N1,N2-dimethyl-N1-{2-[methyl(propan-2-yl)amino]ethyl}-N2-(propan-2-yl)ethane-1,2-diamine

Endpoint summary

Description of key information

The test item tested at two exposure periods of 3 minutes or 1 hour was cytotoxic and, hence, predicted to be corrosive to skin (UN GHS Category 1).

The skin corrosion potential of the test item was assessed with an in vitro membrane barrier assay, using the validated commercial kit CORROSITEX®.
The test item penetrated the bio-barrier after 237.8 minutes. Positive and  negative control results met the acceptance criteria indicating a good functioning of the test system.
According to OECD guideline for testing of chemicals No. 435, the test item is classified as corrosive to the skin, sub-category 1C, based on UN GHS, corresponding to UN packing group III.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 28, 2020 - 2021, March 3, 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 435 (In Vitro Membrane Barrier Test Method for Skin Corrosion)

Version / remarks:

July, 2015

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch No.: UW20226411
Purity: 98%
Expiry Date: 14 February 2022
Storage conditions: Room Temperature

Test system:

artificial membrane barrier model

Remarks:

The test system CORROSITEX® is composed of two components, a synthetic macromolecular bio-barrier and a CDS (Chemical Detection System);

Vehicle:

unchanged (no vehicle)

Details on test system:

SOURCE AND COMPOSITION OF MEMBRANE BARRIER USED
- Was the Corrositex® test kit used: yes, CORROSITEX®, in vitro INTERNATIONAL (17751 Sky Park East, Ste. G. Irvin -
California 92614 - US), batch: CT110419
- Components: two components: a proteinaceous macromolecular aqueous gel and a permeable supporting membrane

- Apparatus and preparation procedures: The permeable supporting membrane provides mechanical support to the proteinaceous gel during the gelling process and exposure to the test item. The supporting membrane prevents
sagging or shifting of the gel and is readily permeable to all test substances. The proteinaceous gel serves as the target for the test item and is placed on the surface of the supporting membrane and allowed to gel, prior to placing the membrane barrier over the indicator solution. The proteinaceous gel should be of equal thickness and density on the entire surface, and with no air bubbles or defects that could affect its functional integrity. The biobarrier will be supplied in the form of a powder to be reconstituted, prepared over a supporting membrane and stored according to the manufacturer’s instructions.

WAS THE COMPATIBILITY TEST PERFORMED: Yes. A Qualify test tube was supplied with the test system, containing the same indicator solution used in theMain Assay. An aliquot of 150 μL of test item was added to the Qualify test tube (amber solution). The tube was shaken to dissolve the test item and the vial was let stand for 1 minute. Colour change (e.g. from amber to red, orange, lightening) or consistency modification (flaking or precipitation) of the CDS was evaluated. In case no change occurred, the substance should not be considered idoneous to proceed with the test.

WAS THE TIMESCALE CATEGORY TEST PERFORMED: Yes. Components of the test item timescale category test (Test Tube A, Test Tube B, Colour Chart and one bottle of Confirmr eagent) were supplied with the test system. An aliquot of 150 μL of test item was added to Tube A (yellow solution) and Tube B (clear solution). Tubes were shaken to mix substances. The colour change in each tube was assigned and recorded according to the Colour chart. Each colour in the colour chart was associated to a letter (Addendum 1). The substance is classified as Category 1 or 2 (time scale category), on the basis of the colour changes and according to the manufacturer’s instructions. When a colour change is not detected either in Tube A or B, a confirm reagent is added to Tube B to confirm the test system suitability.

METHOD OF DETECTION
- Chemical or electrochemical detection system: The indicator solution, which is the same solution used for the compatibility test (qualification), responds to the presence of a test substance. A pH indicator combination of dyes willshow a colour change in response to the presence of the test substance or other types of chemical or electrochemical reactions. The measurement system will be visual.

METHOD OF APPLICATION:
Treatment
A Main Assay was carried out including test item, positive and negative controls. The pre-filled CDS vials were kept at room temperature (17-25°C) before use. A cold biobarrier disc, kept on crushed ice on the bench, was added to the top of each vial. A fixed amount of samples was added to the top within 2 minutes from putting the biobarrier at room temperature /

NUMBER OF REPLICATES: 4 (test item, 500 µL), 1 (negative and positive control, 500 µL)

NUMBER OF INDEPENDENT EXPERIMENTS TO DERIVE FINAL PREDICTION: 2 (Preliminary test and Main Test)

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
The time (in minutes) elapsed between application of the test item to the membrane barrier and barrier penetration was used to classify the test item in terms of corrosivity according to the following formula: CORROSITEX® Time = Detection Time – Start Time Means and standard deviations of CORROSITEX® Time were calculated for replicates of the same sample. Cut-off time values for each of the three corrosive subcategories are to find in the section Any other information on materials and methods incl. tables

Control samples:

yes, concurrent negative control

Duration of treatment / exposure:

A fixed amount of samples was added to the top within 2 minutes from putting the biobarrier at room
temperature

Species:

other: not relevant: in vitro study

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

500 µL for test item and controls

Observation period:

Detection intervals (minutes): 0-5, 55-65,235-245 for test item. Negative control vial was observed at 60 minutes.

