Disodium 6-hydroxy-5-[[4-[[4-(phenylamino)-3-sulphonatophenyl]azo]naphthyl]azo]naphthalene-2-sulphonate

Endpoint summary

Description of key information

The average viability of tissues treated by the test substance Acid Black 26 was, after corection, 78.9 % of negative control average value, i.e. viability was > 50 %. The effect of the test item was negative in EpiDermTM model (tissues were not damaged).

No pathological changes of eyes of all rabbits were observed. During the observation period 48 and 72 hours after application no irritating effects on the eye was observed in all animals. No clinical signs of systemic intoxication were detected. Evaluation of results after single application demonstrated that the test substance, Acid Black 26, is not irritating to eye of rabbit.

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:

experimental study

Adequacy of study:

key study

Study period:

08.12.2015 – 22.01.2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted: 28th July, 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EC) No. 640/2012, 6th June 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: tissue for research puposes from accredited institutions

Source strain:

other: Keratinocyte strain 00267

Vehicle:

water

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUEViable tissues: the reconstructed human epidermal model EpiDerm (EPI-200 ver. 2.0, MatTek, Bratislava, Slovakia); Lot Nos. 23306 and 23308Frozen tissues: the reconstructed human epidermal model EpiDermTM killed by freezing; Lot No.: 16888 FRZN EATEMPERATURE USED FOR TEST SYSTEMculture conditions 37±1°C, 5±1 % CO2, moistened tissueREMOVAL OF TEST MATERIAL AND CONTROLS- Volume and number of washing steps: thoroughly rinsed, blotted to remove the test substance- Observable damage in the tissue due to washing: yes (see Other effects/acceptance ...)MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE- MTT concentration: 1 mg·mL-1- Incubation time: 180 min- Spectrophotometer: Libra S22 at 570 nm. Isopropyl alcohol serves as a blank. FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATABased on Certificate of Analysis the model passed all parametres for viability, barrier function, sterility.NUMBER OF REPLICATE TISSUES: 3CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE- killed tissues- Procedure used to prepare the killed tissues (if applicable): by freezing- N. of replicates : 2- Method of calculation used: see Any other information ...NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:1. Direct MTT reduction - functional check in tubes2. Direct MTT reduction - test in frozen tissues3. MTT test in viable tissues - two experimentsPREDICTION MODEL / DECISION CRITERIAOECD 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%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

The test substance (25 mg) was placed directly atop to the moistened tissue. The material was spread on the tissue surface.NC: sterile PBS (phosphate buffered saline)PC: 5 % SDS (sodium dodecyl sulphate), MatTek, Lot No. 020615TMH in water

Duration of treatment / exposure:

60±5 minutes

Duration of post-treatment incubation (if applicable):

42 hours.

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

78.9

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Criterium #3 for SD of among tissues was not fulfilled in the first experiment. The first experiment was repeated and its results were not taken into evaluation.Difficulties were observed at washing and not all the test substance was removed from tissues. Sometimes, tissues were damaged at removal of the test substance. At the same time, utmost thorough removal of the test substance was very important because the test substance reduces MTT directly. Damage of tissues could be the one of reasons why OD570 values were so different at the first experiment. At the same time, the test substance directly reducing MTT, remained bounded in tissues in different amount.In the second experiment, longer time gaps were let between tissues due to possibility to better removal of the test substance from tissues. Unfortunatelly, it was not very effective, because the test substance was bounded in tissues, so its removal was impossible. Nevertheless, all test conditions were fulfilled and the results of the second experiment were evaluated. In the second experiment the first tissue of negative control was about 25 min lost in internals of laminar box and drained. This tissue was excluded from evaluation.In the repeated experiment all criteria for validity of the test were fulfilled.

Direct MTT reduction: test in frozen tissues

Treatment	tissue 1	tissue 2	mean	SD	%NC	95% conf. interval
PBS 60 min	0.055	0.060	0.058	0.003	100.0	0.053	0.063
446/15 (60 min)	0.096	0.139	0.118	0.022	204.3	0.075	0.161

Average OD₅₇₀value of treated tissues after 60 min treatment (0.118) did not fall into 95% confidence interval of negative control so correction of results of MTT test is necessary.

MTT test: viable tissues

OD₅₇₀values obtained at the MTT test, their averages, standard deviations (%) and relative viabilities

The first experiment:

Treatment	OD570			Mean	SD	%NC
PBS (NC)	1.984	1.910	1.879	1.924	0.044
viability (%NC)	103.1	99.3	97.6	100.0	2.3	100.0
446/15 (C2)	0.878	1.733	0.911	1.174	0.396
viability (%NC)	45.6	90.1	47.3	61.0	20.6	61.0
5% SDS (PC)	0.056	0.077	0.070	0.068	0.009
viability (%NC)	2.9	4.0	3.6	3.5	0.5	3.5

The second experiment:

Treatment	OD570			Mean	SD	%NC
PBS (NC)	0.179	1.855	1.832	1.844	0.012
viability (%NC)	9.7	100.6	99.4	100.0	0.6	100.0
446/15 (C2)	1.999	1.310	1.602	1.637	0.282
viability (%NC)	108.4	71.1	86.9	88.8	15.3	88.8
5% SDS (PC)	0.075	0.065	0.069	0.070	0.004
viability (%NC)	4.1	3.5	3.7	3.8	0.2	3.8

0.179 - tissue excluded from evaluation

PBS - phosphate buffered saline

SDS - sodium dodecyl sulphate

Interference by the test substance (OD₅₇₀= 0.118) was lower than 30% of viable negative control, so correction of second experiment results was made.

