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

ca. 0.33

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

4

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

4

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

4

Irritant / corrosive response data:

Very slight erythema had completely regressed 2 days after application

Prior to the start of the study, all rabbits were examined to ensure that they were physically normal and exhibited normal activity. Throughout the study, the animals were observed daily.

A small area on the application site of two animals was stained purple by the test sample, but this did not prevent the assessment of irritation.

Very slight erythema was observed in all three animals for 1 day after application, but had completely regressed 2 days after application. Very slight oedema was observed in one animai approximately 30 minutes after decontamination, but had completely regressed 1 day later.

There were no additional signs of irritation

Interpretation of results:

other: not irritant

Conclusions:

The tested substance is non-irritant to rabbit skin following a single 4-hour application.

Executive summary:

A group of 3 female rabbits received a single four-hour application of tested Substance to the shorn flank. The animals were assessed for up to 10 days for any signs of skin irritation. Very slight erythema had completely regressed 2 days after application.

The tested substance is non-irritant to rabbit skin following a single 4-hour application.

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

other: read across from supporting substance

Adequacy of study:

key study

Study period:

1997

Reliability:

2 (reliable with restrictions)

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.

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

A total of 3 New Zeland White male rabbits were supplied by B&K Universal GJsl. On arrival at Centro de Investigacion Aplicado SAL the animals weighed 2.0-2.1 kg and were aged approxiamtely 9-10 weeks.
Before administration they were subjected to a prelimianry observation and acclimtization period of at least 7 days. At the start of the study the animals were in perfect health codnitions and weighed 2.2-2.3 kg.

Identification
The rabbits were individually identified using numbered ear tags. The number assigned to each rabbit was stated on the identification card on the cage.

Group size
The substance was tested on one group of three animals. The numbers assigned to the animals were 167, 184 and 195.

Housing
The rabbits were housed individually in stainless steel cages (48x59x37 cm) with a grille floor, placed on racks.
Each cage was identified by a card stating the study code number, administration route, animal number and sex, name or code of the test substance , animal delivery and administration date and study director's name..

Temperature in the animal room was maintained at 17-21°C. Relavtive humidity was between 45-70%.

Lighting was controlled to provide 12 hours light (7:00 to 19:00 hiours) and 12 hours dark per 24 hour period.

The animals were allowed free access to s standard rabbit diet UAR 112 which had been analysed by the manufacturer.

Water was supplied ad libitum by an automatic drinking system. The water, supplied by "Compania de Aguas de Sabadell SA" is periodically checked for the possble presence of contaminants.

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

other: dampened with water

Controls:

other: the untreated skin areas of the test animal serve as the control

Amount / concentration applied:

0.5g placed on a 2.5 x 2.5 cm squares of surgical gauze and applied to the corresponding test area

Duration of treatment / exposure:

4 hours

Observation period:

The reaction of the test zone was evaluated approximately 60 minutes, 24, 48 and 72 hours after the end if the exposure period.

Number of animals:

3 male rabbits

Details on study design:

The animals were shaved approximately 24 hours before administration usign an electric razor, leaving the back and sides exposed from the scapula to the pelvis. One square test zone measuring approximately 6.2 cm squares was delimited on the shaved intatc skin of one side of the animals. The substance was placed on 2.5 x 2.5 cm squares of srugical gauze and applied to the patches in place. The resulting patches were held close to the body using strips of adhesive tape. Finally, a strip of gauze was fixed to the body using hypoallergenic adhesive tape to maintain the semi-occlòusive bandage in place. The aim of this semi-occlusive bandage was to allow good skin absorption of the test substance. Following administration, the animals were observed for a few moments before being returned to their cages. Four hours later, the bandage and patches were removed and the remains of the product were removed with distilled water.

