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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:
20 May 2015-10 July 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD test Guideline No. 431 and EU Method B.40 BIS. Furthermore, functional model conditions and references to historical control data are included in the report.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
Adopted 26 September 2014
Deviations:
no
Qualifier:
according to
Guideline:
other: UE Method B.40 BIS (In Vitro Skin Corrosion: Human Skin Model Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- Name of test material (as cited in study report): Trimellitic anhydride chloride
For more details see Confidential details on test material
Specific details on test material used for the study:
4-chloroformylphthalic anhydride

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
Following the REACH bottom-up strategy, the EpiDerm™ Human Skin Model method was used to assess skin corrosion as recommended in the OECD test guideline No. 431.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi-200-Kit, MatTek Corporation (Ashland, MA, USA).
- Lot number: 22251 Kit AA
- Production Date: no data
- Shipping date: no data
- Delivery date: no data
- Date received: no data
- Date of initiation of testing: May 26, 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 and 60 minutes at 37 °C in a humidified atmosphere of 5% CO2

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: After the exposure period, the tissues were washed with phosphate buffered saline (Invitrogen Corporation, Breda, The Netherlands) to remove residual test substance. Rinsed tissues were kept in 24 well plates on 300 μl DMEM medium until 6 tissues (= one application time) were dosed and rinsed.
- Observable damage in the tissue due to washing: none reported
- Modifications to validated SOP: none reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL MTT solution
- Incubation time: 3 hours at 37 °C, 5% CO2 in air
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm
- Filter: without reference filter
- Linear OD range of spectrophotometer: not reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD (540-570 nm): 1.852 +/- 0.086 [1.0-3.0]
- Barrier function: ET-50: 7.82 hrs [4.77-8.72 hrs]
- Morphology: normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo.
- Contamination: No contamination

NUMBER OF REPLICATE TISSUES: 4 tissues per test substance together with a negative control and positive control

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Not needed

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
A test item is considered corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%.
b) In addition, a test item considered non-corrosive (viability >= 50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test item is decreased below 15%.
A test item is considered non corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%.
b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 27.1 to 29.9 mg of the solid test substance (skin was moistened with 25 μl Milli-Q water to ensure close contact of the test substance)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL of Milli-Q water was used as supplied

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL of 8.0 N Potassium Hydroxide was used as supplied
Duration of treatment / exposure:
3 and 60 minutes.
Duration of post-treatment incubation (if applicable):
not applicable
Number of replicates:
4 tissues per test substance together with a negative control and positive control

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3-minute exposure
Value:
92
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100
Positive controls validity:
valid
Remarks:
14
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1-hour exposure
Value:
3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100
Positive controls validity:
valid
Remarks:
12
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: Not reported
- Direct-MTT reduction: None
- Colour interference with MTT: None

DEMONSTRATION OF TECHNICAL PROFICIENCY: Not included in the report

ACCEPTANCE OF RESULTS:
The in vitro skin corrosion test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 of the two tissues of the negative control should reasonably be within the laboratory historical control data range.
b) The mean relative tissue viability following 3-minute exposure to the positive control should be ≤ 30%.
c) In the range of 20 – 100% viability, the maximum inter-tissue variability (in viability) is ≤ 30% between two tissues treated identically.
d) In the range of 20 – 100% viability, the maximum difference in percentage between the mean viability of two tissues and one of the two tissues is ≤ 15%.

Any other information on results incl. tables

Mean absorption in the in vitro skin corrosion test with Trimellitic anhydride chloride

     3 -minute application              1 -hour application
 A (OD570)  B (OD570)

 Mean (OD570)

   SD  A (OD570)  B (OD570)  Mean (OD570)    SD
 Negative control 1.740 1.649  1.695   +/- 0.065  1.851  1.794  1.823   +/- 0.041 
 Test item 1.520 1.584  1.552   +/- 0.045  0.054  0.041  0.048  +/- 0.009 
 Positive control 0.285 0.174  0.229   +/- 0.078  0.277  0.155  0.216   +/- 0.086 

OD = Optical Density

SD = Standard Deviation

Duplicate exposures are indicated by A and B.

In this table the values are corrected for background absorption (0.0430). Isopropanol was used to measure the background absorption.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (corrosive) based on GHS criteria
Conclusions:
Under the experimental conditions of this study, Trimellitic anhydride chloride is corrosive in the in vitro skin corrosion test under the experimental conditions, therefore, the test substance should be classified category 1 according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.
Executive summary:

An in vitro skin corrosion study was performed according to the most recent OECD Guideline 431, EU Method B.40 BIS and in compliance with GLP, using the EpiDerm™ Human Skin Model.

 

The test item was applied, after moistening with 25 µL of Milli-Q water (approximately 25 mg for 3-minute and 1 -hour exposures) directly on top of the skin tissue.

The positive control had a mean relative tissue viability of 14% after the 3-minute exposure.

The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The acceptability criteria for the maximum inter-tissue variability in viability between two tissues treated identically and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues were met, indicating that the test system functioned properly.

Skin corrosion is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with Trimellitic anhydride chloride compared to the negative control tissues was 92% and 3%, respectively. Because the mean relative tissue viability for Trimellitic anhydride chloride was below 15% after 1 hour treatment it is considered to be corrosive.

Finally, it is concluded that this test is valid and that Trimellitic anhydride chloride is corrosive in the in vitro skin corrosion test under the experimental conditions described in this report

Under the experimental conditions of this study, Trimellitic anhydride chloride is corrosive in the in vitro skin corrosion test under the experimental conditions, therefore, the test substance should be classified category 1 according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.

This study is considered as acceptable and satisfies the requirement for skin corrosion endpoint.