dialkyl-methyldihydro-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:

2019-01-23 to 2019-01-25

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.

This in vitro method is designed to predict and classify the skin irritation potential of a chemical by assessment of its effect on EpiDerm. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 60 min exposure period.

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-incubation period: 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

Irritation / corrosion parameter:

% tissue viability

Remarks:

NSMTT-corrected

Run / experiment:

1

Value:

96.2

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:

Test Acceptance Criteria
- mean absolute OD570 nm of the three negative control tissues is ≥ 0.8 and ≤ 2.8 --> pass (1.752)
- mean relative tissue viability of the three positive control tissues is ≤ 20% --> pass (4.1%)
- standard deviation (SD) of relative tissue viability obtained from each three concurrently tested tissues is ≤ 18%. --> pass (0.2-7.3)

Any other information on results incl. tables

Pre-Experiments

The mixture of 30 µL test item per 1 mL MTT medium showed reduction of MTT compared to the solvent. The mixture turned blue/purple.

For quantitative correction of results, two killed tissues were treated with 30 µL of the test item (KT) and two killed tissues were left untreated as a control (KU), respectively. NSMTT was calculated relative to the negative control of living tissues (NC) according to the following formula:

NSMTT [%] = [(OD_KT- OD_KU)/OD_NC] * 100 = [(0.049-0.045)/1.709] * 100 = 0.2%

Mean OD_KT = 0.049

Mean OD_KU = 0.045

Mean OD_NC = 1.709

NSMTT was ≤ 30% (0.2%) relative to the negative control of livingepidermis, the true MTT metabolic conversion (TOD_TT) of the test item treated living tissues TM was therefore corrected according to the following formula:

TOD_TT = OD_TM– (OD_KT– OD_KU) = 1.648 – (0.049-0.045) = 1.644

The mixture of 30 µL of the test item per 300 µl aqua dest. showed colouring detectable by unaided eye-assessment (no colouring was detected with 300 µl isopropanol). Therefore, the absorption of the chemical in water was measured in the range of 570 ± 30 nm.

The test item in water absorbed light in the relevant range.For quantitative correction of results, the non-specific colour of additional viable tissues (NSC_living) was determined by using additional viable tissues without MTT-staining and calculated according to the following formula:

NSC_living [%] = [OD_TVT/OD_NC]*100 = [0.002/1.709]*100 = 0.1%

Mean OD_TVT = 0.002

NSC_living was ≤ 5% relative to the negative control of living epidermis, therefore no correction of the results was necessary.

For test items which act as non-specific MTT-reducers and show non-specific colouring of living tissues, a third control for non-specific colour in killed tissues (NSC_killed) was performed to avoid a possible double-correction for colour interference. Therefore, two killed tissues were treated with 30 µL of the test item (TKT). The non-specific colour of additional viable tissues (NSC_killed) was then calculated according to the following formula:

NSC_killed [%] = [OD_TKT/OD_NC]*100 = [0.001/1.709]*100 = 0.1%

Mean OD_TKT = 0.001

As correction with the NSC_living control was not necessary, correction with the NSC_killed control was also not required.

Result of the Test Item dialkyl-methyldihydro-heteropolycycle

In the present study the test item was applied topically to the EpiDerm 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 reduction of MTT compared to the solvent. The test item turned blue/purple.

NSMTT was ≤ 30% (0.2%) relative to the negative control of living epidermis.

The mixture of 30 μL of the test item per 300 μl aqua dest. showed colouring detectable by unaided eye-assessment (no colouring was detected with 300 μl isopropanol). Therefore, the absorption of the chemical in water was measured in the range of 570 ± 30 nm.

The test item in water absorbed light in the relevant range. For quantitative correction of results, the non-specific colour of additional viable tissues (NSCliving) was determined by using additional viable tissues without MTT-staining. NSCliving was ≤ 5% relative to the negative control of living epidermis, therefore no correction of the results was necessary.

For test items which act as non-specific MTT-reducers and show non-specific colouring of living tissues, a third control for non-specific colour in killed tissues (NSCkilled) was performed to avoid a possible double-correction for colour interference. Therefore, two killed tissues were treated with 30 μL of the test item (TKT). As correction with the NSCliving control was not necessary, correction with the NSCkilled control was also not required.