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

other: 60 min

Score:

ca. 1

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

other: 24h, 48h, 72h

Score:

0

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

other: 60min, 24h, 48h, 72h

Score:

0

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

other: 60 min

Score:

ca. 1

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

other: 24h, 48h, 72h

Score:

0

Irritation parameter:

edema score

Basis:

animal #1

Time point:

other: 60 min

Score:

0

Irritation parameter:

edema score

Basis:

animal #1

Time point:

other: 24h, 48h, 72h

Score:

0

Irritation parameter:

edema score

Basis:

animal #2

Time point:

other: 60min, 24h, 48h, 72h

Score:

0

Irritation parameter:

edema score

Basis:

animal #3

Time point:

other: 60min, 24h, 48h, 72h

Score:

0

Other effects:

The behaviour and physical condition of the rabbits were normal throughout the study

Interpretation of results:

other: not irritant

Conclusions:

The substance was administered for the study of primary skin irritation in rabbits. After assessment of the alterations caused according to OECD 404 and EU B4, it is considered NON IRRITANT to the skin.

Executive summary:

The degree of skin irritation caused by substance was determined after it was applied to shaved rabbit skin, under semi-occlusive patch and left for 4 hours. After assessment and evaluation of the alterations which it induced according to OECD 404 and EU B4 it is considered NON IRRITANT. In the examinations carried out in the first 60 minutes after removing the semi-occlusive patch, two animals presented very slight erythema (grade 1). In the examinations carried out 24, 48 and 72 hours after treatment, no dermal lesions were recorded in any of the animals. The mean erythem and edema scores for the substance at 24, 48 and 72 hours in the three animals, were both 0.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been conduced in GLP on the registering substance

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

yes

Remarks:

see below "Any other information..."

GLP compliance:

yes (incl. QA statement)

Species:

other: Bovine eyes

Details on test animals or tissues and environmental conditions:

Slaughter house: Butcher Service s.r.l. - Mattatoio no. 2067 M St. Teverina Km. 7800 - 01100 - Viterbo.
Age of animals: 6-12 months.
Killing time: From 8:30 to 12:00 in the morning.
Transport condition: Eyes were maintained in transport solution at approximately +4 °C.
Examination: Eyes were examined for the presence of any defects which could render the eye unsuitable for use (opacity, scratches or pitting
of the corneal surface, vascularisation or pigmentation).
Cornea excision: Each cornea with 2-3 mm of surrounding sclera was dissected from the eye using a scalpel, scissors and forceps and placed
into a Petri dish containing HBSS.
Mounting in the chamber: Each cornea was mounted into a prewarmed testing chamber with the endothelial surface of the cornea placed in contact
with the O-ring of the posterior part of the chamber. Care was taken to ensure the correct position of the cornea minimizing the
presence of pigmented area inside the O-ring and avoiding movements which would damage the cornea. The chamber was then
filled with complete EMEM without phenol red maintained at 321 °C (posterior part of the chamber first to maintain convexity).
Equilibration: The corneas in their holders were incubated in a liquid bath at 321 °C at least for 1 hour to permit metabolic stabilisation. At the end of the pre-dose incubation period, the two chambers were drained (anterior first) and re-filled with complete EMEM without
phenol red maintained at approximately 32 °C (posterior first).
Selection: Basal opacity of corneas was determined by means of a calibrated meter, measuring difference in light transmission between the corneas placed into the beam paths versus air (Opacitometer). During measurement, attention was paid to keep corneas away
from the bath for the minimal time needed. Corneas with a basal value7 arbitrary units were excluded from testing. The mean
opacity of the remaining corneas was determined. Corneas were distributed in treatment groups starting from those nearest
to the mean value for the negative control group and trying to approximately maintain opacity values similar to the mean of
the negative control group in the test item and positive control groups.

Vehicle:

physiological saline

Amount / concentration applied:

In the first experiment the test item was prepared at the concentration of 200 mg/mL in physiological saline (Baxter, batch no. 11D0802), immediately before use without correction for the displacement due to the volume occupied by the test item; thus the concentration obtained was lower than 20% w/v, stated in the study protocol. In the second experiment the final concentration was corrected and according to the protocol and OECD indications exactly at 20 % w/v

Duration of treatment / exposure:

Corneas were exposed in horizontal position for 4 hours+/- 10 minutes, incubated in a liquid bath at 32+/-1 °C.

