4,5-dihydro-5-oxo-4-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]-1-(4-sulphophenyl)-1H-pyrazole-3-carboxylic acid, sodium salt

Endpoint summary

Description of key information

One key skin irritation study and two key eye irritation studies are available.

Skin irritation:

In this in vitro study the skin irritation potential of the test item Reactive Yellow 42 was assessed according to OECD 439 Test Method and EU-Method B.46 and in compliance to GLP. The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 50% (94.3%) after 60 min treatment and 42 h post-incubation.

Eye irritation:

In the first study (2018) that was performed, the eye irritancy potential of Reactive Yellow 42 was investigated in the bovine corneal opacity and permeability assay. The study was performed according to OECD TG 437 and in compliance to GLP. A mean in vitro irritation score of 5.34 was calculated. According to the evaluation criteria of the test guideline, no prediction can be made regarding the classification of the test substance Reactive Yellow 42 (IVIS >3,≤55).

In the second study (2018), the potential of the test item to induce eye irritation was analysed by using the three-dimensional human corneal epithelium model EpiOcular, consisting of normal, human-derived epidermal keratinocytes mimicking characteristics of the corneal epithelium. The study was performed in accordance to OECD TG 492 and in compliance to GLP. In this study, under the given conditions, the test item showed irritant effects. The mean relative tissue viability (% negative control) was ≤ 60% (21.5%).

According to the OECD Guideline 492, the EpiOcular Eye Irritation Test does not allow discrimination between eye irritation/reversible effects on the eye (Category 2) and serious eye damage/irreversible effects on the eye (Category 1).

Taken the precautionary principle into account and in the absence of additional data, the substance will be classified as category 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:

8.-25.11.2016

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:

2015

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2012

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: human-derived epidermal keratinocytes

Source strain:

other: not applicable

Vehicle:

unchanged (no vehicle)

Details on test system:

- Source: MatTek Corporation (82105 Bratislava, Slovakia).
- The EpiDerm™ tissue: normal, human-derived epidermal keratinocytes which have been cultured to
form a multilayered, highly differentiated model of the human epidermis.
- Surface: 0.63 cm.
- Pre-incubation: 60 ± 5 minutes in the incubator (37 ± 1 °C, 5% CO2), then for about 18 ± 3 hours (37 ± 1 °C, 5% CO2).

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 mg + 25 µL DPBS

Duration of treatment / exposure:

60 ± 1 min

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

3

Details on study design:

Details of the test procedure used
- EpiDerm™ tissue of human-derived epidermal keratinocytes was used
- Conditions of exposure: 37 ± 1 °C, 5% CO2
- Washing: inserts gently rinsed with DPBS
- Number of tissue replicates used per test chemical and controls: 3
- MTT assay: incubation with 0.3 mL of MTT solution for 60 minutes at 37 ± 1 °C, 5% CO2)

- Data evaluation:
Irritant potential of the test item was predicted from the relative mean tissue viabilities compared to the negative control tissues concurrently treated with DPBS.
The test item is considered to be irritant to skin in accordance with regulation EC 1272/2008 (UN GHS “Category 2”), if the tissue viability after exposure and post-incubation is less or equal to 50%.

- Historical data positive control: Mean Viability:4.0%; Rel. Standard Deviation: 2.0%

- Historical data negative control: Mean Absorption: 1.830%; Rel. Standard Deviation:0.376%

- The test meets acceptance criteria if:
- mean absolute OD570 nm of the three negative control tissues is ≥ 0.8 and ≤ 2.8
- mean relative tissue viability of the three positive control tissues is ≤ 20%
- standard deviation (SD) of relative tissue viability obtained from each three concurrently tested tissues is ≤ 18%.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Single test with three tissues

Value:

>= 94.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The acceptance criteria were met

