Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From June 25, 2019 to July 11, 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
2,7-Naphthalenedisulfonic acid, 5-(acetylamino)-3-[2-[5-[[4-chloro-6-[[3-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]amino]-1,3,5-triazin-2-yl]amino]-2-sulfophenyl]diazenyl]-4-hydroxy-, sodium salt (1:4)
EC Number:
695-779-2
Cas Number:
80019-35-8
Molecular formula:
C29H21ClN8O17S5.4Na
IUPAC Name:
2,7-Naphthalenedisulfonic acid, 5-(acetylamino)-3-[2-[5-[[4-chloro-6-[[3-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]amino]-1,3,5-triazin-2-yl]amino]-2-sulfophenyl]diazenyl]-4-hydroxy-, sodium salt (1:4)
Test material form:
solid: particulate/powder

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: recoonstituted human epidermis model
Cell source:
other: Adult human-derived epidermal keratinocytes
Source strain:
other: Adult human
Details on animal used as source of test system:
EPISKIN kits are manufactured according to defined quality assurance procedures (certified ISO 9001).
(a) Biological safety aspects: All biological components of the epidermis and the kit culture medium were tested for the presence of viruses, bacteria and mycoplasma.
(b) Quality control aspects: The quality of the final product was assessed by an MTT cytotoxicity test with Sodium Dodecyl Sulphate (SDS) and by histological examination.
Vehicle:
unchanged (no vehicle)
Details on test system:
EPISKIN Standard Model TM is a 0.38 cm^2 reconstituted human epidermis model. Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum (Tinois et al., 1984). Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
The test item was tested for its ability to direct formazan reduction. This pre-test was done on the same day of starting the main study.
-6 well of 12 well plate was filled with 2 mL of MTT solution (0.3 mg/L).
-To above 6 wells, 10uL of the test item was added to 3 wells and 10 µL of buffer to other 3 wells and mixed well.
-Incubated the mixture for 3 hours at 37°C protected from light.
Since, no change in the color (blue or purple) of MTT solution was observed, the test item was identified to have no interaction with MTT.
Duration of treatment / exposure:
Pre-Test
The test item was tested for its ability to direct formazan reduction. This pre-test was done on the same day of starting the main study.
- 6 well of 12 well plate was filled with 2 mL of MTT solution (0.3mg/L).
- To above 6 wells, 10uL of the test item was added to 3 wells and 10uL of buffer to other 3 wells and mixed well.
- Incubated the mixture for 3 hours at 37°C protected from light.
Since, no change in the color (blue or purple) of MTT solution was observed, the test item was identified to have no interaction with MTT.
Duration of post-treatment incubation (if applicable):
Post treatment incubation: 42 hours
Incubated the treated and rinsed epidermis at 37°C, 5% CO2, 95% humidified atmosphere for 42 hours.

MTT Test After the 42 hours Incubation Period
Required number of wells of the assay plate were filled with 2 mL per well of 0.3 mg/mL MTT in assay medium.
Treated units were transferred to the MTT filled wells. Plate was covered with lid and incubated for 3 hours at 37°C, 5% CO2, 95% humidified atmosphere.

2.3 Formazan Extraction (Day 3)
(a) 1.5 mL of conic "safelock" type micro tubes were labelled appropriately for test item, negative control, positive control and their replicate Number (1 tube/epidermis).
(b) Tissue units were placed on absorbent paper to dry the tissues.

(c) Using the special biopsy punch a total biopsy of the epidermis was done.
(d) Gently separated the epidermis from the collagen matrix with the aid of forceps, and both parts (epidermis and collagen matrix) were placed into the labelled micro tubes.
(e) When all the tissues were punched and ready in the micro tubes, 500 µL of acidic isopropanol per tube was added.
(f) Each tube was plugged to avoid evaporation and mixed thoroughly using a vortex mixer.
(g) All the tissues were correctly immersed in the solvent and extraction start time for each tissue was noted.
(h) The tubes were stored for 4 hours at room temperature (recommended 18 to 23°C) and protected from light.
(i) Vortex each tube at the middle of the incubation end time was noted.
(j) Tissues were removed from each tube extraction end time was noted.
(k) Extraction solution was homogenized by pipetting 3 times up and down.
(l) From each well, two replicates with 200 µL solution (each) were pipetted into a 96-well-plate which was read in using spectrophotometer at 560 nm. Acidified isopropanol solution was used as blank.
Number of replicates:
6

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
Main Study
The assay medium supplied with the kits was kept at 2~8°C (refrigerator).
(a) Maintenance medium was pre-warmed to 37°C before using.
(b) EPISKIN kit was kept at room temperature in the Bio safety cabinet until next step.

