Disodium 3-hydroxy-4-[(4-methyl-2-sulphonatophenyl)azo]-2-naphthoate

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 January 2017 - 13 January 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

29 July 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU method B.40 BIS (In Vitro Skin corrosion: Human Skin Model Test)

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

dd 03 November 2015

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 3-hydroxy-4-[(4-methyl-2-sulphonatophenyl)azo]-2-naphthoate

EC Number:

227-497-9

EC Name:

Disodium 3-hydroxy-4-[(4-methyl-2-sulphonatophenyl)azo]-2-naphthoate

Cas Number:

5858-81-1

Molecular formula:

C18H12N2Na2O6S

IUPAC Name:

disodium 3-hydroxy-4-[(4-methyl-2-sulphonatophenyl)azo]-2-naphthoate

Test material form:

solid: particulate/powder

Details on test material:

Name as cited in the report: D&C Red 6
Appearance: red powder
Storage conditions: at room temperature

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Justification for test system used:

Recommended test system in international guidelines (OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Skin Model (EPI-200)
- Tissue Lot number: 24956 kit Q and R
- Tissue surface: 0.6 cm²
- All cells used to produce Eipderm™ are purchased or derived from tissue obtained by MatTek Corporation from acredited institutions.
- Cells are screened for potential biological contaminants (HIV-1, Hepatitis B, Hepatitis C, bacteria, yeast and fungi)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: All incubations, with the exception of the test item incubation of 3 minutes at room temperature, were carried out in a controlled environment at 37.0 ± 1.0°C (actual range 36.5 - 37.0°C).

REMOVAL OF TEST MATERIAL AND CONTROLS
- After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test item.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2 replicates per exposure duration, one negative control, one positive contol.

ACCEPTANCE OF RESULTS:
The in vitro skin corrosion test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 of the two tissues of the negative control should reasonably be within the laboratory historical control data range.
b) The mean relative tissue viability following 3-minute exposure to the positive control should be ≤ 30%.
c) In the range of 20 – 100% viability, the maximum inter-tissue variability (in viability) is ≤ 30% between two tissues treated identically.
d) In the range of 20 – 100% viability, the maximum difference in percentage between the mean viability of two tissues and one of the two tissues is ≤ 15%.

DECISION CRITERIA: see table 1 under 'Any other information on materials and methods'

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

other: concurrent control for MTT reduction by test item

Amount/concentration applied:

30.03 to 39.16 mg of test item on the skins moistened with 25 μl Milli-Q water

Duration of treatment / exposure:

3-minute and 1-hour

Duration of post-treatment incubation (if applicable):

none

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3-minute exposure

Value:

101

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

(100%)

Positive controls validity:

valid

Remarks:

(14%)

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1-hour exposure

Value:

96

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

(100%)

Positive controls validity:

valid

Remarks:

(9%)

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: No
- Colour interference with MTT: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes, mean relative tissue viability following 3-minute exposure was 7%.
- Acceptance criteria met for variability between replicate measurements: No, the Coefficient of Variation between tissue replicates of the negative control substance was 31% at the 1-hour treatment time which is not within the acceptability criteria of the assay (≤30%).

- Because the acceptability criterium for variability between replicate measurements was not met and the absolute OD570 (optical density at 570 nm) of the negative control tissues was not within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤2.8) for one of the skin tissues of the 3-minutes treatment time (0.754) and the absolute mean OD570 was not within the laboratory historical control data range both at the 3-minutes and 1-hour treatment time, a second set of negative controls was used. These negative controls were available from studies performed at the same time which fulfilled all acceptability criteria and could therefore be used for this study. The mean relative tissue viability following the 1-hour exposure to the positive control was 13% (first negative control) or 9% (second negative control). In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was ≤ 25% with the second negative control, indicating that the test system functioned properly.

- Final results for tissue viability were based on the second set of negative controls.

- Table 2 ('Any other information on results') shows the mean tissue viability with the first and the second set of negative controls, table 2 shows the results of individual OD measurements at 570 nm.

Any other information on results incl. tables

Table 2 Mean tissue viability

	3-minute application viability (percentage of control)	1-hour application viability (percentage of control)
Negative control	100	100
D&C Red 6	189 (101)	140 (96)
Positive control	27 (14)	13 (9)

The viability with the second negative control is given between brackets.

_{Table 3 Mean absorption in the in vitro skin corrosion test with D&C Red 6}

	3-minute application			1-hour application
	A (OD 570)	B (OD 570)	Mean (OD570) ±SD	A (OD 570)	B (OD 570)	Mean (OD570) ±SD
Negative control	0.754	0.891	0.822 ±0.097	1.224	0.842	1.033 ±0.270
Negative control (2)	1.707	1.380	1.543 ±0.231	1.546	1.462	1.504 ±0.060
D&C Red 6	1.495	1.610	1.552 ±0.081	1.625	1.270	1.448 ±0.251
Positive control	0.271	0.172	0.222 ±0.070	0.138	0.123	0.130 ±0.011

SD = Standard Deviation

Duplicate exposures are indicated by A and B.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

An in vitro skin corrosion test was conducted with D&C Red 6 according to OECD 431 guideline and GLP principles. It is concluded that this test is valid and that the test substance is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

Executive summary:

In an in vitro skin corrosion test using a human skin model (EpiDerm Skin Model, Lot no.: 24956 kit Q and R), the influence of the test substance on the viability of human skin was tested. At least twenty-five mg (actual dose: 30.03 to 39.16 mg) of D&C Red 6 was applied directly to 0.6 cm² cultured skin. After 3-minute and 1-hour treatments the substance was removed and the viability of the cells was tested by reduction of MTT. Viability of unexposed skin was set at 100%. The relative mean tissue viability after 3 minutes and 1 hour of exposure to the test item were 101% and 96%, respectively. The positive control had a mean cell viability of 9%. Since the mean relative tissue viability was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment, it can be concluded that D&C Red 6 is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.