Reaction products of diazotised 5,5’-methylenedianthranilic acid, coupled with resorcinol, subsequently coupled with diazotised 4-aminobenzene-1,3-disulfonic acid, chelated with copper sulphate, sodium salt

Endpoint summary

Description of key information

Skin Irritation/Corrosion

Non irritant

Eye Irritation/Corrosion

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 July 20 to 27, 2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Reliability of the source study is 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2015

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2009

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-bufferd 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): 23347- 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:

3CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Tissues: freeze-killed tissues
- Procedure used to prepare the killed tissues (if applicable):
- N. of replicates : 2
- Method of calculation used: True viability = %Viability of treated tissue – (%)NS

MTT NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:PREDICTION MODEL / DECISION CRITERIA
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:
-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.
-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

Duration of treatment / exposure:

one hour

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Value:

ca. 75.7

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

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.

Colour interference
25 mg of the test substance was added to 2 mL of isopropyl alcohol. The test substance was insoluble in it. After 125 minutes of shaking at room temperature, mixture was centrifuged; supernatant was decanted and used for measuring of spectra in range of wavelength from 190 to 900 nm.The test substance almost did not absorbed light in range 540-600 nm; OD570 were 0.0-0.008. Nevertheless, colorant control was also included in the test because enough tissues were available.

MTT test
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 placed at room temperature and the remaining 35 minutes at 37±1°C, 5±% CO2. After that the test substance was removed, tissues were rinsed and post-incubated. 1st post-incubation took 24 hours, 2 minutes and 2nd took 18 hours, 39 minutes. Many washing cycles must have been performed due to removal of the test substance from tissue surface. Nevertheless, tissues (and walls of inserts) remained coloured even after extraction with isopropyl alcohol, what means, that the test substance is insoluble in it. After the first post incubation medium was yet slightly coloured with the test substance. After post-incubations the MTT assay was performed by transferring the tissues to 24-well plates containing MTT medium (1 mg•mL-1). After 3 hour MTT incubation, the blue formazan salt formed by cellular mitochondria was extracted with 2.0 ml/tissue of isopropyl alcohol for 2 hours, room temperature and shaking, and the optical density of the extracted formazan was determined using a spectrophotometer at λ=570 nm. In addition, two viable tissue replicates, which underwent the entire testing procedure (treatment for 60 minutes) but were incubated with assay medium instead of MTT solution during the MTT incubation step to generate a non-specific colour (NSCliving) control, were used concurrently with MTT test. OD570 of these tissues were 0.017 and 0.008 so average value was 0.013, what is 0.7 % of negative control OD570.

Data correction for colour interference:
True viability = % Viability of treated tissue – % NSCliving 75.7 % - 0.7 % = 75.0 %

Interpretation of results:

other: Not classified according to the OECD Guideline principles

Conclusions:

Not irritant

Executive summary:

Method

The test item was assayed for the in vitro skin irritation in human epidermal model EpiDermTM. 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 75.7 % (75.0 % after correction).

 

Conclusion

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 05 to 06, 2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Reliability of the source study is 1

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.
- Number of animals:
- 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

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

Duration of treatment / exposure:

4 hours

Duration of post- treatment incubation (in vitro):

1.5 hours

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
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 (red-brown colour). 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:
- Corneal 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.
- Corneal 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 GENESYSTM 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.- Others (e.g, pertinent visual observations, histopathology): (please specify)

SCORING SYSTEM:
In Vitro Irritancy Score (IVIS) 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. 32.68

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

other: No prediction can be made on Non classification based on the OECD Guideline Principles

Conclusions:

IVIS = 32.68

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.

 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 32.68 but this result could be affected by higher opacity values (corneas were coloured by the test substance:red-brown colour).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Skin Irritation

The ECHA Guidance on the application of the CLP Criteria set out that normally, the recommendations 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 (75 %) above the threshold for irritation potential set out in the OECD guideline, therefore the substance is considered as NOT IRRITANT

 

Eye Irritation

The ECHA Guidance on the application of the CLP Criteria set out that a substance can be considered as causing serious eye damage (Category 1) based on positive results in the BCOP test, and a negative results from the BCOP test methods can be used for classification purposes.

According to the OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants),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:

 

- IVIS ≤ 3 – No Classification (UN GHS)

- IVIS > 3; ≤ 55 – No prediction can be made on the non classification

- IVIS > 55 – Category 1 (UN GHS)

 

The test item showed a value of in vitro irritancy score (IVIS ) of 32.68, therefore in the range of no prediction ( 3 > IVIS ≤ 55 ). Using a conservative approach, the test substance has been classified as Irritant to eyes Category 2, according to the CLP Regulation n. 1272/2008.