Details on study design:

Eyes transport solution: Hanks Balanced Salt Solution (HBSS) modified, supplemented with Penicillin sulphate and Streptomycin sulphate both at the
final concentration of 100 IU/mL.
Complete EMEM: EMEM1 (with or without phenol red) supplemented with 1% (v/v) Foetal Bovine Serum. These media were prewarmed in a water
bath at 32 °C during the experimental procedure.
Sodium fluorescein solution: 5 mg/mL solutions of sodium fluorescein in DPBS
Method used: A closed chamber method was used, being the test item a solid non surfactant substance.
Treatment: The medium was completely removed from the anterior chamber. Using the closed chamber method, the treatment was
carried out by pipetting the formulated test item or control items through the dosing holes of the chamber.
Exposure period: Corneas were exposed in horizontal position for 4 hours10 minutes, incubated in a liquid bath at 321 °C.
Washing: After exposure, corneas were rinsed thoroughly with complete EMEM with phenol red. For the test item treated group, the
glass windows of the anterior chambers were removed to allow a more effective removal of the test item from the corneas.
A final wash with prewarmed complete EMEM without phenol red was carried out. Finally, the anterior chamber was re-filled
with prewarmed complete EMEM without phenol red.
Post-exposure period: There was no post-exposure period.

Irritation parameter:

in vitro irritation score

Value:

ca. 16.6

Negative controls validity:

valid

Interpretation of results:

other: The test item could not be clearly classified

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Based on the IVIS of 25.5, according to the OECD Guideline no. 437, the test item is clearly classified as non corrosive, but it could not be clearly classified as not irritant.

Executive summary:

The potential of the test item, Acid Black 210 Na, to cause corrosion/severe irritation was examined by using the Bovine Corneal Opacity and Permeability (BCOP) assay, in agreement with OECD Guideline no. 437 (adopted on 26 July 2013) and the Guidance Document OECD series on testing and assessment no. 160.

Two experiments were performed. In the first experiment, the test item was dissolved at 200 mg/mL in physiological saline and tested on the epithelial surface of three idoneous bovine corneas, for an exposure period of 4 hours. The mean opacity detected with an opacitometer at the end of the test item exposure period was 15.5. However, after the exposure period, the treated corneas appeared green-black coloured and the mean opacity value may have been affected by the colouration. No relevant increases in corneas permeability were observed after treatment with the test item.

Since the test item solution for this experiment was prepared without correction for displacement due to the volume occupied by the test item, in order to comply with the relevant guideline, a second experiment was performed.

In the second experiment the test item was dissolved at 20% (w/v) in physiological saline and tested on the epithelial surface of three idoneous bovine corneas, for an exposure period of 4 hours. The mean opacity detected with an opacitometer at the end of the test item exposure period was 23.7. This was confirmed at the macroscopic observation in which opacity of the treated corneas was noted although the treated corneas appeared green-black coloured. Slight increases in corneas permeability were observed after treatment with the test item.

In both experiments, positive and negative controls (a 20% (w/v) Imidazole solution in physiological saline and physiological saline alone, respectively), were concurrently tested in similar conditions and gave the expected results.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

other: read across from supporting substance

Adequacy of study:

key study

Study period:

1997

Reliability:

2 (reliable with restrictions)

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.

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

A total of 3 animals were supplied by B&K UNIVERSAL GJ. On arrival at Centro de Investigacion y Desarollo Aplicado SAL the animals weighed 2.0-2.1 kg and were aged approxiamtely 9-10 weeks. Before administration, they were subjected to a preliminary observation and acclimatization period of 7 days. At the start of the study, the aniamls were in perfetc health and the bodyweight was between 2.1-2.3 kg.
The new Zeland White Rabbit was chosen as a test species because it is an animal normally accepted and suitable fpr the study of eye irrittion and there is sufficient background information to justify its use.
Identification
The rabbits were individually identified usign numbered tags, placed on the rabbits ears. The number assigned to each rabbit was stated on the identification card on the cage.

