1,1,3-trimethylindan-4-amine

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 Jul - 06 Aug 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Test No. 431: In Vitro Skin Corrosion: Reconstructed Human Epidermis (Rhe) Test Method adopted 14 Jun 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Freie und Hansestadt Hamburg, Behörde für Gesundheit und Verbraucherschutz

Test material

Test material information

Constituent 1

Reference substance name:

1,1,3-trimethyl-2,3-dihydro-1H-inden-4-amine

EC Number:

838-724-1

Cas Number:

94568-76-0

Molecular formula:

C12H17N

IUPAC Name:

1,1,3-trimethyl-2,3-dihydro-1H-inden-4-amine

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiDerm™ (EPI-212)

Source strain:

other: not applicable (human skin model)

Details on animal used as source of test system:

not applicable (human skin model)

Justification for test system used:

The used test system is in line with OECD TG 431.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ (EPI-212) (MatTek In Vitro Life Science Laboratories, Bratislava, Slovak Republic)
- Tissue batch number(s): 30883
- Date of initiation of testing: 17 Jul 2020

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: incubation with MTT assay solution at 37°C (5% CO2 and 95% relative humidity)

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: one: At the end of the exposure period, the test item was carefully washed from skin surface with DBPS (Dulbecco's phosphate buffered saline). No information on volume given.
- Observable damage in the tissue due to washing: not reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL MTT assay solution
- Incubation time: 3 h
- Spectrophotometer: Tecan Infinite 200Pro (Tecan Deutschland GmbH, Crailsheim, Germany)
- Wavelength: 540 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: A MTT cell viability test was performed by MatTek. The results were provided in the Certificate of Analysis of the EpiDerm™ Reconstructed Human Epidermis. The determined OD (540 - 570 nm) was 1.864 ± 0.078 (acceptance criteria: 1.0 - 3.0). The negative control tissue has been shown to be stable in culture (provided similar viability measurements) for the duration of the test exposure period. The tissue employed has been shown to demonstrate reproductivity over time and between laboratories. Moreover, it has been shown to be capable of predicting the corrosive potential of the reference items when used in the testing protocol selected.

- Barrier function: A barrier function test was performed by MatTek. The results were provided in the Certificate of Analysis of the EpiDerm™ Reconstructed Human Epidermis. The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 µL of 1% Triton X-100. The ET-50 value was determined to be 5.7 h (acceptance criteria: 4.77 - 8.72 h).

- Morphology: 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.
- Contamination: A contamination test was performed by MatTek. The cells used to produce the EpiDerm™ tissue were screened for the presence of viruses (HIV-1, Hepatitis B and C), bacteria, yeast and other fungi. No contamination was detected.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
No possible interference with the MTT measurement (OD 540 nm), due to colour changes or direct interacting with the MTT assay reagent, was noted. Therefore, no additional control tissues were needed.

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

PREDICTION MODEL / DECISION CRITERIA (according to CLP and UN-GHS)
- The test item is considered to be corrosive to skin and classified as category 1 (or optional category 1A), if the viability after 3 min 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 min exposure is greater than or equal to 50% and the viability after 1 h exposure is less than 15%;
- The test item is considered to be non-corrosive to skin, if the viability after 3 min exposure is greater than or equal to 50% and the viability after 1 h exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 50 µL

NEGATIVE CONTROL
- Amount(s) applied: 50 µL

POSITIVE CONTROL SUBSTANCE:
- Amount(s) applied: 50 µL
- Concentration: 8 N KOH

Duration of treatment / exposure:

3 and 60 min

Number of replicates:

2 tissues were used per treatment, negative and positive control included, and exposition time (12 tissues in total).

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean value of 2 tissues

Run / experiment:

3 min exposure

Value:

97.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean value of 2 tissues

Run / experiment:

60 min exposure

Value:

34

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Direct-MTT reduction: No direct interaction with the MTT assay reagent was observed.
- Colour interference with MTT: The test substance displayed a yellow discolouration in isopropanol and hence, OD measurement had to be performed. The difference of the OD540 of the test item and the OD540 of isopropanol was 0.009. As this difference was < 0.08, no further colour control using vital EpiDerm was necessary. Hence, the criteria of the test on colour interference were met.
- Visible damage on test system: not reported

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean optical density (OD) of the negative control of 2 tissues was 1.9072 (3 min exposure) or 1.7984 (1 h exposure) and was well within the acceptable range of ≥ 0.8 to ≤ 2.8 (see 'any other information on results incl. tables', table 1 and 2, respectively).
- Acceptance criteria met for positive control: The mean viability of cells treated with the positive reference item 8 N KOH was 5.1% or 4.5% (3 min or 1 h exposure, respectively) of the negative control and, hence well below the 15% cut-off value at the 1 h exposure (see 'any other information on results incl. tables', table 1 and 2, respectively).
- Acceptance criteria met for variability between replicate measurements: The difference of viability between the 2 tissue replicates (at 20 – 100% viability) was below the limit of acceptance of 30%.

