Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 June 2016 until 18 July 2016
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
other: UN GHS (2003, last rev. 2015)
Principles of method if other than guideline:
Based on a “Statement on the Scientific Validity of In Vitro Tests for Skin Irritation” of the European Commission (November 2008), official acceptance of the test method in the EU was achieved and implemented in EU, 2008a, Council Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to EC Regulation No 1907/2006 of the European Parliament and of the Council on REACH; 1st ATP 2009: EC Regulation No 761/2009 of 23 July 2009 amending, for the purpose of its ATP, EC Regulation No 440/2008 laying down test methods pursuant to EC Regulation No 1907/2006 of the European Parliament and of the Council on REACH, section B46.
GLP compliance:
yes (incl. certificate)
Remarks:
Date of inspection: 13-16 July 2015, Date of Signature: 14 September 2015

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

In vitro test system

Test system:
human skin model
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
Each approximately 25 mg (~ 39 mg/cm2 according to guideline) of the test item
Duration of treatment / exposure:
60 minutes.
Number of replicates:
3 tissues

Test animals

Species:
other: reconstituted human epidermis model
Details on test animals and environmental conditions:
Identification: 1-[(4-Methoxy-2-nitrophenyl)azo]-2-naphthol
Batch: 3. April 2010
EINECS No.: 256-458-9
CAS No.: 49744-28-7
Purity: 98.50% (w/w), dose calculation was not adjusted to purity
Appearance: Red powder
Expiry Date: 03 April 2020 (statement of producer)
Storage Conditions: At room temperature
Certificate of Analysis: AZ 575/Toxd2, dated 11 December 2015

Test system

Type of coverage:
other: Topical
Preparation of test site:
other: Not applicable
Vehicle:
other: No vehicle used
Controls:
yes
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
Each approximately 25 mg (~ 39 mg/cm2 according to guideline) of the test item were applied to the tissues, wetted with 25 µL DPBS, and spread to match the surface of the tissue for a complete treatment time of 60 minutes.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight):
30 µL DPBS (MatTek) were used as negative control per tissue


POSITIVE CONTROL
- Amount(s) applied (volume or weight):
30 µL of a 5% SLS solution in deionised water (MatTek) were used a positive control per tissue.
Duration of treatment / exposure:
60 minutes
Observation period:
Not applicable
Number of animals:
Not applicable

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
The mean relative absorbance value of the test item after correction, corresponding to the cell viability, achieved 92.4% (threshold for irritancy: ≤ 50%) compared to the result of the negative control.
Value:
> 1.673 - < 1.687
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of irritation

In vivo

Irritant / corrosive response data:
Compared to the relative absorbance value of the negative control the corrected mean relative absorbance value achieved 92.4% after exposure of the skin tissues to the test item. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.
Other effects:
No

Any other information on results incl. tables

Results after treatment with 1-[(4-Methoxy-2-nitrophenyl)azo]-2-naphthol and the controls (exposure interval of 60 minutes):

Dose

Group

Tissue

No 

 Absorb.

570 nm
Well 1

Absorb.

570 nm
Well 2
 

 Absorb.

570 nm
Well 3

Mean

Absorb.
of 3 Wells
 

Mean

Absorb.

of

3 Wells

corrected 

 Mean Absorb.

of 3

tissues

after

blank

corr.*

Rel.

Absorb.
[%] Tissue
1, 2 + 3

** 

Relative

Standard Deviation 

Mean Rel.

Absorb.

[% of Negative Control]

*** 

Blank   0.038 0.047 0.037 0.041 0        

Negative

Control

1 1.972 1.935 1.892 1.933 1.892 1.81 104.5 4.9 100
2 1.881 1.831 1.874 1.862 1.821 100.6
3 1.788 1.738 1.746 1.757 1.717 94.8

Test

Item

1 1.547 1.514 1.552 1.538 1.497 1.687 82.7 9.8 93.2
2 1.811 1.828 1.81 1.816 1.776 98.1
3 1.822 1.829 1.837 1.829 1.789 98.8

Positive

Control 

1 0.093 0.091 0.091 0.092 0.051 0.05 2.8 7.6 2.8
2 0.093 0.094 0.094 0.093 0.053 2.9
3 0.087 0.086 0.086 0.086 0.046 2.5

Dose Group Tissue No. Absorb.
570 nm
Well 1
Absorb.
570 nm
Well 2
Absorb.
570 nm
Well 3
Mean
Absorb.
of 3
Wells

Mean
Absorb.
of 3

Wells
blank
corr.

Rel.

Absorb.
[%]**

OD ***

Corr.

Mean
Rel.
Absorb.
[% of
Neg.

Contr.]
****

Blank   0.038 0.047 0.037 0.041 0.000   1.673 92.4
Negative Control 1 0.038 0.038 0.039 0.038 -0.002 100.0
Test Item 1 0.053 0.053 0.058 0.055 0.014 -652.3

*   Mean of three replicate wells after blank correction

**  relative absorbance per tissue [rounded values]:

*** OD=ODcoloured tissue(MTT assay)– ODcoloured tissue (no MTT assay)

****relative absorbance per treatment group [rounded values]

The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water led to a change in colour. An additional test with one viable tissue was necessary. The result was used for data correction of the results in the main experiment.

Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour. An additional test with freeze-killed tissues was not necessary.

The mean relative absorbance value of the test item after correction, corresponding to the cell viability, achieved 92.4% (threshold for irritancy: ≤ 50%) compared to the result of the negative control, consequently the test item was not irritant to skin.

Applicant's summary and conclusion

Interpretation of results:
other: Not irritant
Conclusions:
In conclusion, it can be stated that in this study and under the experimental conditions reported, 1-[(4-Methoxy-2-nitrophenyl)azo]-2-naphthol is not irritant to skin according to UN GHS and EU CLP regulation.
Executive summary:

This in vitro study was performed to assess the irritation potential of 1-[(4-Methoxy-2-nitrophenyl)azo]-2-naphthol by means of the Human Skin Model Test.

The test item passed the MTT interference pre-test. Due to its intensive colour, an additional test with one viable tissue (without MTT addition) was necessary to correct the result in the main experiment.

Approximately 25 mg of the test item and each 30 µL of the negative control (DPBS) or the positive control (5% SLS) were applied to triplicate tissue each.

The test item and the positive and negative controls were washed off the skin tissues after 60 minutes treatment. After further incubation for about 43.3 hours the tissues were treated with the MTT solution for 3 hours following nearly 69 hours extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm.

The acceptance criteria were met:

  • Tissue viability was meeting the acceptance criterion if the mean OD570of the negative control tissues was > 0.8 and ≤2.8 (values between 1.738 and 1.972).
  • The mean relative tissue viability of the positive control was ≤ 20% (2.8).
  • The relative standard deviations between the % variability values of the test item and the controls in the main test were below 10% (threshold of the "OECD Guideline for the Testing of Chemicals 439: In vitro Skin Irritation: Reconstructed Human Epidermis Test Method”: < 18%), thus ensuring the validity of the study.

Compared to the relative absorbance value of the negative controlthe corrected mean relative absorbance value achieved 92.4% after exposure of the skin tissues to the test item. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.