N-Phenyl-diethanolamine, reaction products with formaldehyde

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 30 March 2020 to 17 April 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study is conducted in accordance with the relevant OECD test guideline and GLP. All validity criteria were met.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: EU Method B.40 BIS (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

31 May 2008

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

18 June 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Test material form:

liquid

Details on test material:

Storage conditions: At room temperature protected from light
Physical description: Light orange liquid

In vitro test system

Test system:

human skin model

Remarks:

EpiDerm Skin Model

Source species:

other: human-derived epidermal keratinocytes

Cell type:

non-transformed keratinocytes

Cell source:

other: human-derived epidermal keratinocytes, purchased or derived from tissue obtained by MatTek Corposation from accredited institutions

Source strain:

other: Keratinocyte strain 00267

Details on animal used as source of test system:

N/A

Justification for test system used:

The test is based on the experience that corrosive chemicals show cytotoxic effects following short-term exposure to the stratum corneum of the epidermis. The test is designed to predict and classify the skin corrosion potential of a test item by assessment of its effect on a three-dimensional human epidermis model (1-4).
The test consists of topical application of the test item on the skin tissue for 3-minute and
1-hour. After exposure the skin tissue is thoroughly rinsed to remove the test item followed by immediate determination of the cytotoxic (corrosive) effect.
Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) at the end of the treatment.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model
- Tissue batch number(s): 33008 kit G+B and 33018 kit G+F
- Production date: Not specified
- Shipping date: Not specified
- Delivery date: Not specified
- Date on Certificate of Analysis: 1 April 2020
- Date of initiation of testing: Not specified

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C ± 1.0°C
- Temperature of post-treatment incubation (if applicable): 37°C ± 1.0°C

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 item. The skin inserts were carefully dried. Rinsed tissues were kept in 24 well plates on 300 µL DMEM until 6 tissues (= one application time) were dosed and rinsed
- Observable damage in the tissue due to washing: Not specified
- Modifications to validated SOP: Not specified

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 5 mg/mL diluted (1:5) with MTT diluent (supplemented DMEM)
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 4

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1, although initially the tissues treated for 3 minutes with the test item did not fulfill the acceptability criteria. This part of the experiment was rejected and repeated.

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or 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 substance 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%.
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439: N/A

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

50 µL

Duration of treatment / exposure:

3 minutes, 1 hour

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

4

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute application

Value:

74

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100% tissue viability

Positive controls validity:

valid

Remarks:

8.2% tissue viability

Remarks on result:

other: no indication of corrosion

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour application

Value:

27

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100% tissue viability

Positive controls validity:

valid

Remarks:

8.8% tissue viability

Remarks on result:

other: no indication of corrosion

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: None reported
- Direct-MTT reduction: The solutions did not turn blue/purple nor a blue/purple precipitate was observed so it was concluded that the test item did not interfere with the MTT endpoint
- Colour interference with MTT: The solutions did not turn blue/purple nor a blue/purple precipitate was observed so it was concluded that the test item did not interfere with the MTT endpoint

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes - the absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit 2.8) and the laboratory historical control data range.
- Acceptance criteria met for positive control: Yes - the mean relative tissue viability following the 1-hour exposure to the positive control was 8.8%.
- Acceptance criteria met for variability between replicate measurements: Yes - in the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was  27%, indicating that the test system functioned properly

Any other information on results incl. tables

Table 1          
Mean Absorption in the in vitro Skin Corrosion Test with N-Phenyl-diethanolamine, reaction products with formaldehyde EC: 942-131-4

	3-minute application					1-hour application
	A (OD570)	B (OD570)	Mean (OD570)		SD	A (OD570)	B (OD570)	Mean (OD570)		SD
Negative control	1.769	1.737	1.753	±	0.022	1.733	1.616	1.674	±	0.083
Test item	1.398	1.189	1.294	±	0.148	0.383	0.527	0.455	±	0.102
Positive control	0.155	0.134	0.144	±	0.015	0.124	0.169	0.147	±	0.032

SD = Standard deviation

Duplicate exposures are indicated by A and B.

In this table the values are corrected for background absorption (0.0436 and 0.0424 for the  3-minute and 1 hour application, respectively).

Isopropanol was used to measure the background absorption.

Table 2          
Mean Tissue Viability in the in vitro Skin Corrosion Test with N-Phenyl-diethanolamine, reaction products with formaldehyde EC: 942-131-4

	3-minute application viability (percentage of control)	1-hour application viability (percentage of control)
Negative control	100	100
Test item	74	27
Positive control	8.2	8.8

 

Table 3          
Coefficient of Variation between Tissue Replicates

	3 minute	1 hour
Negative control	1.8	6.8
Test item	15	27
Positive control	14	27

CV (%) = 100 - [(lowest OD₅₇₀/highest OD₅₇₀) x 100%]

Individual OD Measurements at 570 nm

	3-minute application (OD570)   A           B		1-hour application (OD570)   A           B
Negative control OD570 measurement 1 OD570 measurement 2 OD570 measurement 3
	1.8317	1.7845	1.8072	1.6497
	1.8198	1.7895	1.7597	1.6520
	1.7851	1.7682	1.7583	1.6720
Test item OD570 measurement 1 OD570 measurement 2 OD570 measurement 3
	1.4371	1.2239	0.4353	0.5674
	1.4380	1.2386	0.4108	0.5683
	1.4499	1.2365	0.4292	0.5719
Positive control OD570 measurement 1 OD570 measurement 2 OD570 measurement 3
	0.1972	0.1786	0.1699	0.2097
	0.1992	0.1761	0.1654	0.2136
	0.1987	0.1771	0.1645	0.2121

OD = Optical density

Duplicate exposures are indicated by A and B.

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate the test item for its ability to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200)). The possible corrosive potential of the test item was tested through topical application for 3 minutes and 1 hour.

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

The test item was a light orange liquid. The test item was applied undiluted (50 µL) directly on top of the skin tissue. At the end of the treatment period. Cell viability was assessed using the MTT assay.

The positive control had a mean relative tissue viability of 8.8% after the 1-hour exposure. The absolute mean OD₅₇₀(optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤2.8) and the laboratory historical control data range. In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was  ≤27%, indicating that the test system functioned properly.

Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 74% and 27%, respectively. Because the mean relative tissue viability for the test item was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment the test item is considered to be not corrosive.

In conclusion, the test item is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.