Group size
The substance CARBON DERMA AF 135 was tested on a group of three animals. The number assigned to the animals were 169, 191 and 194.+

Housing
The rabbits were housed individually in stainless steel cages with a grille floor (48x59x37 cm) placed on racks. Each cage was idenitified by a card stating the study code number, animal number and sex, the quantity administered and name or code of the test substance, study director's name, route and animal arrival and administration dates.
Temperature in the animal room was maintained at 17-21°C and the relative humidity was between 45-70%.
Lighting was controlled to provide 12 hours light (7:00 to 19:00 hours) and 12 hours dark per 24-hour period. Throughout the study, the animals had free access to a standard rabbit diet UAR 112 which has been analysed by the manufacturer. Water was supplied ad libitum by an automatic drinking system. The water, supplied by "Compania de Aguas de Sabadell SA" is periodically checked for possible contaminants.

Vehicle:

water

Controls:

other: the untreated left eye was used as a control

Amount / concentration applied:

The quantity of test substance administered to each animal was 0.1ml, not exceeding a mass of 100 mg.

Duration of treatment / exposure:

The test substance was instilled in a single application.

Observation period (in vivo):

The behaviour of the animals was observed immediately following instilaltion of the test substance and they were then returned to their cages and observed at regular intervals so as not to miss any serious effect of the test substance. The degree of eye irritation was evaluated for each rabbit 1, 24,48 and 72 hours after treatment according to the criteria outlined below. Additional observations were carried oput after 6 days to check the reversibility or irreversibility of the observed reactions.

Number of animals or in vitro replicates:

3 animals

Details on study design:

Both eyes of each animal were examined before treatment in order to reject any animal showing eye alterations. The animal was immobilised and the product was poured into the right conjunctival sac, made perfectly accessible by carefully pulling outwards the lower lid.After application of the test substance, the lids of the treated eye were held closed for a few seconds.

Irritation parameter:

cornea opacity score

Remarks:

(corneal opacity)

Basis:

animal #1

Time point:

other: mean at 24, 48 and 72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Remarks:

(corneal opacity)

Basis:

animal #2

Time point:

other: mean at 24, 48 and 72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Remarks:

(corneal opacity)

Basis:

animal #3

Time point:

other: mean at 24, 48 and 72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 h

Irritation parameter:

iris score

Basis:

animal #1

Time point:

other: mean at 24, 48 and 72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

animal #2

Time point:

other: mean at 24, 48 and 72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

animal #3

Time point:

other: mean at 24, 48 and 72 h

Score:

0

Max. score:

4

Irritation parameter:

conjunctivae score

Remarks:

(hyperaemia)

Basis:

animal #1

Time point:

other: mean at 24, 48 and 72h

Score:

0.78

Max. score:

4

Reversibility:

fully reversible within: 72 h

Irritation parameter:

conjunctivae score

Remarks:

(hyperaemia)

Basis:

animal #2

Time point:

other: mean at 24, 48 and 72 h

Score:

0.78

Max. score:

4

Reversibility:

fully reversible within: 72 h

Irritation parameter:

conjunctivae score

Remarks:

(hyperaemia)

Basis:

animal #3

Time point:

other: mean at 24, 48 and 72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 6 d

Irritation parameter:

conjunctivae score

Remarks:

(Edema)

Basis:

animal #1

Time point:

other: mean at 24, 48 and 72 h

Score:

0

Max. score:

4

Irritation parameter:

conjunctivae score

Remarks:

(edema)

Basis:

animal #2

Time point:

other: mean at 24, 48 and 72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 h

Irritation parameter:

conjunctivae score

Remarks:

(edema)

Basis:

animal #3

Time point:

other: mean at 24,48 and 72 h

Score:

0

Max. score:

4

Other effects:

The behaviour and physical condition of the rabbits were normal throughout the study.

Interpretation of results:

other: not irritant

Conclusions:

The substance was administered for the study of Primary Eye Irritation. After assessment and evalutaion of the alterations caused by the substance, according to OECD 405 and EU B5 it is considered to cause NO IRRITATION to the eye.

