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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 JUNE 2018 to 4 SEPTEMBER 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
OECD 431 (July 2016) In Vitro Skin Corrosion: Reconstructed Human Epidermis (Rhe) Test Method
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from multiple donors
Source strain:
not specified
Justification for test system used:
Reconstructed Human Epidermis (RhE; EpiSkinTM-SM).
This is a three-dimensional human skin model comprising a reconstructed epidermis with a functional stratum corneum. Differentiated and stratified epidermis model comprises the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Three-dimensional RhE model, comprised of non-transformed, humanderived epidermal keratinocytes, which have been cultured to form a multi-layered, highly differentiated
model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multilayered stratum corneum containing intercellular lamellar lipid layers representing main lipid classes analogous to those found in vivo.
The RhE test method is based on the premise that corrosive chemicals are able to penetrate the stratum corneum by diffusion or erosion, and are cytotoxic to the cells in the underlying layers. Cell viability is measured by enzymatic conversion of the vital dye MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide], into a blue formazan salt that is quantitatively measured after extraction from tissues. Corrosive chemicals are identified by their ability to decrease cell viability below defined threshold levels. The RhE-based skin corrosion test methods have shown to be predictive of in vivo skin corrosion effects assessed in rabbits according to the OECD guideline 404.
Vehicle:
other: Apply topically to the epidermal surface
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Reconstructed Human Epidermis (RhE; EpiSkinTM-SM) (Ref EKINJ13).
- Tissue batch number(s): 18-EKIN-030
- Production date: Expiration date 30/07/2018
- Date of initiation of testing: 24/07/2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 min, 1h (±5 min) or 4h (±10 min) at room temperature
- Temperature of post-treatment incubation (if applicable): culture conditions

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: RhE inserts were rinsed with DPBS (1x)
- Observable damage in the tissue due to washing: none
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL ready to use solution
- Incubation time: 3h ± 5 min under culture conditions
- Spectrophotometer:
- Wavelength: The optical densities (OD) were measured between 540 to 600 nm
- Filter: none
- Filter bandwidth: none
- Linear OD range of spectrophotometer:

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability:
- Barrier function:
- Morphology:
- Contamination:
- Reproducibility:

NUMBER OF REPLICATE TISSUES: 2 replicates (RhE inserts (2 inserts per exposure time))

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues: 36/12
- Procedure used to prepare the killed tissues (if applicable):
- N. of replicates : 2
- Method of calculation used:
Calculation of Non-Specific MTT reduction (NSMTT)
With the killed tissues treated with the Negative control, the ODku (Mean OD of the two replicate killed
tissues (2 OD values per tissue already corrected with ODblank mean) treated with the Negative Control, with
MTT incubation (similar calculations as for ODNc)) was calculated as follows:
− Individual OD Negative control killed insert 1 (ODku1) = ODku1 raw - ODblank mean
− Individual OD Negative control killed insert 2 (ODku2) = ODku2 raw - ODblank mean
Mean ODku = (ODku1 + ODku2)/2
With the killed tissues treated with the test item the ODKT was calculated as follows:
− Individual OD Test item killed insert 1 (ODku1) = ODkT1 raw - ODblank mean
− Individual OD Test item killed insert 2 (ODku2) = ODkT2 raw - ODblank mean
Mean ODkT = (ODkT1 + ODkT2)/2
Calculation of individual NSMTT (%):
− Tissue 1: % NSMTT1 = 100 x [(ODkT1-ODku)/ ODNc]
− Tissue 2: % NSMTT2 = 100 x [(ODkT2-ODku)/ ODNc]
Mean NSMTT (%):
% NSMTT = (% NSMTT1 + % NSMTT2) / 2

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:
3 (3 min, 1h, 4h)

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if Corrosive class I 1A If viability < 35% after 3 min exposure, Corrosive class II 1B If viability ≥ 35% after 3 min and < 35% after 1
hour exposure OR Corrosive class III 1C If viability ≥ 35% after 1 hour and < 35% after 4
hours exposure
- The test substance is considered to be non-corrosive to skin ifIf viability ≥ 35% after 1 hour and < 35% after 4 hours exposure
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439:
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 20 mg
- Concentration (if solution):

VEHICLE
- Amount(s) applied (volume or weight with unit): none
- Concentration (if solution):
- Lot/batch no. (if required):
- Purity: none

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): Saline solution B Braun (0.9% w/v NaCl)
- Concentration (if solution): 0.9% w/v NaCl

POSITIVE CONTROL
- Amount(s) applied (volume or weight): Glacial acetic acid
- Concentration (if solution): 99.7%
Duration of treatment / exposure:
3 min
1 hour
4 hours
Duration of post-treatment incubation (if applicable):
3h (± 15 min) under culture conditions
Number of replicates:
2 replicatesfor each

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Run 1 (3 min), Replicate 1 (3 min)
Run 2 (1h), Replicate 2 (1h)
Run 3 (4h), Replicate 3 (4h)

Mean final relative viability (TTNSC) % ± SD 98.0±7.0
Value:
>= 91 - <= 105
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Taking into account the results obtained in the current study, it is concluded that, under the assayed
experimental conditions and according to the OECD guideline for the Testing of Chemicals Nº. 431, the test
item 1,4-bis(isopropylamino)anthraquinone can be categorised as Non-Corrosive according to the United
Nations Globally Harmonized System of Classification and Labelling of Chemicals.