Sodium 4-amino-5-hydroxy-3-(4-nitrophenylazo)-6-(phenylazo)naphthalene-2,7-disulphonate

Endpoint summary

Description of key information

Skin irritation/corrosion:

Not Irritant

Eye irristation/corrosion:

Not irritant

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From October 4 to 7, 2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The test was conducted on a Similar Substance. The reliability of the original report is 1. The complete justification for the read across approach is attached at section 13.

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

other: normal human-derived epidermal keratinocytes

Vehicle:

other: PBS (phosphate buffered saline)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: reconstructed human epidermal model EpiDerm™ (EPI-200, MatTek, Bratislava, USA)
- Tissue batch number(s): Lot No. 23361- Tissue surface: 0.63 cm²The EpiDerm™ tissues are cultured on specially prepared cell culture inserts and shipped as kits, containing tissues on shipping agarose together with the necessary amount of culture media.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
The tissues were rinsed with PBS, blotted to remove the test substances, and then transferred to fresh medium.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/ml
- Incubation time: 3h
- Spectrophotometer: Libra S22
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES:
Three tissues are used per the test substance, three for the the positive (PC) and three for the negative (NC) controls.

DIRECT MTT REDUCTION - functional check in tubes
- the test substance is added to 1 mL MTT medium (red) and incubated in the incubator at culture conditions for 1 hour.
- At the end of the exposure time, the presence and intensity of the staining (if any) is observed. If the solution changes colour from red to blue, other steps to correction have to be done.

COLOUR INTERFERENCE - coloured substances
25 mg of the test substance are added to 2 ml isopropanol/water and placed on an orbital plate shaker for 2 to 3 hours at room temperature. After centrifugation, two 200 μl aliquots of supernatant and pure isopropanol (blank) are transferred to a 96-well plate and the absorbance is measured with a plate reader at OD570.
If, after subtraction of the OD for isopropanol, the OD of the test article solution is
> 0.08 the material has to be considered as possibly interacting with the MTT measurement and an additional test on colorant controls has to be performed.
Each coloured test article is applied to two additional tissues (the colorant controls (CC)) and is treated in the same way. Instead in MTT medium, these tissues are kept in assai medium. The following steps are identical as in the MTT test.
The true tissue viability is calculated as the percent tissue viability obtained with living tissues exposed to the interfering test chemical and incubated with MTT solution minus the percent non-specific colour obtained with living tissues exposed to the interfering test chemical and incubated with medium without MTT, run concurrently to the test being corrected (%NSCliving).
True viability = %Viability of treated tissue – %NSCliving

EVALUATION OF THE RESULTS AND CLASSIFICATION
For further classification the relative cell viability is calculated for each tissue as % of the mean of the negative control tissues viability, which is set at 100 %. The cut-off values for the prediction of irritation are given below; these values are stated in OECD Test Guideline No. 439 (1), par. 36:
a. In case the test chemical is found to be non-corrosive (e.g., based on TG 430, 431 or 435), and shows tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50%, the test chemical is considered to be irritant to skin in accordance with UN GHS (3) Category 2.
b. The test chemical may be considered as non-irritant to skin in accordance with UN GHS No Category if the tissue viability after exposure and post-treatment incubation is more than (>) 50%.
A single testing run composed of three replicate tissues should be sufficient for a test chemical when the classification is unequivocal. However, in cases of borderline results, such as non-concordant replicate measurements and/or mean percent viability equal to 50 ± 5%, a second run should be considered, as well as a third one in case of discordant results between the first two runs.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 mg of thr test substance

Duration of treatment / exposure:

1 hour

Duration of post-treatment incubation (if applicable):

approximately 42 hours

Number of replicates:

3
reconstructed human epidermal model EpiD

Irritation / corrosion parameter:

% tissue viability

Value:

ca. 85.5

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Colour interference
25 mg of the test substance were added to 2 mL of water and another 25 mg were added to 2 mL of isopropyl alcohol in the test tube. The test substance was partially soluble in water (red solution) and a little soluble in isopropyl alcohol (pink solution).
Both tubes were shaken for 120 minutes at room temperature and then centrifuged. Two 200 μl aliquots of every supernatant and pure isopropanol (blank) were transferred to a 96-well plate and OD was measured at 570 nm.
Water extract was non-measurable. OD570>3.
Pure OD570 of isopropyl-alcohol extract was 0.029 what is <0.08.
It means that, at good rinse of the dissolved test substance from tissues, colorant control has not to be performed.

Direct MTT reduction-functional check in tubes
25 mg of the test substance was added to 1.0 mL of MTT medium. Solution was incubated for 1 hour (37±1°C, 5±1 % CO2, humidified). The test substance changed colour
of MTT medium to red,). The test substance is not directly-reducing.

