methyl-bis(2-arylpropyl)dihydro-heteropolycycle

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-01-22 to 2019-04-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

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:

other: Commission Regulation (EC) No. 640/2012, L 193, Part B.46. “In vitro Skin Irritation: Reconstructed Human Epidermis Test Method” 06-Jul-2012

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation Protocol for: In Vitro EpiDermTM Skin Irritation Test (EPI-200-SIT) For use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm (EPI-200-SIT)

Version / remarks:

Version 07-Nov-2014

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Test material

Test material information

Constituent 1

Test material form:

liquid

In vitro test system

Test system:

human skin model

Remarks:

EpiDerm

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.

Vehicle:

unchanged (no vehicle)

Details on test system:

The test was carried out with the reconstituted three-dimensional human skin model EpiDerm (MatTek). This skin model consists of normal human epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK are cultured on chemically modified, collagen-coated cell culture inserts (Millicell). The EpiDerm epidermis model exhibits in vivo-like morphological and growth characteristics which are uniform and highly reproducible. It consists of organised basal, spinous and granular layers and a multi-layered stratum corneum analogous to patterns found in vivo.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Dose Groups
1. Negative control 30 µL Dulbecco’s phosphate buffered saline
2. Positive control 30 µL 5% sodium dodecyl sulfate solution
3. Test Item 30 µL (undiluted) / 25 mg + 25 µL Dulbecco’s phosphate buffered saline

Duration of treatment / exposure:

60 ± 1 min

Duration of post-treatment incubation (if applicable):

Post-treatment incubation: 42 ± 2 h
MTT incubation: 3h

Number of replicates:

The test was performed on a total of 3 tissues per dose group.

Test system

Details on study design:

The test was performed on EpiDerm, an organotypic reconstructed three-dimensional model of the human epidermis. 3 replicate tissues are dosed with the test item, the negative control (30 µL DPBS) and the positive control (30 µL 5% SDS), respectively. After 60 ± 1 min treatment period at 37°C (35 min) and room temperature (25 min) the test item and the controls are rinsed off with DPBS and the tissues are post-incubated for 42 +/- 1 h. Then the tissues are stained via MTT for 3 hours. The extracts are measured photometrically at 570 nm.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

48.9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

57.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3

Value:

72.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The controls confirmed the validity of the study. The mean absolute OD570 of the three negative control tissues was ≥ 0.8 and ≤ 2.8 (1.883 [exp.1]; 1.798 [exp.2]; 2.121 [exp.3]). The mean relative tissue viability (% negative control) of the positive control was ≤ 20% (4.9% [exp.1]; 3.5% [exp.2]; 4.5% [exp.3]). Standard deviation of viability of replicate tissues of all dose groups was ≤ 18% (0.2% - 17.9% [exp.1]; 0.5% - 6.5% [exp.2]; 0.3% - 7.7% [exp.3]).

Any other information on results incl. tables

The mixture of 30 μL test item per 1 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%.

The mixture of 30 μL of the test item per 300 μl aqua dest. and per 300 μL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, NSC equalled 0%.

The test item showed no non-specific reduction of MTT and no relevant colouring potential after mixture with aqua dest. and with isopropanol. Therefore, no additional controls for correction of possible false-negative results were necessary.

Experiment 1: The test item showed irritant effects. The mean relative tissue viability (% negative control) was ≤ 50% (48.9%) after 60 min treatment and 42 h post-incubation.

Experiment 2: The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 50% (57.7%) after 60 min treatment and 42 h post-incubation.

Experiment 3: The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 50% (72.7%) after 60 min treatment and 42 h post-incubation.

Result of the Test Item methyl-bis(2-arylpropyl)dihydro-heteropolycycle [exp.1]

Name	Negative Control			Positive Control			Test Item
Replicate Tissue	1	2	3	1	2	3	1	2	3
Absolute OD570	1.991	1.766	1.859	0.134	0.134	0.132	1.293	0.852	0.674
	2.017	1.787	1.878	0.134	0.131	0.143	1.325	0.840	0.673
Mean Absolute OD570	1.883****			0.135			0.943
OD570(Blank Corrected)	1.947	1.721	1.815	0.089	0.089	0.088	1.248	0.807	0.629
	1.972	1.742	1.833	0.089	0.086	0.098	1.281	0.795	0.628
Mean OD570of the Duplicates (Blank Corrected)	1.959	1.732	1.824	0.089	0.087	0.093	1.264	0.801	0.629
Total Mean OD570of the 3 Replicate Tissues (Blank Corrected)	1.838*			0.090			0.898
SD of Mean OD570of the 3 Replicate Tissues (Blank Corrected)	0.115			0.003			0.329
Relative Tissue Viability [%]	106.6	94.2	99.2	4.8	4.8	5.1	68.8	43.6	34.2
Mean Relative Tissue Viability [%]	100.0			4.9**			48.9
SD of Relative Tissue Viability [%]***	6.2			0.2			17.9
CV [% Viabilities]	6.2			3.2			36.6

^*          Blank-corrected mean OD_{570 nm} of the negative control corresponds to 100% absolute tissue viability.

^**         Mean relative tissue viability of the three positive control tissues is ≤ 20%.