Preparation and Pre-incubation (Day 0)
This step was conducted in a Biosafety cabinet
(a) 3 wells of a 12 well assay plates were filled with pre-warmed maintenance medium (2mL per well).
(b) EPISKIN kit was opened and epidermis units were transferred into maintenance medium filled wells, using sterile forceps avoiding air bubbles.
Labelled plate lids by replicate treatment order as follows:
Negative control Positive control Test item
Rep 1 (NCR1) Rep 1 (PCR1) Rep 1 (9243 R1)
Rep 2 (NCR2) Rep 2 (PCR2) Rep 2 (9243 R2)
Rep 3 (NCR3) Rep 3 (PCR3) Rep 3 (9243 R3)
Key: Rep: Replicate; NC: Negative control; PC: Positive control; 9243 = Study number for test item CR SR94

(c) Incubated at 37°C, 5% CO2, in a >95% humidified atmosphere for 24 hours.
Duration of treatment / exposure:
Application of test item and rinsing (Day 1)
(a) Pre-warmed maintenance medium (2 mL per well) was filled by taking into account of the 3 wells per replicate. Plate lids were labeled with test item (3 wells per test item), or negative control (3 wells) or positive control (3 wells).

Topical applications: 15 minutes treatment
(b) 5 µL of Distilled water was applied to the epidermal surface in order to improve further contact between the test item and epidermis.
(c) 10 µL of negative control, 10 mg of test item and 10 µL of positive control were applied on the top of the epidermis.
(d) Followed the defined application order (Rep 1, Rep 2 and Rep 3).
(e) After application, plate containing the treated epidermis was closed with lid and placed in the same biosafety cabinet for 15 minutes at room temperature.
Only for positive control, re-spread after 7 minutes contact time. And each tissue applications were done at 1 minute interval.

End of the treatment and removal of the test chemical: After 15 minutes exposure:
(f) Treated epidermal units were removed using forceps, and rinsed thoroughly with 25 mL sterile DPBS by filling and emptying the tissue inserts to remove residual material from the epidermal surface.
(g) Epidermal units were placed on an absorbent paper and remaining DPBS were removed from the epidermal surface by gently taping, and swept the epidermal surface with a cotton-bud without damaging the epidermis.
(h) Blotted tissue units were transferred to the new maintenance medium pre-filled wells.
Details on study design:
The assay medium supplied with the kits was kept at 2-8°C (refrigerator).
(a) Maintenance medium was pre-warmed to 37°C before using.
(b) EPISKIN kit was kept at room temperature in the Bio safety cabinet until next step.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Remarks:
Mean
Value:
81.217
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
85.306
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2
Value:
79.26
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3
Value:
79.084
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the results, the mean percent viability of the tissue treated with test item CR SR94 was 81.217% and it was greater than 50% of the negative control. Hence, under the conditions of the study the test item was found to be 'Non Irritant' to skin in accordance with UN GHS as specified in the OECD Guideline for the Testing of Chemicals.
Executive summary:

Test item, CR SR94 was applied topically to the EPISKIM TM epidermal model (three epidermis units are used per each test item, positive and negative controls) for 15 minutes. Exposure to the test item was terminated by rinsing with Dulbecco's phosphate buffered saline (DPBS). Epidermis was then incubated at 37for 42 additional hours. The viability was assessed by incubating the tissues for 3 hours with MTT solution in a 12 well plate (0.3 mg/mL in assay medium; 2 mL per well). The precipitated formazan was then extracted using acidified isopropanol (0.5 mL) for 4 hours at room temperature and quantified spectrophotometrically at 560nm using 96 well plates (200µL/well). SDS5% and DPBS treated epidermis were used as positive (PC) and negative controls (NC) respectively. Each treated tissue % viability was obtained by comparing with the mean of the negative control tissues.

The negative controls mean Optical Density (OD) was found to be 0.852, which is well within the acceptability range. The mean percent viability of the tissue treated with positive control was 18.763% and it was less than 50% of the negative control, which reflects the irritant nature of positive control and the sensitivity of the tissues used in the study.

The mean percent viability of the tissue treated with test item was 81.217% and it was greater than 50% of the negative control. The variation within the replicates was found to be less than 18%. Hence, under the conditions of the studym, the test item was found to be Non Irritant.

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