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Diss Factsheets

Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted in 2014 study at recognised contract research organisation.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
4-isopropylbenzonitrile
EC Number:
237-492-3
EC Name:
4-isopropylbenzonitrile
Cas Number:
13816-33-6
Molecular formula:
C10H11N
IUPAC Name:
4-(propan-2-yl)benzonitrile
Test material form:
other: liquid
Details on test material:
-Batch: SC00008199
-Purity: 99.5%
-Expiry Date: 19 August 2016

Test animals

Species:
other: N/a: EPISKIN reconstructed human epidermis model (In Vitro)
Strain:
other: n/a
Details on test animals or test system and environmental conditions:
EPISKIN Reconstructed Human Epidermis Model Kit:
-Supplier : SkinEthic Laboratories, Lyon, France
-Date received : 02 September 2014


Test system

Type of coverage:
other: N/a
Preparation of test site:
other: N/a
Vehicle:
unchanged (no vehicle)
Controls:
other: N/a
Amount / concentration applied:
The test substance was used as supplied (100% concentration).



A 3 mg/mL MTT stock solution was prepared in DPBS. The stock solution was diluted to 0.3 mg/mL with assay medium when required. A 0.04 N
solution of hydrochloric acid in isopropanol was prepared when required.

Application of Test Item and Rinsing
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the second column of 3 wells of the 12-well plate. Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was applied topically to the corresponding tissues ensuring
uniform covering. 10 μL (26.3 μL /cm2) of the test item was applied to the epidermis surface. Triplicate tissues treated with 10 μL of DPBS served as
the negative controls and triplicate tissues treated with 10 μL of SDS 5% w/v served as the positive controls. To ensure satisfactory contact with the
positive control item the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the
centre). After 7-Minutes contact time the SDS solution was re-spread with a pipette tip to maintain the distribution of the SDS for the remainder of the
contact period. The plate(s) were kept in the biological safety cabinet at room temperature for 15 minutes.

At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++
and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream
of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 hours.
Duration of treatment / exposure:
a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours
Observation period:
n/a
Number of animals:
Tissues were tested in triplicate
Details on study design:
Preparation of Negative and Positive Control Substances, MTT and Acidified Isopropanol:
The negative control substance, PBS, was used as supplied. The positive control substance, SDS, was prepared as a 5% w/v aqueous solution. A 3 mg/mL MTT stock solution was prepared in DPBS. The stock solution was diluted to 0.3 mg/mL with assay medium when required. A 0.04 N solution of hydrochloric acid in isopropanol was prepared when required.

Test for Direct MTT Reduction:
As specified, a test substance may interfere with the MTT endpoint, if it is able to directly reduce MTT and at the same time is present on or in the tissues when the MTT viability test is performed. To identify this possible interference, the test substance is checked for the ability to directly reduce MTT according to the following procedure: 10 μL of the test substance was added to 2 mL of a 0.3 mg/mL MTT solution freshly prepared inassay medium. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control. If the MTT solution containing the test substance turns blue, the test substance is presumed to have reduced the MTT and the determination of skin irritation potential would be performed in parallel onviable and water-killed tissues for quantitative correction of the results.

Pre-incubation (Day 0: Tissue Arrival):
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the first column of 3 wells of a pre-labeled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test substance and
each control substance. The tissues were incubated at 37 °C, 5% CO2 in air overnight.

Main Test

Application of Test Substance and Rinsing (Day 1):
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the second column of 3 wells of the 12-well plate. Triplicate tissues were treated with the test substance for an exposure period of 15 minutes. The test substance was applied topically to the corresponding tissues ensuring uniform covering. 10 μL (26.3μL /cm2) of the test substance was applied to the epidermis surface. Triplicate tissues treated with10 μL of DPBS served as the negative controls and triplicate tissues treated with 10 μL of SDS5% w/v served as the positive controls. To ensure satisfactory contact with the positive control substance the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the centre). After 7-Minutes contact time the SDS solution was re-spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period. The plate(s) were kept in the biological safety cabinet at room temperature for 15 minutes. At the end of the exposure period,
each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by
filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test substance. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were
incubated at 37 °C, 5% CO2 in air for 42 hours.

MTT Loading/Formazan Extraction (Day 3):
Following the 42-Hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenize the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer at -14 to -30 ºC for possible inflammatory mediatordetermination. 2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plate(s). The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37 °C, 5% CO2in air. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labeled 1.5 mL micro tubes containing 500 μL ofacidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction offormazan crystals out of the MTT-loaded tissues.

Absorbance/Optical Density Measurements (Day 6):
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous colored solution. For each tissue, duplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. 200 μL of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at 562 nm (without a reference filter) using the Anthos 2001 microplate reader.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other: other: Relative mean tissue viability (%)
Value:
84.5
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 42 hours. Reversibility: other: not applicable. (migrated information)

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information EU DSD & CLP Not classified for Irritation. UN GHS Not classified for Irritation (category 3 can not be determined). Criteria used for interpretation of results: other: GHS EU
Conclusions:
The relative mean viability of the test item treated tissues was 84.5% after the 15-Minute exposure period and 42 hours post-exposure incubation period therefore the test item was classified as non-irritant.