4,5,6,7-tetrahydro-1H-benzotriazole

Skin irritation / corrosion

Administrative data

Endpoint:

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Test guideline 2

Qualifier:

according to guideline

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

Test material information

Constituent 1

Reference substance name:

4,5,6,7-tetrahydro-1H-benzotriazole

EC Number:

229-858-6

EC Name:

4,5,6,7-tetrahydro-1H-benzotriazole

Cas Number:

6789-99-7

Molecular formula:

C6H9N3

IUPAC Name:

4,5,6,7-tetrahydro-1H-1,2,3-benzotriazole

Details on test material:

Physical state, appearance: solid, white
Storage conditions: room temperature

In vitro test system

Test system:

human skin model

Remarks:

Three dimensional human epidermis model EpiDermTM

Source species:

human

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

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean of three samples

Value:

99

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

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 identification		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

Conclusions:

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.