Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
adopted: 28th July, 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Test material form:
solid: particulate/powder
Details on test material:
Batch No.: 7012/2007
Appearance: brown powder
Composition of test substance:
Main component:
2-(4-aminoanilino)-5-nitrobenzenesulphonic acid 5.5 area %

4-[[2,4-dihydroxy-3,5-bis[[4-(4-nitro-2-sulphonatophenyl)amino]phenyl]azo]phenyl]azo]-5-
hydroxynaphtalen-2,7-disulphonate, sodium salts 13.9 area %

4-[[2,4-dihydroxy-3-[(4-methoxyphenyl)azo]-5-[[4-[(4-nitro-2-sulphonatophenyl)amino]phenyl]azo]phenyl]azo]-
5-hydroxynaphthalene-2,7-disulphonate, sodium salts 42.4 area %

4-[[2,4-dihydroxy-3,5-bis[(4-methoxyphenyl)azo]phenyl]azo]-5-
hydroxynaphthalene-2,7-disulphonate, sodium salts 18.2 area %
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Test substance name: Acid Brown 235
- Other name: Korostan Brown DGR
- Source and lot/batch No. of test material: 7012/2007
- Expiration date of the lot/batch: May 2018
- Appearance: dark brown powder

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Vehicle:
physiological saline
Details on test system:
TEST SYSTEM
The reconstructed human epidermal model EpiDerm (EPI-200, MatTek, Bratislava) consists of normal human-derived epidermal keratinocytes, which have been cultured to form a multilayered highly differentiated model of the human epidermis. The EpiDerm system is manufactured according to defined quality assurance procedures. The EpiDerm tissues (surface 0.63 cm²) are cultured on specially prepared cell culture inserts and shipped as kits, containing tissues on shipping agarose together with the necessary amount of culture media.

DIRECT MTT REDUCTION - FUNCTIONAL CHECK IN TUBES
Some test substances may interfere with the MTT endpoint if it is able to directly reduce MTT. Therefore, functional check for this possibility was performed as follows: 25 mg of the test substance was added to 1 mL MTT medium (red) and incubated in the incubator (37±1°C, 5±1 % CO2, moistened) for 1 hour. At the end of the exposure time, the presence and intensity of the staining (if any) were observed. If the solution changed colour from red to blue, other steps (test in frozen tissues) to correction have to be done.

COLOUR INTERFERENCE
To identify potential interference by coloured test chemical and decide on the need for additional controls, OD570 (optical density at 570 nm) of the test chemical in water (environment during exposure) and/or isopropyl alcohol (extracting solution) is measured.
25 mg of the test substance was mixed with 2 mL of water for injection and shaken for 2-3 hours. The same procedure was performed for isopropyl alcohol. After that, test tubes were centrifuged and decanted. OD570 of supernatant was then measured. If, after subtraction of the OD for isopropanol, the OD of the test article solution is > 0.08 (approximately 5% of the mean viability of the negative control) the test substance has to be considered as possibly interacting with the MTT measurement and an additional test on colorant controls has to be performed.