True viability (OD₅₇₀) = 1.637 - 0.118 = 1.519

True viability (% of negative control) is 78.9.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the above-described experimental design, average viability of tissues treated by the test substance Acid Black 26 was, after correction, 78.9 % of negative control average value, i.e. viability was > 50 %. The effect of the test item was negative in EpiDermTM model (tissues were not damaged).

Executive summary:

The test item, Acid Black 26, 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: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method test (2015) and Protocol for: In Vitro EpiDerm^TMSkin Irritation Test For use with MatTek Corporation’s Reconstructed Human Epidermal Model EPI-200-SIT (2014).

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.

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.

As the test substance was suspected to be direct reducing, test with frozen tissues was performed to detection if the tets substance remaining in tissues reduces MTT directly. Direct reduction in tissues was confirmed and result in viable tissue was corrected.

Under the above-decribed experimental design, average viability of tissues treated by the test substance was 78.9 %, i.e. viability was > 50 %.

The effect of the test substance was negative in EpiDerm^TMmodel (tissues were not damaged).

According to the classification criteria, the test substance is considered to have no category in regard to skin irritation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15.02. – 24.02.2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

Adopted October 2, 2012

Deviations:

yes

Remarks:

(see Any other information ...)

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS- Source: breeding farm VELAZ s.r.o., Lysolaje, Czech Republic, RČH CZ 21760118- Weight at study initiation: 2.70 – 3.40 kg- Housing: conventional animal room – individually in metallic cages- Diet (e.g. ad libitum): ad libitum- Water (e.g. ad libitum): ad libitum- Acclimation period: 5 days, no signs of disease were observedENVIRONMENTAL CONDITIONS- Temperature (°C): 20 ± 3°C, permanently monitored- Humidity (%): 30 – 70%, permanently monitored- Photoperiod (hrs dark / hrs light): 12 h/12 h

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

A dose of 100 mg of the test substance was applied to the test site in supplied form.

Duration of treatment / exposure:

The test substance was placed into the conjunctival sac of one eye of animals after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second in order to prevent loss of the substance. The substance was solid and has not been removed from the eye of the test animal by physiological mechanisms at the first observation time point of 1 hour after treatment. The eye was rinsed with physiological saline solution.

Observation period (in vivo):

examined at 1, 24, 48 and 72 hours after application

Number of animals or in vitro replicates:

3

Details on study design:

REMOVAL OF TEST SUBSTANCE - Washing (if done): rinsed with physiological saline solution - Time after start of exposure: 1h SCORING SYSTEM: To the ocular reactions observed at each time interval the grades were assigned according to the grading system given in OECD Test Guideline No. 405 Acute Eye Irritation/Corrosion. Adopted October 2, 2012. TOOL USED TO ASSESS SCORE: hand-slit lamp

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal #5

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

iris score

Basis:

animal #5

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 24h

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

3

Irritation parameter:

conjunctivae score

Basis:

animal #5

Time point:

24/48/72 h

Score:

0

Max. score:

3

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

chemosis score

Basis:

animal #5

Time point:

24/48/72 h

Score:

0

Max. score:

4

No alterations of control eyes were observed during the whole study.

Interpretation of results:

GHS criteria not met

Conclusions:

Redness of conjunctivae (some blood vessels of conjunctivae were hyperaemic (injected)) and chemosis of lids (some swelling above normal) were observed in all animals 1 hour after application of test substance. Due to coloration of the test substance the evaluation of irritation was difficult.No changes were observed on eye at 24 hour after application in animals 3 and 5. Only in animal No. 1: Conjunctivae - Some blood vessels were hyperaemic (injected). No pathological changes of eyes of all rabbits were observed for the rest of the study. During the observation period (48 and 72 hours after application) no irritating effects on the eye was observed in all animals. No clinical signs of systemic intoxication were detected. Evaluation of results after single application demonstrated that the test substance, Acid Black 26, is not irritating to eye of rabbit.

Executive summary:

The test substance, Acid Black 26, was tested for the assessment of eye irritation/corrosion effects using albino rabbit (New Zealand Albino breed).

The test was performed according to the OECD Test Guideline No. 405 Acute Eye Irritation/Corrosion. Adopted October 2, 2012.

The strategy given in this OECD test guideline was respected, initial consideration and weight-of-the-evidence analysis was performed before in vivo testing.

The test was performed initially using one animal (No. 1). Because no corrosive or severe irritating effects were observed in initial test, the response was confirmed using two additional animals (No. 3 and No. 5).

Redness of conjunctivae (some blood vessels of conjunctivae were hyperaemic (injected) and chemosis of lids (some swelling above normal) were observed in all animals 1 hour after application of test substance. Due to coloration of the test substance the evaluation of irritation was difficult.

No changes were observed on eye at 24 hour after application in animals 3 and 5. Only in animal No. 1: Conjunctivae - Some blood vessels were hyperaemic (injected). No pathological changes of eyes of all rabbits were observed for the rest of the study. During the observation period (48 and 72 hours after application) no irritating effects on the eye was observed in all animals. No clinical signs of systemic intoxication were detected.

Evaluation of results after single application demonstrated that the test substance, Acid Black 26, is not irritating to the eye of rabbit.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Under the above-described experimental design, average viability of tissues treated by the test substance Acid Black 26 was, after correction, 78.9 % of negative control average value, i.e. viability was > 50 %. The effect of the test item was negative in EpiDermTM model (tissues were not damaged).

No pathological changes of eyes of all rabbits were observed. During the observation period 48 and 72 hours after application no irritating effects on the eye was observed in all animals. No clinical signs of systemic intoxication were detected. Evaluation of results after single application demonstrated that the test substance, Acid Black 26, is not irritating to eye of rabbit.