HISTORICAL CONTROL DATA
See 'Any other information on results incl. tables', table 3

Any other information on results incl. tables

Table 1: Results in vitro Skin Corrosion Test after 3 min exposure

	Negative control		Positive control		Test item
Tissue sample	1	2	1	2	1	2
OD540	2.0636 2.0489 1.6127	1.9325 1.9252 1.8602	0.0924 0.0935 0.0931	0.1000 0.0991 0.0979	1.7584 1.7840 1.8056	1.9456 1.9301 1.9260
Mean OD540 (CV%)	1.9084 (13.4)	1.9060 (2.1)	0.0930 (0.6)	0.0990 (1.1)	1.7827 (1.3)	1.9339 (0.5)
Mean OD540  values of replicates (PD%)	1.9072 (0.1)		0.0960 (6.3)		1.8583 (8.1)
Individual Viability [%]	100.1	99.9	4.9	5.2	93.5	101.4
Mean Viability of 2 replicates [%] (DV%)	100.0 (0.2)		5.1 (0.3)		97.5 (7.9)

CV: Coefficient of variation

PD: Percent difference

DV: Difference of viability

 

 

Table 2: Results In Vitro Skin Corrosion test after 60 min exposure

	Negative control		Positive control		Test item
Tissue sample	1	2	1	2	1	2
OD540	1.7190 1.7808 1.7738	1.8480 1.8246 1.8440	0.0817 0.0795 0.0808	0.0799 0.0809 0.0800	0.8170 0.8109 0.7797	0.4173 0.4250 0.4146
Mean OD540 (CV%)	1.7579 (1.9)	1.8389 (0.7)	0.0807 (1.4)	0.0803 (0.7)	0.8025 (2.5)	0.4190 (1.3)
Mean OD540 values of replicates (PD%)	1.7984 (4.5)		0.0805 (0.5)		0.6108 (62.8)
Individual Viability [%]	97.7	102.3	4.5	4.5	44.6	23.3
Mean Viability of 2 replicates [%] (DV%)	100.0 (4.6)		4.5 (0.0)		34.0 (21.3)

CV: Coefficient of variation

PD: Percent difference

DV: Difference of viability

 

Table 3: Historical data of negative and positive controls: most recent background data from GLP studies of the years 2017 - 2019 (n = 18)

Material	Average OD (mean% difference ± SD)	Average viability [%] (mean% difference ± SD)	Range		No. of unqualified experiments
			Viability [%]	% difference
Short time incubation – 3 min
Negative control (Non-corrosive)	1.503 (3.0 ± 2.7)	100 (3.0 ± 2.7)	91.7 – 108.3	0.57 – 10.7	0^#1
8 N KOH (Corrosive)	0.086 (7.5 ± 13.9)	5.8 (7.5 ± 13.9)	3.4 – 11.0	0.29 – 48.8	0
Long time incubation – 60 min
Negative control (Non-Corrosive)	1.603 (3.6 ± 2.8)	100 (3.6 ± 2.8)	92.3 – 107.7	0.21 – 10.0	0^#1
8 N KOH (Corrosive)	0.070 (2.5 ± 2.8)	4.5 (2.5 ± 2.8)	2.8 -  6.5	0.32 – 11.7	0^#2

^#1 unqualified results = if the mean OD of the negative control tissues is < 0.8 or > 2.8; if difference in viability for duplicate tissues > 30%

^#2 unqualified results = 8 N KOH: viability > 15% (1 h exposure)

Applicant's summary and conclusion

Interpretation of results:

other: non-corrosive

Conclusions:

Under the present test conditions, 1,1,3-trimethylindan-4-amine tested at two exposure periods of 3 min or 1 h was non-corrosive to reconstructed human epidermis tissue in the EpiDerm™ model.
There is regulatory acceptance in the EU that a substance can be considered corrosive (Skin Corrosive Cat.1A) based on a positive result in the human epidermis model test. Negative in vitro corrosivity responses are not conclusive with respect to non-classification or classification as irritant and shall therefore be subject to further evaluation.