MTT TEST
Application:
25 mg of the test substance (25 mg of substance/surface ratio 39.7 mg/cm2) was dosed directly on tissue moistened with 25 µL of PBS (phosphate buffered saline). The substance was spread on the tissue surface. A single test, composed of three replicate tissues, was run.
Procedure:
On the day of receipt, EpiDerm tissues were conditioned by incubation to release transport stress related compounds and debris. Duration of pre-incubations before treatment was 60 minutes.
After pre-incubations, tissues were topically exposed to the test chemicals for 60±1 minutes (25 minutes at room temperature and the remaining 35 minutes at culture conditions). Three tissues were used per the test substance, three for the positive (PC) and three for the negative (NC) controls. Tissues are then thoroughly rinsed with PBS. Inserts were then transferred to fresh medium.
After 23 hours and 48 minutes of post-incubation period, the medium was replaced by fresh one. Tissues were incubated for another 18 hours, 21 minutes. Afterwards, the MTT assay was performed by transferring the tissues to 24-well plates containing MTT medium (1 mg/mL). After 185±5 minutes MTT incubation, the blue formazan salt formed by cellular mitochondria was extracted with 2.0 ml/tissue of isopropyl alcohol (for minim. 120 minutes, room temperature, shaking) and the optical density of the extracted formazan was determined using a spectrophotometer at 570 nm.
OD570 measuring:
OD570 was measured on a spectrophotometer Libra S22. Isopropyl alcohol served as a blank. Allowed band width is 2-3 nm. No external filter was used.
Viability calculation:
Relative cell viability is calculated for each tissue as % of the mean of the negative control tissues. Than the mean relative tissue viability of three individual tissues exposed to the test substance is calculated – this value is used for the comparison with limit.
Control samples:
yes, concurrent vehicle
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg Acid Brown 235

VEHICLE
- Amount(s) applied (volume or weight with unit): 25 µL phosphate buffered saline

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): phosphate buffered saline

POSITIVE CONTROL
- Concentration (if solution): 5% sodium dodecyl sulphate
Duration of treatment / exposure:
60 min
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
94
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
DIRECT MTT REDUCTION - FUNCTIONAL CHECK IN TUBES
25 mg of the test substance was added to 1.0 mL of MTT medium. Suspension was incubated for 1 hour at culture conditions. After incubation, the medium was brown-red coloured. The test substance does not reduce MTT directly.

COLOUR INTERFERENCE
The test substance is soluble in water for injection. OD570 of solution in water for injection was >3. The test substance is not soluble in isopropyl alcohol. Average OD570 value from 2 wells was 0.008 what is < 0.08. On the basis of results obtained, it was decided do not use concurrent colorant control in the MTT test.

ACCEPTANCE CRITERIA FULFILMENT
The mean OD570 of the NC tissue was 2.373 ± 0.115 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 is 3.3 % which meets the acceptance criterion of ≤ 20 %.
The SD calculated from individual % tissue viabilities of the 3 identically treated replicates for the positive control, negative control and test substance was < 18 %.
All study acceptance criteria were fulfilled.

Any other information on results incl. tables

Table 1: Optical density and viability

 

Treatment

OD570

Avg

SD

Average viability

(% NC)

1

2

3

NC

PBS

2.450

2.458

2.211

2.373

0.115

100.0

%

103.2

103.6

93.2

100.0

4.8

C4

390/16

2.412

2.187

2.096

2.232

0.133

94.0

%

101.6

92.2

88.3

94.0

5.6

PC

5% SDS

0.063

0.087

0.082

0.077

0.010

3.3

%

2.7

3.7

3.5

3.3

0.4

NC - negative control

PC - positive control

C4 - test substance

SD - standard deviation

SDS - sodium dodecyl sulphate

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the test conditions, average viability of tissues treated by the test substance, Acid Brown 235, was 94.0 % of negative control average value i.e. viability was > 50 %.
The effect of the test substance was negative in EpiDerm model.
According to the classification criteria given in this assay, the test substance, Acid Brown 235, is considered to have no category in accordance with UN GHS and is therefore considered a non-irritant.
Executive summary:

The test substance, Acid Brown 235, was assayed for in vitro skin irritation in the human epidermal model EpiDerm.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 EpiDerm Skin Irritation Test For use with MatTek Corporation’s Reconstructed Human Epidermal Model EPI-200-SIT.

In preliminary experiment neither colour interference with the endpoint nor direct MTT reduction were found.

After pre-incubation of tissues, 25 mg of the test substance was placed directly on previously moistened tissue and 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 experimental design average viability of treated tissues was 94.0%, i.e. viability was >50 %.

The effect of the test substance was negative in EpiDerm model (tissues were not damaged).

According to the classification criteria, thetest substance, Acid Brown 235, is considered to have no category in regard to skin irritation.