Details on study design:

not relevant: in vitro study

Irritation / corrosion parameter:

penetration time (in minutes)

Run / experiment:

main experiment

Value:

237.8

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: indication of corrosion

Other effects / acceptance of results:

Preliminary test: Results of Qualification and Categorisation tests are shown in the attachment under Overall remarks. The test item issuitable for this test system since red colour was recorded after adding the test item. The ime category was 1 since Tube B content changed the colour to deep purple, after the addition of the test item.

Main Assay: After addition of the test item to the bio-barrier, a colour change to red was observed in the Chemical Detection System after a mean time of 237.8 minutes. Negative control was not corrosive within 60 minutes. Positive controls gave the expected
penetration response time (13 minutes and 32 seconds), within the range based on historical control data.

Interpretation of results:

Category 1C (corrosive) based on GHS criteria

Conclusions:

The skin corrosion potential of the test item was assessed with an in vitro membrane barrier assay, using the validated commercial kit CORROSITEX®.
The test item penetrated the bio-barrier after 237.8 minutes. Positive and negative control
results met the acceptance criteria indicating a good functioning of the test system.
According to OECD guideline for testing of chemicals No. 435, the test item is classified as corrosive to the skin, sub-category 1C, based on UN GHS, corresponding to
UN packing group III.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

other: artificial skin model

Justification for test system used:

Skin corrosion refers to the production of irreversible tissue damage in the skin following the application of a test item [as defined by the Globally Harmonised System for the Classification and Labelling of Chemical Substances and Mixtures (GHS)]. The OECD Guideline 431 does not require the use of live animals or animal tissue for the assessment of skin corrosivity.
The test described in the OECD Guideline 431 allows the identification of corrosive chemical substances and mixtures. It further enables the identification of non-corrosive substances and mixtures when supported by a weight of evidence determination using other existing information (e.g., pH, structure-activity relationships, human and/or animal data). It does not normally provide adequate information on skin irritation, but provides an in vitro procedure allowing the identification of non-corrosive and corrosive substances and mixtures in accordance with UN GHS (1). It also allows a partial sub-categorisation of corrosives.
For a full evaluation of local skin effects after single dermal exposure, it is recommended to follow the sequential testing strategy as appended to Test Guideline 404 and provided in the Globally Harmonised System. This testing strategy includes the conduct of in vitro tests for skin corrosion (as described in the OECD guideline 431) and skin irritation before considering testing in live animals.

Vehicle:

unchanged (no vehicle)

Details on test system:

The test item was applied topically to a three-dimensional human skin model, comprising at least a reconstructed epidermis with a functional stratum corneum. Corrosive materials are identified by their ability to produce a decrease in cell viability (as determined, for example, by using the MTT reduction assay) below defined threshold levels at specified exposure periods. The principle of the human skin model assay is based on the hypothesis that corrosive chemicals are able to penetrate the stratum corneum by diffusion or erosion, and are cytotoxic to the underlying cell layers. Prior to testing the assay was validated, using the Epidermis Model EpiDermTM (MatTek), by demonstrating the technical proficiency as detailed in OECD guideline 431.
6.1 Human skin model
The following Reconstructed Human Epidermis Model was used:
EpiDerm™ (EPI-200-SCT, Lot no. 30846) MatTek In Vitro Life Science Laboratories, s.r.o, Mlynské Nivy 73, 821 05 Bratislava II, Slovak Republic.
This human skin model used for this test complied with the following:
6.1.1 Basic procedure
EpiDerm tissues were conditioned by pre-incubation (1 hour) in Maintenance Medium1 for release of transport stress related compounds and debris in the incubator (37°C, 5% CO2, 95% relative humidity). After pre-incubation tissues were transferred to fresh Maintenance Medium and topically exposed with the test chemicals for 3 min and 1 h, respectively. Two tissues were used per treatment, negative and positive control and exposition time (12 tissues in total). After exposure tissues were rinsed, blotted and assay medium was replaced by MTT assay medium2 (final concentration: 1 mg MTT3 (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue)/mL medium). After 3-h incubation, tissues were washed with Dulbecco's phosphate buffered saline (D-PBS)4, blotted and the blue formazan salt was extracted with isopropanol5. The optical density of the formazan extract was determined spectrophotometrically at 540 nm and cell viability was calculated for each tissue as % of the mean of the negative control tissues. Skin corrosion potential of the test materials was classified according to the remaining cell viability obtained after 3 minutes or 1 hour exposure with the test chemical.
6.1.2 General model conditions
Human keratinocytes were used to construct the epithelium. Multiple layers of viable epithelial cells were present under a functional stratum corneum. The skin model also had a stromal component layer. The stratum corneum was multi-layered with the necessary lipid profile to produce a functional barrier with robustness to resist rapid penetration of cytotoxic markers. The containment properties of the model prevented the passage of material around the stratum corneum to the viable tissue. Passage of test chemicals around the stratum corneum would lead to poor modelling of the exposure to skin. The skin model was free of contamination with bacteria (including mycoplasma) or fungi.
6.1.3 Functional model conditions
The magnitude of viability was quantified by using MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue). In these cases the optical density (OD) of the extracted (solubilised) dye from the negative control tissue was at least 20-fold greater than the OD of the extraction solvent alone. The negative control tissue has been shown to be stable in culture (provide similar viability measurements) for the duration of the test exposure period. The stratum corneum has been shown to be sufficiently robust to resist the rapid penetration of certain cytotoxic marker chemicals (e.g. 1% Triton X-100). This property was estimated by the exposure time required to reduce cell viability by 50% (ET50) (for the EpiDerm™model this is >4 hours). The tissue employed has been shown to demonstrate reproducibility over time between laboratories. Moreover, it has been shown to be capable of predicting the corrosive potential of the reference chemicals when used in the testing protocol selected.
6.2 Test for interference of chemicals with the assay procedures
Optical properties of the test item or its chemical action on the MTT may interfere with the assay leading to a false estimate of viability, as the test item may prevent or reverse the colour generation as well as cause it. This may occur when a specific test item is not completely removed from the tissue by rinsing or when it penetrates the epidermis.
Prior to the testing, the test item was evaluated for colour changes. Concurrent negative controls (sterile deionised water6) were run in parallel.
50 μL of the test item were mixed with 300 μL sterile deionised water and incubated in the dark at 37°C, 5% CO2 and 95% relative humidity for 60 minutes. No discolouration of the test item was noted. In addition, 50 μL of the test item were added to 2 mL isopropanol and incubated at room temperature for three hours. No discolouration of the test item was noted.
Furthermore, the test item was evaluated for the potential to interfere with the MTT assay reagent (e.g. reduction). A concurrent negative control (sterile deionised water) was run in parallel. 50 μL of the test item were added to 1 mL of the MTT medium (1 mg MTT/mL) and incubated at 37°C, 5% CO2, and 95% relative humidity for 60 minutes. Untreated MTT solution was used as control. A discoloration of the test item to orange with black precipitation was noted. Hence, due to interacting of the test item with the MTT measurement (reduction of MTT) an additional test with freeze-killed tissues had to be performed.
6.3 Administration of the test, negative and positive reference items
The test item was used as supplied.
50 μL of test item was applied to the skin model with a surface area of 0.63 cm2 to uniformly cover the skin surface. A minimum of 70 μL substance applied per cm2 is required by the guidelines. Two replicate tissues for each treatment (exposure periods) were employed. At the end of the exposure period, the test item was carefully washed from the skin surface with D-PBS. The positive control item was 8 N KOH7 and the negative control was sterile deionised water. 50 μL of negative and positive controls were used.
6.4 Additional test for interference (reduction) of the test item with MTT
The frozen tissues were stored in the freezer (-20 ± 5°C).
The test item was applied to two freeze-killed tissues. In addition, two freeze-killed tissues were left untreated. The entire assay protocol was performed on the frozen tissues in parallel to the assay performed with the live EpiDermTM tissues. Then the data were corrected as described in section 6.7.
6.5 Cell viability measurements
MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS number 298-93-1) reduction, which had been shown to give accurate and reproducible results, was used to measure cell viability. Each skin sample was placed in an MTT assay solution of 1 mg/mL (37°C incubation temperature, 5% CO2, 95% relative humidity) for 3 hours. The precipitated blue formazan product was extracted using the solvent isopropanol and the concentration of the formazan was measured by determining the optical density (OD) at a wavelength of 540 nm in a spectrophotometer (Tecan Sunrise Magellan Version 6.48).
Cell viability measurements were carried out for both exposure periods (1st period: 3 min; 2nd period: 60 min). The measurements were made for each of the two tissues in triplicate.
6.6 Barrier function and quality controls (QC) of the model
The EpiDerm™ System was manufactured according to defined quality assurance procedures. All biological components of the epidermis and the culture medium were tested by the manufacturer for viral, bacterial, fungal and mycoplasma contamination. MatTek corporation determines the ET50 value following exposure to Triton X-100 (1%) for each EpiDerm™ lot. The ET50 must fall within a range established based on a historical database of results.
6.7 Data correction procedure
For the MTT reducing test item, calculations were corrected by values of correspondent additional controls.
True viability = Viability of treated tissue – Interference from test chemical
= OD tvt – OD kt
where OD kt = (mean OD tkt – mean OD ukt)
tvt = treated viable tissue kt = killed tissues
tkt = treated killed tissue ukt = untreated killed tissue (NC treated tissue)
If the interference by the test substance is greater than 30% of the negative control value, additional steps must be taken into account or the test substance may be considered incompatible with this test.
6.8 Assay acceptability criteria
Assay acceptance criterion 1: Negative control
The absolute OD of the negative control (NC) tissues (treated with sterile deionised water) in the MTT test is an indicator of tissue viability obtained in the testing laboratory after shipping and storing procedures and under specific conditions of use. The tissues treated with the negative control should not be below historically established boundaries.
The assay meets the acceptance criterion if the mean ODof the NC tissues is ≥ 0.8 and ≤ 2.8.
Assay acceptance criterion 2: Positive control
A 8 N KOH was used as positive control (PC) and tested concurrently with the test chemicals. Concurrent means here the PC has to be tested in each assay.
Tissues treated with the PC, should reflect the ability of the tissues to respond to a corrosive chemical under the conditions of the test method (viability after 1 hour exposure: < 15%).
Assay acceptance criterion 3: variability between tissue replicates
Associated and appropriate measures of variability between tissue replicates should not exceed 30% (in the range of 20 – 100% viability).
6.9 Interpretation of results
The OD values obtained for each test sample were used to calculate a percentage viability relative to the negative control, which is arbitrarily set at 100%. The cut-off percentage cell viability value distinguishing corrosive from non-corrosive test items or discriminating between different corrosive classes, e.g. subcategories 1A, 1B and 1C, or the statistical procedure(s) used to evaluate the results and identify corrosive materials, is clearly defined and documented, and shown to be appropriate. The criteria of corrosivity associated with the EpiDermTM model are as follows:
- the test item is considered to be corrosive to skin and classified as category 1 (or optional category 1A9), if the viability after 3 minutes exposure is less than 50%;
- the test item is considered to be corrosive to skin and classified as sub-category 1B-and-1C, if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is less than 15%;
- the test item is considered to be non-corrosive to skin, if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3-minute exposure