^***        Standard deviation (SD) obtained from the three concurrently tested tissues is ≤ 18%

****     The mean absolute OD₅₇₀ of the negative control is ≥ 0.8 and ≤ 2.8.

Result of the Test Item methyl-bis(2-arylpropyl)dihydro-heteropolycycle [exp. 2]

Name	Negative Control			Positive Control			Test Item
Replicate Tissue	1	2	3	1	2	3	1	2	3
Absolute OD570	1.663	1.848	1.876	0.099	0.098	0.113	0.952	1.081	1.086
	1.671	1.871	1.859	0.099	0.099	0.116	1.021	1.085	1.107
Mean Absolute OD570	1.798****			0.104			1.055
OD570(Blank Corrected)	1.621	1.805	1.833	0.056	0.056	0.071	0.909	1.038	1.043
	1.628	1.828	1.816	0.056	0.056	0.073	0.978	1.042	1.065
Mean OD570of the Duplicates (Blank Corrected)	1.624	1.816	1.825	0.056	0.056	0.072	0.943	1.040	1.054
Total Mean OD570of the 3 Replicate Tissues (Blank Corrected)	1.755*			0.061			1.012
SD of Mean OD570of the 3 Replicate Tissues (Blank Corrected)	0.113			0.009			0.060
Relative Tissue Viability [%]	92.5	103.5	104.0	3.2	3.2	4.1	53.7	59.2	60.0
Mean Relative Tissue Viability [%]	100.0			3.5**			57.7
SD of Relative Tissue Viability [%]***	6.5			0.5			3.4
CV [% Viabilities]	6.5			15.0			5.9

          Blank-corrected mean OD_{570 nm} of the negative control corresponds to 100% absolute tissue viability.

^**         Mean relative tissue viability of the three positive control tissues is ≤20%.

^***        Standard deviation (SD) obtained from the three concurrently tested tissues is ≤ 18%

****      The mean absolute OD₅₇₀ of the negative control is ≥ 0.8 and ≤ 2.8.

Result of the Test Item methyl-bis(2-arylpropyl)dihydro-heteropolycycle [exp. 3]

Name	Negative Control			Positive Control			Test Item
Replicate Tissue	1	2	3	1	2	3	1	2	3
Absolute OD570	1.938	2.161	2.238	0.135	0.136	0.143	1.512	1.681	1.425
	1.951	2.167	2.269	0.139	0.137	0.149	1.540	1.734	1.431
Mean Absolute OD570	2.121****			0.140			1.554
OD570(Blank Corrected)	1.893	2.115	2.192	0.089	0.090	0.098	1.467	1.635	1.380
	1.906	2.121	2.224	0.094	0.091	0.103	1.494	1.688	1.386
Mean OD570of the Duplicates (Blank Corrected)	1.899	2.118	2.208	0.092	0.091	0.100	1.481	1.662	1.383
Total Mean OD570of the 3 Replicate Tissues (Blank Corrected)	2.075*			0.094			1.508
SD of Mean OD570of the 3 Replicate Tissues (Blank Corrected)	0.159			0.005			0.142
Relative Tissue Viability [%]	91.5	102.1	106.4	4.4	4.4	4.8	71.3	80.1	66.6
Mean Relative Tissue Viability [%]	100.0			4.5**			72.7
SD of Relative Tissue Viability [%]***	7.7			0.3			6.8
CV [% Viabilities]	7.7			5.7			9.4

^*                 Blank-corrected mean OD₅₇₀ of the negative control corresponds to 100% absolute tissue viability.

^**         Mean relative tissue viability of the three positive control tissues is ≤ 20%

^***        Standard deviation (SD) obtained from the three concurrently tested tissues is ≤ 18%.

^****       The mean absolute OD₅₇₀ of the negative control is ≥ 0.8 and ≤ 2.8.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item showed no irritant effects. The test item is therefore classified as “non-irritant” in accordance with UN GHS “No Category”.

Executive summary:

In the present study, in three independent experiments, methyl-bis(2-arylpropyl)dihydro-heteropolycycle

was applied topically to the EpiDerm^TM Tissue for 60 min followed by a 42 h post-incubation period and immediate determination of cytotoxic effects via MTT reduction assay.

Irritant potential of the test item was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with DPBS.

The mixture of 30 µL test item per 1 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%.

The mixture of 30 µL of the test item per 300 µl aqua dest. and per 300 µL isopropanol showed no colouring detectable by unaided eye-assessment.Therefore, NSC equalled 0%.

The test item showed no non-specific reduction of MTT and no relevant colouring potential after mixture with aqua dest. and with isopropanol. Therefore, no additional controls for correction of possible false-negative results were necessary.

Experiment 1:

The test item showed irritant effects. The mean relative tissue viability (% negative control) was ≤ 50% (48.9%) after 60 min treatment and 42 h post-incubation.

Experiment 2:

The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 50% (57.7%) after 60 min treatment and 42 h post-incubation.

Experiment 3:

The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 50% (72.7%) after 60 min treatment and 42 h post-incubation.

The controls confirmed the validity of the study. The mean absolute OD₅₇₀of the three negative control tissues was >= 0.8 and ≤ 2.8 (1.883 [exp.1]; 1.798 [exp.2]; 2.121 [exp.3]).

The mean relative tissue viability (% negative control) of the positive control was ≤ 20% (4.9% [exp.1]; 3.5% [exp.2]; 4.5% [exp.3]).

Standard deviation of viability of replicate tissues of all dose groups was ≤ 18% (0.2% - 17.9% [exp.1]; 0.5% - 6.5% [exp.2]; 0.3% - 7.7% [exp.3]).