Formaldehyde, oligomeric reaction products with 3-aminomethyl-3,5,5-trimethylcyclohexylamine and 4-tert-butylphenol

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 February 2018 - 02 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

3-aminomethyl-3,5,5-trimethylcyclohexylamine

EC Number:

220-666-8

EC Name:

3-aminomethyl-3,5,5-trimethylcyclohexylamine

Cas Number:

2855-13-2

Molecular formula:

C10H22N2

IUPAC Name:

3-(aminomethyl)-3,5,5-trimethylcyclohexanamine

Constituent 2

Reference substance name:

4-tert-butylphenol

EC Number:

202-679-0

EC Name:

4-tert-butylphenol

Cas Number:

98-54-4

Molecular formula:

C10H14O

IUPAC Name:

4-tert-butylphenol

Constituent 3

Reference substance name:

2-(((5-amino-1,3,3-trimethylcyclohexyl)methylamino)methyl)-4-tert-butylphenol

Molecular formula:

C21H36N2O

IUPAC Name:

2-(((5-amino-1,3,3-trimethylcyclohexyl)methylamino)methyl)-4-tert-butylphenol

Test material form:

liquid: viscous

In vitro test system

Test system:

human skin model

Source species:

hamster

Cell type:

non-transformed keratinocytes

Justification for test system used:

recommended by guideline

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Reconstructed Human Epidermis Model Kit
- Tissue batch number(s): 25883
- Delivery date: 27 February 2018
- Date of initiation of testing: 28 February 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C
- Temperature of post-treatment incubation (if applicable): 37 °C, 5% CO2

REMOVAL OF TEST MATERIAL AND CONTROLS
- Rinsing was achieved by filling and emptying each tissue under a constant soft stream of Dulbecco’s Phosphate Buffered Saline (DPBS) (without Ca++ Mg++) for approximately 40 seconds, to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper. Each tissue was placed into the prepared holding plate until all tissues were rinsed.
- Observable damage in the tissue due to washing: no


MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Labtech LT-4500 microplate reader

NUMBER OF REPLICATE TISSUES: duplicates

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- killed tissues
- Procedure used to prepare the killed tissues (if applicable): freeze killed
- N. of replicates : duplicates
- Method of calculation used: True viability = mean OD tvt-(OD tkt-OD ukt)

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- 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%.
< 25% after 3 min exposure: Sub-category 1A
≥ 25% after 3 min exposure: Combination of sub-categories 1B-and-1C
- 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%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 μL
- Concentration (if solution): 100%

NEGATIVE CONTROL
- Sterile distilled water
- Amount(s) applied (volume or weight): 50 μL

POSITIVE CONTROL
- Potassium Hydroxide
- Amount(s) applied (volume or weight): 50 μL
- Concentration (if solution): 8.0 N

Duration of treatment / exposure:

3-Minutes and 60-Minutes

Duration of post-treatment incubation (if applicable):

not applicable

Number of replicates:

2

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes exposure

Value:

45.2

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes exposure

Value:

17

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system:
no
- Direct-MTT reduction:
The MTT solution containing the test item turned purple and therefore the test item was demonstrated to directly reduce MTT. Therefore, an additional procedure using freeze-killed tissues was performed. The results of the freeze-killed tissues were subtracted from the mean OD of the test item treated viable tissues to obtain the true amount of MTT reduction that reflects metabolic conversion only:
True viability = mean OD tvt-(OD tkt-OD ukt)
- Colour interference with MTT:
The solution containing the test item was a yellow color, therefore additional color correction tissues were incorporated into the testing procedure. However, the results obtained showed that negligible color interference occurred. It was therefore considered unnecessary to use the results of the color correction tissues for quantitative correction of results. The results generated from the color correction tissues are presented below.
3 minutes exposure:
Mean of test item treated color correction tissues = 0.033 OD570
Mean of untreated color correction tissues = 0.007 OD570
Color interference by the test item relative to the negative control value:
0.033 – 0.007 / 1.134 (mean of negative control) = 2.3%