Value:

28.1

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1-hour exposure

Value:

14.1

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

Category 1 (corrosive) based on GHS criteria

Conclusions:

The EpiDerm™ model was employed. The test item, the negative control sterile deionised water or the positive reference item 8 N KOH were applied to the skin model surface at two exposure periods of 3 minutes or 1 hour.
Two tissues were used for each treatment and concurrent control groups. Cell viability was determined by using the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue) reduction assay and expressed as relative percentage of viability of the negative control-treated tissues.
The test item was applied topically as supplied (liquid). Sterile deionised water was used as the negative control. 8 N KOH was used as the positive reference item. The test item and the reference items were applied to the skin model surface at two exposure periods of 3 minutes or 1 hour.
In comparison to the negative control, the mean viability of cells exposed to the test item was 28.1% after a 3-minute exposure period and 14.1% after a 1-hour exposure (corrected viability calculated for MTT reducing test items using freeze-killed control tissues). The 3-minute exposure value was less than the cut-off percentage cell viability value of 50%, distinguishing corrosive from non-corrosive test items. Hence, the test item was corrosive in this skin model and is predicted to be corrosive to human skin (UN GHS Category 1).
The mean optical density (OD) of the negative control of 2 tissues was 1.480 (3-minute exposure) or 1.578 (1-hour exposure) and was well within the acceptable range of ≥ 0.8 to ≤ 2.8. The mean viability of cells treated with the positive reference item 8 N KOH was 5.7% or 5.1% (3 minute or 1-hour exposure) of the negative control and, hence well below the 15% cut-off value at the 1-hour exposure.
The difference of viability between the two tissue replicates (at 20 - 100% viability) was below the limit of acceptance of 30%. Hence, all acceptance criteria were fulfilled.

Conclusion
Under the present test conditions the test item tested at two exposure periods of 3 minutes or 1 hour was cytotoxic and, hence, predicted to be corrosive to skin (UN GHS Category 1) in an experiment employing an artificial three-dimensional model of human skin.

Executive summary:

Under the present test conditions the test item tested at two exposure periods of 3 minutes or 1 hour was cytotoxic and, hence, predicted to be corrosive to skin (UN GHS Category 1) in an experiment employing an artificial three-dimensional model of human skin.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (corrosive)