MTT test
Duration of pre-incubations before treatment was 60 minutes and 19 hours and 58 minutes.
25 mg of the test substance was placed directly atop the tissue moistened with 25 µL of PBS. The material was then spread on the tissue surface. Length of exposition was 60 minutes of which 25 minutes were tissues kept at room temperature and the remaining 35 minutes at culture conditions. After that the test substance was removed, tissues were rinsed and post-incubated. 1st post-incubation took 23 hours 48 minutes and 2nd took 18 hours, 21 minutes.
The other handling was performed as described in paragraph 4.3.3.
The dissolved test substance was rinsed from tissues - no colour was eluted to medium at post-incubations. Small amount of the test substance remained in tissues and coloured them red. Tissues remained coloured red even after extraction with isopropyl alcohol (see Figure 2).
The third tissue was perforated together with its pad at removal of the test substance from tissue surface what markedly influenced on tissue character at the experiment and decrease of OD570.

Interpretation of results:

other: CLP criteria not met

Conclusions:

Not irritant

Executive summary:

Method

The test substance, was assayed for the in vitro skin irritation in human epidermal model EpiDerm^TM.The test was performed according to the OECD Test Guideline No. 439.

After pre-incubation of tissues, 25 mg of the test substance was placed directly atop to the previously moistened tissue and it was spread on the entire tissue surface. Length of exposition was 60 minutes. Three tissues were used for the test substance and every control. Two tissues more were used as colorant control to correction of possible colour interference, which undergo the entire testing procedure excepting of incubation with MTT medium.

After removal of the test substance, tissues were post-incubated for approximately 42 hours due to leave of damage reparation. Three hours incubation with MTT and two hours extraction period with shaking followed then. Optical density (OD₅₇₀) of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

 

Results

Under the above-described experimental design average viability of treated tissues was 85.5 %, i.e. viability was > 50 %.

Conclusion

The test substance is considered to have no category in regard to skin irritation.

Not irritant

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From October 5 to 6, 2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The test was conducted on a Similar Substance. The reliability of the original report is 1. The complete justification for the read across approach is attached at section 13.

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

2013

GLP compliance:

yes (incl. QA statement)

Species:

other: Bovine

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Breeding service CHOVSERVIS a.s. division TORO® Hlavečník, Hradec Králové, Czech Republic
- Characteristics of donor animals: 12 to 30 months old
- Time interval prior to initiating testing: typically collected and used on the same day
- Indication of any existing defects or lesions in ocular tissue samples: The eyes, once they arrive at the laboratory, were carefully examined for defects including scratched, and neovascularisation. Only corneas from eyes free of such defects were used.
- Indication of any antibiotics used: penicillin at 100 IU/mL and streptomycin at 100 μg/mL and streptomycin at 100 μg/mL

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
Amount applied: 2 g
Concentration 0.2 g/ml

VEHICLE
Amount applied: 10 ml of 0.9% NaCl solution

Duration of treatment / exposure:

4 hours

Duration of post- treatment incubation (in vitro):

1.5 hour

Number of animals or in vitro replicates:

3 replicates

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Eyes were collected by slaughterhouse employees. The eyes were enucleated as soon as possible after death. Only healthy animals considered suitable for entry into the human food chain were used as a source of corneas for use in the BCOP test.

QUALITY CHECK OF THE ISOLATED CORNEAS
The eyes, once they arrive at the laboratory, were carefully examined for defects including scratched, and neovascularisation. Only corneas from eyes free of such defects were used. The isolated corneas, after achieve normal metabolic activity (inductive incubation at 32 ± 1°C for one hour), were examined again. The corneas that show macroscopic tissue damage (e.g., scratches, pigmentation, neovascularization) or a baseline opacity >7 opacity units were discarded. From 25 eyes the 9 eyes were eliminated after inductive incubation, because the baseline opacity values were >7. Nine corneas were used for the study (the corneas No. 6, 7, 9, 10, 11, 14, 15, 16 and 20), 4 eyes was superfluous and remaining 3 were used for the testing of another substance.

NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
0.9% NaCl

POSITIVE CONTROL USED
20% Imidazole

APPLICATION DOSE AND EXPOSURE TIME
2g of test substance in 10 ml of physiological saline for 4 hours

TREATMENT METHOD
Closed chamber was used, because the test substance was applicable by micropipette. The test substance (750 µL of application form) to cover the epithelial side of the cornea is introduced into the anterior chamber through the dosing holes on the top surface of the chamber, and the holes were subsequently sealed with the chamber plugs during the exposure.

REMOVAL OF TEST SUBSTANCE
After the exposure period, the negative control and the positive control substance was removed from the anterior chamber with EMEM (containing phenol red - the effectiveness of rinsing acidic or alkaline materials). The corneas were given a final rinse with EMEM (without phenol red). The EMEM (without phenol red) was used as a final rinse to ensure removal of the phenol red from the anterior chamber prior to the opacity measurement. The anterior chamber was then refilled with fresh EMEM without phenol red. The opacity and permeability of each cornea were recorded. The test substance was removed from the anterior chamber with EMEM – repeatedly, because the test substance is coloured . Subsequently the test substance was removed mechanically using a cotton swab and brush. The corneas (applied the test substance) were also rinsed with EMEM (containing phenol red). Lastly EMEM (without phenol red) was used for final rinsing. The test substance was complete removal, but corneas stayed coloured by the test substance. The anterior chamber was then refilled with fresh EMEM without phenol red. The opacity and permeability of each cornea were recorded.

