Reaction mass of 2-[(4-methyl-2-nitrophenyl)diazenyl]-3-oxo-N-phenylbutanamide and 2-[(4-nitrophenyl)diazenyl]-3-oxo-N-phenylbutanamide

Endpoint summary

Description of key information

- Study No. 266/17/4AI: Pigment Yellow 4 - In vitro Skin Irritation Test (EpiDermTM Model); VUOS-CETA Report No. 18 -73, 2018.

Result: no category in regard to skin irritation

- Study No. 88.1872: Hansa-Gelb G03 gran. - Prüfung auf Augenreizung am Kaninchem (Pigment Yellow 1)

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:

18.01.2017 - 26.01.2018

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. 761/2009, 23rd July 2009

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: MatTek Protocol for: INVITRO EpiDermTM SKIN IRRITATION TEST For use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm

Version / remarks:

Model EPI-200-SIT, Rev. 26/3/2012,1-37

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) TISSUETissues: the reconstructed human epidermal model EpiDerm (EPI-200 ver. 2.0, MatTek, Bratislava, Slovakia); Lot No. 25875TEMPERATURE 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 with PBS, blotted to remove the test substanceMTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE- MTT concentration: 1 mg·mL-1- Incubation time: 180±5 mins- 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: 3NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:A single testing, composed of three replicate tissues, was run.Direct MTT reduction - functional check in tubes => the test substance is not directly-reducing.1. Colour interference => Average value of blank (isopropyl alcohol) was 0.046. Average value of the test substance isopropyl alcohol extract was 0.065. The difference was 0.019 what is < 0.08, so colorant controls were not employed.2. Direct MTT reduction - functional check in tubesThe test substance did not reduce MTT directly.3. MTT testThe test substance was well washable with PBS. No changes in tissue appearance were observed during the experiment.PREDICTION 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:

25 mg of the test substance was placed directly on tissue moistened with 25 µL of PBS and spread on the entire tissue surface. A single testing, composed of three replicate tissues, was run.NC: PBS (phosphate buffered saline), prepared in laboratory 30/08/2016, exp. 02/03/2017PC: 5 % SDS (sodium dodecyl sulphate), MatTek, Lot No. 012616TMB, exp. 26/01/2017

Duration of treatment / exposure:

60±1 minutes

Duration of post-treatment incubation (if applicable):

After 25 hours and 7 minutes of post-incubation period, the medium was replaced by fresh one. Tissues were incubated for another 18 hours, 37 minutes.

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

96.2

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

All study acceptance criteria were fulfilled. The mean OD570 of the NC tissue was 1.963 which meets the acceptance criteria of ≥ 0.8 and ≤ 2.8.The mean viability of the PC tissues expressed as % of the negative control tissues was 2.6 % which meets the acceptance criterion of ≤ 20 %. The SD calculated from individual % tissue viabilities of the 3 identically treated replicates was 0.1 % for the positive control, 2.2 % for negative control and 1.6 % for the test substance what is < 18 % in all cases.

MTT test

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

Treatment	OD570			Avgn	SD	%NC
PBS (NC)	2.021	1.950	1.917	1.963	0.043
viability (%NC)	103.0	99.4	97.7	100.0	2.2	100.0
266/17 (C1)	1.857	1.874	1.931	1.887	0.032
viability (%NC)	94.6	95.5	98.4	96.2	1.6	96.2
5% SDS (PC)	0.047	0.051	0.053	0.050	0.002
viability (%NC)	2.4	2.6	2.7	2.6	0.1	2.6

PBS - phosphate buffered saline

SDS - sodium dodecyl sulphate

Interpretation of results:

GHS criteria not met

Conclusions:

Under the above-described experimental design average viability of treated tissues was 96.2 %, i.e. viability was > 50 %.The effect of the test substance was negative in EpiDermTMmodel (tissues were not damaged).According to the classification criteria the test substance, Pigment Yellow 4, is considered to have no category in regard to skin irritation.

Executive summary:

The test substance, Pigment Yellow 4, was assayed for in vitro skin irritation in the 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 (2015) and Protocol for: In Vitro EpiDermTM Skin Irritation Test For use with MatTek Corporation’s Reconstructed Human Epidermal Model EPI-200-SIT (see par. 1.4).

In initial experiments either direct MTT reduction or colour interference were not found. 

In main experiment after pre-incubation of tissues, 25 mg of the test substance was placed directly on previously moistened tissues and it was spread on the entire tissue surface. The length of exposure was 60 minutes. Three tissues were used for the test substance and for positive and negative controls.

After removal of the test substance, tissues were post-incubated for approximately

42 hours. 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.

Under the above-described experimental design average viability of treated tissues was 96.2 %, 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, Pigment Yellow 4, 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:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Structure similarity.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Vehicle:

water

Observation period (in vivo):

72 h

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.33

Max. score:

2

Reversibility:

fully reversible within: 48h

Irritation parameter:

iris score

Basis:

animal: 2 and 3

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: not applicable

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1.67

Max. score:

3

Reversibility:

fully reversible within: 7 days

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1.33

Max. score:

3

Reversibility:

fully reversible within: 7 days

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.67

Max. score:

3

Reversibility:

fully reversible within: 72 h

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.67

Max. score:

4

Reversibility:

fully reversible within: 48 h

Irritation parameter:

chemosis score

Basis:

animal: 2 and 3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Interpretation of results:

GHS criteria not met

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available