Result of the Test Item

Name	NK			PC			TM
Tissue	1	2	3	1	2	3	1	2	3
absolute OD570	2.021	1.928	1.971	0.105	0.107	0.113	1.895	1.886	1.790
	2.020	1.959	2.045	0.114	0.110	0.118	1.888	1.932	1.888
OD570(blank-corrected)	1.978	1.885	1.928	0.062	0.064	0.070	1.852	1.843	1.747
	1.977	1.916	2.002	0.071	0.067	0.075	1.845	1.889	1.845
mean OD570of the duplicates (blank-corrected)	1.978	1.900	1.965	0.067	0.066	0.073	1.849	1.866	1.796
total mean OD570of 3 replicate tissues (blank-corrected)	1.948*			0.068			1.837
SD OD570	0.042			0.004			0.036
relative tissue viability [%]	101.5	97.6	100.9	3.4	3.4	3.7	94.9	95.8	92.2
mean relative tissue viability [%]	100.0			3.5**			94.3
SD tissue viability [%]***	2.1			0.2			1.9
CV [% viabilities]	2.1			5.5			2.0

 

^* Blank-corrected mean OD_{570 nm}of the negative control corresponds to 100% absolute tissue viability.

^**Mean relative tissue viability of the three positive control tissues is£ 20%.

^***Standard deviation (SD) obtained from the three concurrently tested tissues is≤ 18%

NK negative control of living tissues

PC positive control

TM test item treated living tissues

Interpretation of results:

other: the test item showed no irritant effects

Conclusions:

In this study under the given conditions the test item showed no irritant effects. The test item is therefore classified as “non-irritant” in accordance with UN GHS “No Category”.

Executive summary:

In this in vitro study the skin irritation potential of the test item Reactive Yellow 42 was assessed according to

OECD 439 Test Method and EU-Method B.46 and in compliance to GLP.

The potential of the test item to induce skin irritation was analysed by using the three-dimensional human epidermis model EpiDerm (MatTek) comprising a reconstructed epidermis with a functional stratum corneum.

In the present study Reactive Yellow 42 was applied topically to the EpiDerm tissue for 60 min followed by a 42 h post-incubation period and immediate determination of cytotoxic effects via MTT reduction assay.

Irritant potential of the test item was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with DPBS.

The test item showed no non-specific reduction of MTT and no colouring potential after mixture with isopropanol. The mixture of the test item with aqua dest. showed colouring in the relevant wavelength range, therefore NSC_livingwas determined. Since NSC_livingwas ≤ 5% (0.4%), no correction of results was necessary.

The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 50% (94.3%) after 60 min treatment and 42 h post-incubation.

The controls confirmed the validity of the study. The mean absolute OD₅₇₀of the three negative control tissues was >= 0.8 and ≤ 2.8. The mean relative tissue viability (% negative control) of the positive control was ≤ 20% (3.5%). Standard deviation of viability of replicate tissues of all dose groups was ≤ 18% (0.2% - 2.1%).

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:

weight of evidence

Study period:

17.-18.01.2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

28 July 2015

GLP compliance:

yes (incl. QA statement)

Species:

human

Strain:

other: normal, human-derived epidermal keratinocytes

Details on test animals or tissues and environmental conditions:

- Test system: reconstructed human cornea-line epithelium (RhCE) model, cultured to form a stratified, highly differentiated squamous epithelium morphologically similar
to that found in a human cornea
- Source: EpiOcular™ tissue kits (e.g. OCL-200-EIT; MatTek)

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

- Negative Control 50 μL Aqua dest.
- Positive Control 50 μL methyl acetate
- Test Item 50 mg

Duration of treatment / exposure:

6 h

Duration of post- treatment incubation (in vitro):

18 h

Number of animals or in vitro replicates:

2 tissues per dose group

Details on study design:

Experimental Procedure:
- Equilibration: incubated for 1 h in a humidified incubator at 37 ± 1 °C, 5.0% CO2 / 95% air in assay medium
- Pre-incubation: in a humidified incubator at 37 ± 1 °C, 5.0% CO2 / 95% air for 16 - 24 h
- Pre-treatment: with 20 μL of DPBS-buffer and incubated for 30 ± 2 min in a humidified incubator at 37 ± 1 °C, 5.0% CO2 / 95% air
- Treatment (exposure): incubated for 6 ± 0.25 h at 37 ± 1 °C, 5.0% CO2 / 95% air
- Post-treatment: incubated for 18 ± 0.25 h at 37 ± 1°C, 5.0% CO2 / 95% air

MTT assay:
Incubation with 0.3 mL of MTT solution for 3 h ± 10 min at 37 ± 1 °C, 5.0% CO2 / 95% air
After incubation with MTT the inserts were incubated with isopropanol and shaken for 2-3 hours at room
temperature.

Data Analysis:
Ocular irritation potential of the test item was predicted from the relative mean tissue viabilities
obtained after treatment compared to the negative control tissues concurrently treated with
Aqua dest.

Test Acceptance Criteria:
The test meets acceptance criteria if:
- mean absolute OD570 nm of the negative control is > 0.8 and < 2.5
- mean relative tissue viability of the positive control is < 50%
- relative tissue viability difference of replicate tissues is < 20%.

Deviations:
The deviations did not influence the quality or integrity of the present study.

Irritation parameter:

other: Mean Tissue Viability [%]

Run / experiment:

mean of two tissues

Value:

21.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

The controls confirmed the validity of the study.

Result of the Test Item Reactive Yellow 42

Name	Negative control		Positive control		Test item
Tissue	1	2	1	2	1	2
OD570values	1.488	1.419	0.657	0.619	0.232	0.458
	1.513	1.411	0.685	0.622	0.234	0.466
OD570values (blank-corrected)	1.445	1.376	0.613	0.576	0.189	0.415
	1.47	1.368	0.642	0.579	0.191	0.423
Mean of the duplicates	1.457	1.372	0.628	0.577	0.19	0.419
Mean OD	1.414*		0.602		0.304
TOTTT	-		-		0.299
Mean sd OD	0.06		0.04		0.16
Tissue Viability (%)	103	97	44.4	40.8	13.4	29.6
Relative tissue viability difference (%)***	6		3.6		16.2
Mean tissue viability (%)	100		42.6**		21.5

*Corrected mean OD570 of the negative control corresponds to 100% absolute tissue viability

**Mean relative tissue viability of the positive control is < 50%

***Relative tissue viability difference of replicate tissues is < 20%

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

In this study under the given conditions the test item showed irritant effects.

According to the OECD Guideline 492, the EpiOcular Eye Irritation Test does not allow discrimination between eye irritation/reversible effects on the eye (Category 2) and serious eye damage/irreversible effects on the eye (Category 1).
For these purposes, further testing with other suitable test methods may be required.

Executive summary:

The potential of the test item to induce eye irritation was analysed by using the three-dimensional human corneal epithelium model EpiOcular, consisting of normal, human-derived epidermal keratinocytes mimicking characteristics of the corneal epithelium. The study was performed in accordance to OECD TG 492 and in compliance to GLP.

In the present study Reactive Yellow 42 was applied topically to the EpiOcular tissue for 6 h followed by 25 min post-soaking incubation after removal of the test item. After a 18 h post-treatment period cytotoxic effects were determined via MTT reduction assay.

Ocular irritation potential of the test item was predicted from the relative mean tissue viabilities compared to the negative control tissues concurrently treated with Aqua dest.

The mixture of 50 mg test item per 1 mL MTT medium showed no reduction of MTT as compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%.

The controls confirmed the validity of the study. The mean absolute OD570 of the two negative control tissues was > 0.8 and < 2.5 (1.458). The mean relative tissue viability (% negative control) of the positive control was < 50% (42.6%). The maximum inter tissue difference of replicate tissues of all dose groups was < 20% (16.2%).

The mixture of 50 mg test item per 1 mL A. dest. showed colouring as compared to the solvent.

Since the mean relative tissue viability of the test item treated tissues (TM) was below the 60% threshold value no coloured tissue controls were performed, since the test item has to be classified as irritant in any case.