METHODS FOR MEASURED ENDPOINTS
- Opacity: the amount of light transmission through the cornea. Corneal opacity was measured quantitatively with the aid of an opacitometer (Opacitometer, MC2 - Le spécialiste du laboratoire – France) resulting in opacity values measured on a continuous scale. Opacity values of treated corneas were corrected by subtracting individual background opacity values and the mean opacity is calculated.
- Permeability: the amount of sodium fluorescein dye that penetrates all corneal cell layers (i.e., the epithelium on the outer cornea surface through the endothelium on the inner cornea surface) measured indirectly using visible light spectrophotometry. 1 mL sodium fluorescein solution (5 mg/mL) was added to the anterior chamber of the corneal holder, which interfaced with the epithelial side of the cornea, while the posterior chamber, which interfaced with the endothelial side of the cornea, is filled with fresh EMEM. The holder was incubated in horizontal position for 1.5 hours at 32 ± 1 ºC. The amount of sodium fluorescein that crosses into the posterior chamber was quantitatively measured with the aid of UV/VIS spectrophotometry (Spectrophotometer GENESYS TM 10 UV/VIS Scanning). The values of absorbance measured at 490 nm were recorded as optical density (OD490) values. This term was used because the measuring is performed with visible light spectrophotometer using a standard 1 cm path length. Mean OD value of treated corneas was corrected by subtracting the mean OD value of negative control and the mean permeability is calculated.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS) calculation
Resulting mean opacity and OD490 values for each treatment group was combined in an empirically-derived formula to calculate an in vitro irritancy score (IVIS) for each treatment group as follows:
IVIS = mean opacity value + (15 x mean permeability OD490 value)

DECISION CRITERIA
The IVIS cut-off value for identifying the test substance as including serious eye damage (UN GHS Category 1) and the test substance not requiring classification for eye irritation or serious damage (UN GHS No Category) will be given hereafter:
IVIS:
≤ 3 UN GHS No Category: Chemicals that do not meet the requirements for classification as: UN GHS Category 1 or 2. Interchangeable with “Not Classified”
> 3; ≤ 55 No prediction can be made
> 55 UN GHS Category 1: “Serious eye damage”

Irritation parameter:

in vitro irritation score

Value:

ca. 7.45

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

other: No prediction can be made on test substance potential to cause eye irritation or serious eye damage based on the OECD Guideline Priciples

Conclusions:

IVIS = 7.45

Executive summary:

Method

The test substance was tested for the evaluation the potential ocular corrosivity or severe irritancy as measured by its ability to induce opacity and increased permeability in an isolated bovine cornea.

The test was performed according to theOECD Test Guideline No. 437 (Adopted 26^thJuly 2013).

The test was performed using nine isolated bovine corneas. The testing was performed on three groups of corneas: test substance treatment group, positive control group and negative control group. Three corneas per group were used. Closed-chamber method was used, because the test substance was applicable by micropipette. The opacity and permeability of each cornea were measured. The In Vitro Irritancy Score (IVIS) was calculated from the values of opacity and permeability.

Results

The In Vitro Irritancy Score (IVIS) for the test substance was 7.45 but this result could be affected by higher opacity values (mild colouring of corneas after removing the test substance: light-pink touch).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

The test substance, was assayed for the in vitro skin irritation in human epidermal model EpiDerm^TM.The test was performed according to the OECD Test Guideline No. 439. Under the above-described experimental design average viability of treated tissues was 85.5 %, i.e. viability was > 50 % Therefore the substance is considered as not irritant

Eye irritation

Two tests were considered in order to complete the assessment.

Based on the BCOP results on Similar substance 2 no prediction can be made; in addition, the IVIS score could be affected by the mild colouring of corneas left after removing the test substance (light pink

touch). Therefore, the results on similar substances were considere for the assessment. Taking into account the whole data set of eye irration, and the hystorical data for the endpoint on the

chemical family, Acid Black 001 is considered as not classified according to the CLP Regulation.

Justification for classification or non-classification

Skin Irritation

The ECHA Guidance on the application of the CLP Criteria set out that normally, there commendations for classification according to GHS criteria based on the results of an in vitro test are mentioned in the corresponding OECD test guideline. According to the OECD Guideline 439 the cut-off values for the prediction of irritation are:

 -  The test chemical is considered to be irritant to skin in accordance with UN GHS Category 2 if the tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50 %.

- The test chemical may be considered as non-irritant to skin in accordance with UN GHS No Category if the tissue viability after exposure and post-treatment incubation is more than (>) 50 %.

 

The test item showed a value of the relative absorbance (85.5 %) above the threshold for irritation potential set out in the OECD guideline, therefore the substance is considered as NOT IRRITANT

 

Eye Irritation

Taking into account the whole data set of eye irration, and the hystorical data for the endpoint on the chemical family, Acid Black 001 is considered as not classified according to the CLP Regulation.