60 minutes exposure:
Mean of test item treated color correction tissues = 0.036 OD570
Mean of untreated color correction tissues = 0.005 OD570
Color interference by the test item relative to the negative control value:
0.036 – 0.005 / 1.426 (mean of negative control) = 2.2%

Double Correction Check
The results of the color correction tissues were not used for quantitative correction of results. Therefore it was unnecessary to use the results of the killed color correction tissues as no double correction for color interference would have occurred. The results generated from the non-viable color correction tissues are presented below.
3 minutes exposure:
Mean of test item treated killed color correction tissues = 0.021 OD570
Mean of untreated killed color correction tissues = 0.012 OD570
Potential double correction by the test item relative to the negative control value:
0.021 – 0.012 / 1.134 (mean of negative control) = 0.8%

60 minutes exposure:
Mean of test item treated killed color correction tissues = 0.021 OD570
Mean of untreated killed color correction tissues = 0.014 OD570
Potential double correction by the test item relative to the negative control value:
0.021 – 0.014 / 1.426 (mean of negative control) = 0.5%

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
The mean OD570 for the negative control treated tissues was 1.634 for the 3 Minute exposure period and 1.426 for the 60 Minute exposure period. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control:
The relative mean tissue viability for the positive control treated tissues was 5.1% relative to the negative control following the 60 Minute exposure period. The positive control acceptance criterion was therefore satisfied.
- Acceptance criteria met for variability between replicate measurements:
In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.

Any other information on results incl. tables

Mean OD570 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Tissue	Exposure Period	MeanOD570of individual tissues	Mean OD570of duplicate tissues (tvt)	Corrected Mean OD570(tvt-[tkt-ukt])	Standard Deviation	Coefficient of Variation (%)	Relative Mean Viability (%)
Negative Control	3 Minutes	1.699	1.634		0.092	5.6	100*
		1.569
	60 Minutes	1.461	1.426		0.050	3.5
		1.390
Positive Control	3 Minutes	0.078	0.081		0.004	na	4.9
		0.083
	60 Minutes	0.070	0.073		0.004	na	5.1
		0.075
Test Item	3 Minutes	1.101	1.134	0.739	0.047	4.1	45.2
		1.167
	60 Minutes	0.604	0.613	0.243	0.012	2.0	17.0
		0.621

 

Correction factor:
3 minute exposure corrected mean OD570 =     0.507   (tkt) – 0.112      (ukt) = 0.395

60 minute exposure corrected mean OD570 =   0.491   (tkt) – 0.121      (ukt) = 0.370

OD= Optical density

tvt =Treated killed tissues

tkt= Untreated killed tissues

ukt =Untreated killed tissues

*=   The mean % viability of the negative control tissue is set at 100%

na=  Not applicable

Applicant's summary and conclusion

Interpretation of results:

Category 1B (corrosive) based on GHS criteria

Conclusions:

In this test, the test item Mannich base IPDA-PTBP was considered to be corrosive (Category 1B/C).

Executive summary:

The purpose of this test is to evaluate the corrosivity potential of Mannich base IPDA-PTBP using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes.

Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test item.

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. The test item was found to directly reduce MTT and therefore additional non-viable tissues were incorporated into the testing for correction purposes. The test item was found to have the possibility to cause color interference with the MTT endpoint therefore, as a precaution, additional tissues were incorporated into the testing to correct for this. A third set of controls was included, comprising non-viable tissues, in order to prevent a double correction from a colored test item that also reduces MTT. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT‑loading. After MTT-loading each tissue was placed in 2 mL of Isopropanol for MTT extraction.

At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 mL samples were transferred to the appropriate wells of a pre-labeled 96‑well plate. The optical density (OD) was measured at 570 nm (OD570).

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

 

The relative mean viabilities for each treatment group were as follows:

Exposure Period		Percentage Viability
		Negative Control		Positive Control		Test Item
3 minute		100*		4.9		45.2
60 minute		100*		5.1		17.0

 

*The mean viability of the negative control tissues is set at 100%

The quality criteria required for acceptance of results in the test were satisfied.

In this test, Mannich base IPDA-PTBP was considered to be corrosive.