Executive summary:

The test substance was administered for the study of primary eye irritation. After assessment and evalutaion of the alterations caused by the substance, according to OECD 405 and EU B5 it is considered to cause NO IRRITATION to the eye.

In the course of the first hour after instillation, the test substance induced in one animal slight swelling (grade 1) of the eyelids and nictating membrane and slightly congestive iris (grade 1). Similarly, all the treated animals presented slight lacrimation. Evaluation of hyperaemia in teh conjunctivae was not possible due to the colouring caused by the product in them.

24hours after treatment, all the animals presented some blood vessels definitely hyperaemic (grade 1) in the conjunctivae, accompanied by slight swelling (grade 1) of the eyelids and nictating membrane in one animal and diffuse areas of opacity in the cornea ( grade 1), affecting at least a quarter of the corneal area in another animal.

In the reading made 48 hours after administration, the observed lesions had remitted and by the end of 72 hours after treatment only one animal continued to present some blood vessels definitely hyperaemic ( grade 1) in the conjunctivae. This lesion had completely disappeared by the addtional reading made 6 days after administration.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

For eye irritation/corrosion:

Classification for corrosion was based on a study presented in the framework of DSD notification of new substances, reporting eye damage not reversible within 28 days (Stahl Europe B.V.); few information is available on the study and several attempts to recover the original reports have been made, without success, either by the owner of the notification or the responsible member state (France, presumably).

 

 

According to article 3.3.2.6 of CLP Regulation the proposed classification is justified in the following cases:

Case 1)

“These observations include animals with grade 4 cornea lesions and other severe reactions (e.g., destruction of cornea) observed at any time during the test, as well as persistent corneal opacity,discoloration of the cornea by a dye substance, adhesion, pannus, and interference with the function of the iris or other effects that impair sight. In this context, persistent lesions are considered those which are not fully reversible within an observation period of normally 21 days.”

Case 2)

Substances are also classified in Category 1 if, when applied to the eye of an animal, a substance produces:

–at least in one animal effects on thecornea, iris orconjunctiva that are not expected to reverse or have not fully reversed within an observation period of normally 21 days;

and/or

– at least in 2 of 3 tested animals, a positive response of:

– corneal opacity ≥ 3 and/or

– iritis > 1.5

calculated as the mean scores following grading at 24, 48 and 72 hours afterinstallation of the test material.

 

Case 1) doesn’t apply since the reports cite the following:

Coloration noire de la membrane nictitante réversible à J14 chez 2 animaux et non réversible à 28J chez 1 animal. Cette coloration a empeché l'appreciation de l'erytheme conjonctival jusque J14.

But discoloration is not referred to cornea.

 

Case 2) applies since the report indicates for conjunctiva score that for all animals the maximum value of redness has been observed at 28 days, while for all the other end points very slight effect has been reported.

 

Based on the available information the report is missing of many important details, like the identification of the substance and the related impurities and potential additives in case a commercial sample has been tested. No detail comments on controls and animal housing have been reported.

 

The result is in contradiction with the following complete studies performed on the substance or on the analogous salts of the substance.

Studies on analogous salts hare taken into full consideration since the counter ion has no influence on the outcome of the eye irritation study.Furthermore some of the specific salts present as impurities (Litium sulphate, Lithium chloride, Potassium sulphate) have been actually classified for eye irritation in the framework of REACH registration, letting then consider them as a conservative case in respect to sodium salt

 

 

Study 1: Key study – reliability 2 – Read across study from sodium/potassium salt

J. Zapatero (1997). Acute Oral Toxicity test in Rats. Fixed Dose Method. Test Substance: Carbon Derma AF 135. Testing laboratory: CENTRO DE INVESTIGATION Y DESAROLLO APLICADO S. A. L. Report no.: CD-96/5303T-1. Owner company: CLARIANT PRODUCTIONS S. A. Report date: 1997-07-03.

 

The study has been assigned Reliability 2 only because it has been performed on the sodium/potassium salt, but the study is complete and well conducted, following GLP and performed according to OECD 405

The substance has been identified with a purity of about 65% and impurities indicated as the following salts: sodium sulphate, sodium chloride and potassium chloride.

