Reaction mass of isomers 4-[[2-[(Aminocarbonyl)amino]-4-[(2,6-difluoro-4-pyrimidyl)-amino]phenyl]azo]-1,3-benzenedisulfonic acid, sodium salt and 4-[[2-[(Aminocarbonyl)amino]-4-[(4,6-difluoro-2-pyrimidyl)-amino]phenyl]azo]-1,3-benzenedisulfonic acid, sodium salt

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-04-09 to 2013-04-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: EU Method B.46 (Skin Irritation)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Test material

Test material information

Constituent 1

Reference substance name:

Kilo 10&12

IUPAC Name:

Kilo 10&12

Constituent 2

Reference substance name:

Reactive Golden Yellow HF-RN 1331

IUPAC Name:

Reactive Golden Yellow HF-RN 1331

Details on test material:

Name: Reactive Golden Yellow HF-RN 1331
Batch No.: Kilo 10&12
Chemical Name: 4-[[2-[(Aminocarbonyl)amino]-4-[(2,6-difluoro-4 pyrimidinyl)-amino]phenyl]azo]-1,3-benzenedisulfonic acid, sodium salt
Formula: C17H11F2N7Na2O7S2
Physical State: solid (powder)
Colour: reddish orange
pH: 4.5 at 5%
Molecular Weight: 573.42 g/mol
Purity: 100 % (composition see AZ. No. 12-1327/2473 v.2, dated 2012-10-08)
Storage Conditions: room temperature
Expiry Date: 17.01.2014

Test system

Amount / concentration applied:

10 mg + 15 µL aqua dest.

Duration of treatment / exposure:

15 minutes

Observation period:

42 +/- 1 h

Details on study design:

The test was performed on EpiSkin, an organotypic reconstructed three-dimensional model of the human epidermis. 3 replicate tissues are dosed with the test item (10 mg + 15 µL A. dest. to ensure good contact and even distribution)S, the negative control (10µL PBS) and the positive control (10µL 5% SDS), respectively. After 15 minutes treatment period at room temperature the test item and the controls are rinsed off with PBS and the tissues are post-incubated for 42 +/- 1 h. Then the tissues are stained via MTT for 3 hours. Isopropanol extracts are measured photometrically at 550 nm.

Results and discussion

In vivo

Other effects:

The test item showed no direct MTT reducing capability. The mixture of 10 mg of the test item per 90 µl aqua dest. showed colouring detectable by unaided eye-assessment. Since the colour turned yellow/orange which is not detected by measurement at 550 nm, no quantitative correction of results was necessary.

Any other information on results incl. tables

Name	Negative Control				Positive Control				Test Item
Tissue	1		2	3	1		2	3	1		2	3
absolute OD550	1.056		0.935	1.002	0.134		0.092	0.104	0.912		0.988	0.888
	1.002		0.872	0.963	0.130		0.091	0.099	0.888		0.927	0.869
OD550 (blank-corrected)	1.014		0.894	0.960	0.092		0.050	0.062	0.871		0.947	0.846
	0.960		0.830	0.922	0.088		0.049	0.057	0.846		0.886	0.827
mean OD550of the duplicates (blank-corrected)	0.987		0.862	0.941	0.090		0.050	0.060	0.859		0.916	0.837
total mean OD550of 3 replicate tissues (blank-corrected)	0.930^*				0.066				0.870
SD OD550	0.064				0.019				0.043
relative tissue viabilities [%]	106.2	92.7		101.2	9.7	5.4		6.4	92.3	98.5		89.9
mean relative tissue viability [%]	100.0				7.1^**				93.6
SD tissue viability [%]^***	6.8				2.3				4.4
CV [% viability]	6.8				31.5				4.7

*         Corrected mean OD550 of the negative control corresponds to 100% absolute tissue viability.

**       Mean relative tissue viability of the three positive control tissues is ≤ 40%.

***     The standard deviation (SD) obtained from the three concurrently tested tissues is < 18%.

Applicant's summary and conclusion

Interpretation of results:

other: non irritant

Remarks:

Criteria used for interpretation of results: other: OECD 439

Conclusions:

The test item is classified as "non-irritant" (No Category).

Executive summary:

In the in vitro skin irritation test using the EpiSkin human epidermis model 10 mg test item + 15 µL A. dest. were applied topically for 15 minutes. After 42 h post-incubation cytotoxic effects were determined via MTT reduction assay.

The mean relative tissue viability (% negative control) was > 50%.

The test item is therrefore classified as "non-irritant" (EU CLP and UN GHS: No Category).

This study is classified as acceptable:

OD₅₅₀ of the blank is < 0.1.

Mean OD₅₅₀ of the three negative control tissues is >= 0.6 and <= 1.5.

Mean relative tissue viability of the three positive contol tissues is <= 40%.

Standard deviation (SD) of relative tissue viability obtained from each three concurrently tested tissues is < 18%.

This study satisfies the requirement for Test Guideline OECD 439 for in vitro skin irritation.