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Diss Factsheets

Toxicological information

Skin irritation / corrosion

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Administrative data

skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
13 Dec 2016 - 23 Nov 2017
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
according to guideline
other: Commission Regulation (EU) No 640/2012 of 6 July 2012 amending, for the purpose of its adaptation to technical progress, Regulation (EC) Nº 440/2008, B.46 B.46: In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
Physical state, appearance: solid, white
Storage conditions: room temperature

In vitro test system

Test system:
human skin model
Three dimensional human epidermis model EpiDermTM
Source species:
Cell type:
other: human-derived epidermal keratinocytes which have been cultured to form a multi layered, highly differentiated model of the human epidermis
Cell source:
other: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
Source strain:
other: Tissue model: EPI-200
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
25 μL sterile PBS was applied first. Thereafter, a bulk volume of ca. 25 μL solid ground test material was applied with a sharp spoon and homogeneously distributed together with the fluid.
Duration of treatment / exposure:
The tissues were kept under the laminar flow hood at room temperature for 25 minutes overall and for 35 minutes in the incubator. The tissues were washed with sterile PBS to remove residual test material 1 hour after start of application.
Duration of post-treatment incubation (if applicable):
Rinsed tissues were blotted on sterile absorbent paper and transferred into new 6-well plates pre-filled with 0.9 mL fresh medium. When all tissues were rinsed the surface of each tissue was carefully dried with a sterile cotton swab. Subsequently, the tissues were placed into the incubator at 37°C for 24 ± 2 hours. After 24 ± 2 hours, the tissues were transferred into new 6-well plates pre-filled with 0.9 mL fresh medium and placed into the incubator for an additional 18 ± 2-hour post-incubation period. After the post-incubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours.
Number of replicates:
Three tissues were treated with the test substance, the PC and NC, respectively.

Results and discussion

In vitro

Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean of three samples
Vehicle controls validity:
not applicable
Negative controls validity:
Positive controls validity:
Remarks on result:
no indication of irritation

Any other information on results incl. tables

Individual and mean OD570 values, individual and mean viability values and standard deviations

Test substance
  tissue 1 tissue 2 tissue 3 mean SD CV [%]
NC mean OD570 1.980 1.997 1.873 1.950    
viability [% of NC] 101.5 102.4 96.1 100.0 3.4 3.4
test substance mean OD570 1.838 1.978 1.975 1.930
viability [% of NC] 94.2 101.4 101.3 99.0 4.1 4.2
PC mean OD570 0.047 0.046 0.052 0.049    
viability [% of NC] 2.4 2.4 2.7 2.5 0.2 6.6

The test substance is not able to reduce MTT directly, as determined in a pretest.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
The test article does not show a skin irritation potential in the EpiDerm™ in vitro skin irritation test strategy under the test conditions chosen.
Executive summary:

The potential of the test item to cause dermal corrosion/irritation was assessed by a single topical application of ca. 25 μL bulk volume (about 15 mg) of the undiluted test substance to a reconstructed three-dimensional human epidermis model (EpiDerm™). The irritation test was performed with three EpiDerm™ tissues which were incubated with the test substance for 1 hour followed by a 42-hour post-incubation

period. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation by using a colorimetric test. The reduction of mitochondrial dehydrogenase activity measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the test substance treated epidermal tissues is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability. The following results were obtained in the EpiDerm™ skin irritation test: The test substance is not able to directly reduce MTT. The final mean relative viability of the tissues treated with the test substance determined after an exposure period of 1 hour with an about 42-hour post-incubation was 99.0%. Based on the results observed it was concluded that 4,5,6,7-tetrahydro-1H-benzotriazole does not show a skin irritation potential in the EpiDerm™ in vitro skin irritation test strategy under the test

conditions chosen.