 

All the effects have been properly assessed and described in detail. No effect has been reported for any end point after 6 days in any animal

 

Study 2: supporting study – reliability 2 – Read across study from litium/potassium salt

BASF AG (1984). Report on the acute irritation to the eye of the white rabbit based on OECD 405. Testing laboratory: BASF Department of Toxicology. Report no.: 83/259. Owner company: BASF SE. Report date: 1984-03-01 (Original language: German).

 

The study has been rated Klimish 2 either becauseit has been performed on the lithium/potassium salt and it is not a GLP study even if the study is complete and performed according the OECD guideline 405 without deviations.

The substance is well identified and a certificate has been provided for exact composition: the tested substance consists of a 67% of the dye, considered as the mixture of potassium and lithium salt, 30% of water and a remaining 3% of inorganic salts (Lithium chloride).

 

All the effects have been properly assessed and described in detail. No effect has been reported for any end point after 72 hours in any animal

 

Study 3: Key study study – reliability 1 -

S. Cinelli (2014). ACID BLACK 210 Na, ASSESSMENT OF OCULAR CORROSION/SEVERE IRRITATION, BOVINE CORNEAL OPACITY/PERMEABILITY (BCOP) ASSAY. Testing laboratory: RTC. Report no.: XXXX. Owner company: REACH & Colours Kft . Report date: 2014-02-XX (Original language: English).

 

In order to completely assess the end point the Lead Registrant REACH & Colours Kft performed an in vitro test according to OECD 437, on the registered substance, which composition has been well defined in the composition IUCLID section of the registration dossier and consists of 66.5% of the dye, 16% of water, about 10% of inorganic salts (sodium sulphate and sodium chloride) and a remaining 3% of identified impurities.

 

The BCOP test method is recommended as an initial step within a tiered-testing strategy to identify chemicals inducing serious eye damage, i.e. chemicals to be classified as UN GHS Category 1, without further testing. A chemical that is not predicted as causing serious eye damage or as not classified for eye irritation/serious eye damage with the BCOP test method would require additional testing (in vitro and/or in vivo) to establish a definitive classification

 

According to the updated OECD 437 guideline of 17 September 2012, the IVIS cut-off values for identifying test chemicals as inducing serious eye damage (UN GHS Category 1) and test chemicals not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given hereafter:

 

IVIS	UN GHS
< 3	No Category
>3; < 55	No prediction can be made
> 55	Category 1

 

The test resulted in a medium IVIS of25.3 and alterations of the mean cornea opacity were observed. However, since the test item was coloured, the mean opacity value is affected by the substance remaining on the corneal surfaces. No relevant increases in corneas permeability were recorded after treatment with the test item when compared to those of negative control.

 

Conclusions

Two Klimish 1 studies (J. Zapatero, 1997 and BASF, 1984) have been performed on a mixture of different salts of the dye showing no effect that can trigger to a classification. They are well performed, the substance is well identified and their results are consistent. An in vitro study, according to OECD 437 as been also performed on the registering substance. The result according to the new update of OECD 437 of the 17 September 2012 cannot lead to a conclusion about the classification, but it can be considered as a weight of evidence, together with the two previous cited study for the removal of the classification.

For skin irritation/corrosion:

No skin irritant following EU B4 for rabbits, semiocclusive, 4h exposure, observations at 1, 24, 48 and 72h.

Two test are available, One study (Stahl, 1996) on acid Black 210 sodium salt and one test (Clariant, 1997), on Acid Black 210 potassium salt. Both tests have consistent results and show no skin irritation.

Justification for selection of skin irritation endpoint:

Both reliable in vivo tests. one performed in the registered substance, sodium salt (Stahl, 1996) and one on Acid Black 210, potassium salt (Clariant, 1997) are complete and detailed.

Justification for classification or non-classification

No classification for skin irritation/corrosion is warranted under Regulation 1272/2008

No classification for eye irritation/corrosion is warranted under Regulation 1272/2008