Reaction mass of trimethylolpropane triacrylate and hexamethyleneimine

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 May 2018 to24 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

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)

Version / remarks:

28 July 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.46 “In vitro Skin Irritation: Reconstructed Human Epidermis Model Test ". Official Journal of the European Union No. L142; Amended by EC No. 640/2012 OJ No. L193, 20 July 2012.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of trimethylolpropane triacrylate and hexamethyleneimine

EC Number:

946-043-7

IUPAC Name:

Reaction mass of trimethylolpropane triacrylate and hexamethyleneimine

Test material form:

liquid

Specific details on test material used for the study:

Identification: Photomer 4250
Appearance: Colourless to pale yellow liquid
Purity/Composition: 100% (UVCB)
Test item storage: At room temperature protected from light

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen

Vehicle:

unchanged (no vehicle)

Details on test system:

Test for the Interference of the Test Item with the MTT Endpoint:
A test item may interfere with the MTT endpoint if it is colored and/or it is able to directly reduce MTT. The cell viability measurement is affected only if the test item is present on the tissues when the MTT viability test is performed.

Photomer 4250 was checked for possible direct MTT reduction and color interference in the Skin corrosion test using EpiDerm as a skin model (Test Facility Study No. 20142197). Because solutions did not turn blue / purple and a blue / purple precipitate was not observed it was concluded that Photomer 4250 did not interfere with the MTT endpoint.

Test System Set Up:
On the day of receipt the tissues were transferred to 12-well plates and pre-incubated with pre-warmed Maintenance Medium for 21 hours at 37°C (Figure 1). Maintenance medium and Assay medium were supplied by Skinethic Laboratories, Lyon, France.

MTT medium:
MTT concentrate (Sigma Aldrich, Zwijndrecht, The Netherlands; 3 mg/mL in PBS) diluted (10x) in Assay medium (final concentration 0.3 mg/mL).

Environmental conditions:
All incubations, with the exception of the test item incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 59 - 84%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.5 - 37.2°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Test Item Preparation:
No correction was made for the purity/composition of the test item. The liquid test item was applied undiluted (25 µL) directly on top of the tissue.

Application/Treatment of the Test Item:
The test was performed on a total of 3 tissues per test item together with negative and positive controls. Twenty five µL of the undiluted test item was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 µL PBS (negative control) and 3 tissues with 25 µL 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time.

After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item. After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.

Cell Viability Measurement:
After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 mL MTT-solution (0.3 mg/mL in PBS). The tissues were incubated for 3 h at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in pre-labeled microtubes and extracted with 500 µL isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for 68 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.

Amount/concentration applied:

The liquid test item was applied undiluted (25 µL) directly on top of the tissue.

Duration of treatment / exposure:

15 ± 0.5 minutes

Duration of post-treatment incubation (if applicable):

After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 mL MTT-solution (0.3 mg/mL in PBS). The tissues were incubated for 3 h at 37°C.

Number of replicates:

3 replicates

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other: Mean absorption

Value:

0.074

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

Basis: other: Optical density Remarks: Negative control = 1.003; Positive control = 0.046

Results 2

Irritation / corrosion parameter:

other: Mean tissue viability

Value:

7.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

Basis: other: percentage of control. Remarks: Negative control = 100%; Positive control = 4.6%. (migrated information)

Other effects / acceptance of results:

Photomer 4250 was checked for possible direct MTT reduction and color interference in the Skin corrosion test using EpiDerm as a skin model (Test Facility Study No. 20142197). Because no color changes were observed it was concluded that Photomer 4250 did not interact with the MTT endpoint.

The mean absorption at 570 nm measured after treatment with Photomer 4250 and controls are presented in Appendix 1, Table 1.

The individual OD570 measurements are presented in Appendix 2.

Table 2 shows the mean tissue viability obtained after 15 ± 0.5 minutes treatment with Photomer 4250 compared to the negative control tissues. Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with Photomer 4250 compared to the negative control tissues was 7.4%. Since the mean relative tissue viability for Photomer 4250 was below 50% it is considered to be irritant.

The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 4.6%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range (See Appendix 3). The standard deviation value of the percentage viability of three tissues treated identically was < 4%, indicating that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

In conclusion, Photomer 4250 is irritant in the in vitro skin irritation test under the experimental conditions described in this report and should be classified category 2 according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.

Executive summary:

The objective of this study was to evaluate Photomer 4250 for its ability to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small model (EPISKIN-SMTM)).  The possible skin irritation potential of Photomer 4250 was tested through topical application for 15 minutes.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

Photomer 4250 was a colourless to pale yellow liquid.  Photomer 4250 was applied undiluted (25 µL), directly on top of the skin tissue for 15 ± 0.5 minutes.  After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed.  Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

Skin irritation is expressed as the remaining cell viability after exposure to the test item.  The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with Photomer 4250 compared to the negative control tissues was 7.4%.  Since the mean relative tissue viability for Photomer 4250 was below 50% after 15 ± 0.5 minutes treatment it is considered to be irritant.

The positive control had a mean cell viability of 4.6% after 15 ± 0.5 minutes exposure.  The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range.  The standard deviation value of the percentage viability of three tissues treated identically was < 4%, indicating that the test system functioned properly.

In conclusion, Photomer 4250 is irritant in the in vitro skin irritation test under the experimental conditions described in this report and should be classified category 2 according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.