The test item showed irritant effects. The mean relative tissue viability (% negative control) was ≤ 60% (21.5%).

In this study under the given conditions the test item showed irritant effects. The test item is classified as “irritant“ in accordance with UN GHS “Category 1” or “Category 2”.

According to the OECD Guideline 492, the EpiOcular Eye Irritation Test does not allow discrimination between eye irritation/reversible effects on the eye (Category 2) and serious eye damage/irreversible effects on the eye (Category 1). For these purposes, further testing with other suitable test methods may be required.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

03.05.2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Test system:
Freshly isolated bovine cornea obtained as a by-product from animals freshly slaughtered
- Source: A. Moksel AG, Buchloe, Germany

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

750 µL of the test item preparation, the negative or positive control

Duration of treatment / exposure:

- 4 hours ± 5 minutes incubation at 32 ± 1 °C, washed at least three times with MEM (containing phenol red)

Duration of post- treatment incubation (in vitro):

- After the illuminance measurement the corneas were incubated for 90 minutes with RPMI and
sodium fluorescein solution at 32 ± 1 °C for the optical density determination

Number of animals or in vitro replicates:

3 corneas/group

Details on study design:

Preparation of the Corneas:
The assay uses isolated corneas obtained as a by-product from animals freshly slaughtered.
On the test day, fresh eyes were collected from the slaughterhouse. Immediately cornea preparation was initiated. The eyes were carefully examined for defects and any defective eyes were discarded. The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera. The corneas were mounted in corneal holders with the endothelial side against the O-ring of the posterior chamber. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with RPMI (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI). The corneas were incubated for one hour at 32 1 °C.

Treatment of the Corneas:
After the equilibration period, the medium was removed from both chambers and replaced with fresh complete RPMI. An initial measurement was performed on each of the corneas using the opacitometer. Three corneas with illuminance readings
approximately equivalent to the median illuminance of all corneas were selected as negative-control corneas. The illuminance of each cornea was read and recorded. Only corneas that had an initial illuminance reading I > I0/1.1651 lux were used for the assay. The medium was removed from the anterior chamber and replaced with the test item or control. 750 µL of the test item preparation or the control substance was introduced into the anterior chamber (closed-chamber method).
After 4 hours ± 5 minutes incubation at 32 ± 1 °C either the test substance or the control substance was removed and the epithelium washed at least three times with MEM (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red). The anterior chamber was refilled with complete RPMI and an illuminance measurement was performed. Also, each cornea was observed visually and pertinent observations were recorded. After the illuminance measurement was performed, the medium was removed from both chambers of the holder. The posterior chamber was refilled with fresh complete RPMI. 1 mL of a 5 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes at 32 ± 1 °C. Then the medium from the posterior chamber was removed and its optical density at 490 nm (OD490) was determined, using a spectrophotometer (Jenway 6405 UV/VIS).

Validity of the assay:
The BCOP assay is considered to be valid if the in vitro irritation score obtained with the positive control falls within the two standard deviations of the current historical mean. The negative control responses should result in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

Evaluation of Results:
The following formula was used to calculate the opacity, whereas the values a and b are equipment-specific variables empirically determined by the manufacturer: Opacity= ( I0/I-b)/a; with a = 0.025 and b = 0.9894.

The change in opacity for each cornea was calculated by subtracting the initial opacity reading from the final opacity reading. These values were corrected by subtracting from each the average change in opacity observed for the negative-control corneas. The mean opacity value for each treatment was calculated by averaging the corrected opacity values of each cornea for a given treatment.

The mean OD490 for the blank cuvettes was calculated. The mean blank OD490 was subtracted from the OD490 of each cuvette (corrected OD490). Any dilutions that were made to bring the OD490 values into the linear range of the spectrophotometer (OD490 should be less than 1.500), were taken into account by multiplying the OD490 value of the dilution by the dilution factor. The final-corrected OD490 of the test article and the positive control were calculated by subtracting the average-corrected OD490 of the negative-control corneas from the corrected OD490 value of each treated cornea: Final-corrected OD490 = (OD490 – mean blank OD490) – average-corrected negative control OD490.
The mean OD490 value of each treatment group was calculated by averaging the final corrected OD490 values of the treated corneas for that treatment condition.

The following formula was used to determine the in vitro irritation score (IVIS):
IVIS = mean opacity value + (15 x mean permeability OD490 value)

Acceptance criteria:
A test is considered acceptable if the positive control gives an IVIS that falls within two standard deviations of the current historical mean. The negative or solvent/vehicle control responses should result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneas treated with the respective negative or solvent/vehicle control. A single testing run composed of at least three corneas should be sufficient for a test chemical when the resulting classification is unequivocal. However, in cases of borderline results in the first testing run, a second testing run should be considered (but not necessarily required), as well as a third one in case of discordant mean IVIS results between the first two testing runs.

Deviations:
Revised Certificate of Analysis. This deviation did not influence the quality or integrity of the present study.

Irritation parameter:

in vitro irritation score

Run / experiment:

mean of 3 corneas / 4 h incubation time

Value:

5.34

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No prediction can be made regarding the classification of the test substance

Other effects / acceptance of results:

The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid.
The negative control responses should result in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

In Vitro Irritation Score

Cornea no.	Test item	Corrected opacity	Corrected OD490 value	IVIS
1	Negative control	0.89	0.009	0.59
2		0.04	0.009
3		0.04	0.036
MV		0.32	0.018
4	Positive control	90.91	1.417	121.72
5		92.93	2.397
6		89.52	2.307
MV		91.12	2.04
7	Test item	7.04	-0.002	5.34
8		2.06	0.003
9		7.06	-0.01
MV		5.39	-0.003

MV = mean value

Interpretation of results:

study cannot be used for classification

Conclusions:

A mean in vitro irritation score of 5.34 was calculated. No prediction can be made regarding the classification of the test substance Reactive Yellow 42 according to the evaluation criteria.

Executive summary:

The eye irritancy potential of Reactive Yellow 42 was investigated in the bovine corneal opacity and permeability assay.

The study was performed according to OECD TG 437 and in compliance to GLP.

One cornea treated with Reactive Yellow 42 showed an overall but slight opacity of the tissue.

Another cornea showed a slight yellowish discoloration of the tissue.

The following mean in vitro irritation score was calculated: 5.34.

The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid.

The negative control responses should result in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

No prediction can be made regarding the classification of the test substance Reactive Yellow 42 according to the evaluation criteria.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

All three key studies are recent, well documented, GLP and according to the respective OECD TG.

Skin irritation:

In the performed OECD439, the test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 50% (94.3%) after 60 min treatment and 42 h post-incubation. Thus, based on the available data on skin irritation, the test item does not meet the criteria for classification according to Regulation (EC) 1272/2008 (CLP).

Eye irritation:

In the first study (2018) that was performed, the BCOP study (according to OECD TG 437 & GLP), a mean in vitro irritation score of 5.34 was calculated. According to the evaluation criteria of the test guideline, no prediction can be made regarding the classification of the test substance Reactive Yellow 42 (IVIS >3,≤55).

In the second study (2018), the EpiOcular Eye Irritation Test (according to OECD TG 492 & GLP), the test item did show irritant effects. The mean relative tissue viability (% negative control) was ≤ 60% (21.5%). According to the OECD Guideline 492 however, the EpiOcular Eye Irritation Test does not allow discrimination between eye irritation/reversible effects on the eye (Category 2) and serious eye damage/irreversible effects on the eye (Category 1).

Thus, based on the available data on eye irritation and taking the precautionary principle into account (EpiOcular assay does not allow discrimination between Category 2 and Category 1), the test item is classified as “irritant” in accordance with the criteria for classification according to Regulation (EC) 1272/2008 (CLP) and classified "Category 1".