The test item showed no irritant effects. The mean relative tissue viability (% negative control) was >50% (96.2%, NSMTT-corrected) after 60 min treatment and 42 h post-incubation.

Name	Negative Control			Positive Control			Test Item
Replicate Tissue	1	2	3	1	2	3	1	2	3
Absolute OD570	1.689	1.794	1.726	0.115	0.109	0.112	1.492	1.789	1.698
	1.685	1.844	1.773	0.117	0.110	0.114	1.618	1.816	1.731
Mean Absolute OD570	1.752****			0.113			1.691
OD570(Blank Corrected)	1.646	1.751	1.683	0.072	0.066	0.069	1.449	1.746	1.655
	1.642	1.801	1.730	0.074	0.067	0.071	1.575	1.773	1.688
Mean OD570of the Duplicates (Blank Corrected)	1.644	1.776	1.707	0.073	0.066	0.070	1.512	1.759	1.671
Total Mean OD570of the 3 Replicate Tissues (Blank Corrected)	1.709*			0.070			1.648
TODTTNSMTT	-			-			1.644
TODTTNSCliving	-			-			no correction necessary
SD of Mean OD570of the 3 Replicate Tissues (Blank Corrected)	0.066			0.003			0.125
Relative Tissue Viability [%]	96.2	103.9	99.9	4.3	3.9	4.1	88.5	103.0	97.8
Mean Relative Tissue Viability [%]	100.0			4.1**			96.4
Mean Relative Tissue Viability [%] - NSMTT Corrected	-			-			96.2
Mean Relative Tissue Viability [%] - NSClivingCorrected	-			-			no correction necessary
True Tissue Viability [%]- NSMTT. NSClivingand NSCkilledCorrected	-			-			double correction not necessary
SD of Relative Tissue Viability [%]***	3.9			0.2			7.3
CV [% Viabilities]	3.9			4.6			7.6

^*          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 NSMTT control

NSMTT	KU		KT		Negative Control
Tissue	1	2	1	2	1	2	3
Absolute OD570 -values	0.083	0.093	0.088	0.093	1.689	1.794	1.726
	0.084	0.093	0.091	0.096	1.685	1.844	1.773
OD570(Blank Corrected)	0.040	0.050	0.045	0.050	1.646	1.751	1.683
	0.041	0.050	0.048	0.053	1.642	1.801	1.730
Mean OD570of the Duplicates (Blank Corrected)	0.041	0.050	0.047	0.052	1.644	1.776	1.707
Total Mean OD570of the 2 or 3 Replicate Tissues (Blank Corrected)	0.045		0.049		1.709
SD of Mean OD570of the Duplicates (Blank Corrected)	0.007		0.004		0.066
NSMTT [%]	0.2				-
Relative Tissue Viability [%]	-				96.2	103.9	99.9
Mean Relative Tissue Viability [%]	-				100.0
SD of Relative Tissue Viability [%]	-				3.9
CV [% Viabilities]	-				3.9

Result of the NSC_living control

NSCliving	TVT		Negative Control
Tissue	1	2	1	2	3
Absolute OD570	0.045	0.045	1.689	1.794	1.726
	0.044	0.045	1.685	1.844	1.773
OD570(Blank Corrected)	0.002	0.002	1.646	1.751	1.683
	0.001	0.002	1.642	1.801	1.730
Mean OD570of the Duplicates (Blank Corrected)	0.002	0.002	1.644	1.776	1.707
Total Mean OD570of the 2 or 3 Replicate Tissues (Blank Corrected)	0.002		1.709
SD of Mean OD570of the Duplicates (Blank Corrected)	0.000		0.066
NSCliving[%]	0.1		-
Relative Tissue Viability [%]	-		96.2	103.9	99.9
Mean Relative Tissue Viability [%]	-		100.0
SD of Relative Tissue Viability [%]	-		3.9
CV [% Viabilities]	-		3.9

Result of the NSC_killed control

NSCkilled	TKT		Negative Control
Tissue	1	2	1	2	3
Absolute OD570	0.044	0.044	1.689	1.794	1.726
	0.044	0.045	1.685	1.844	1.773
OD570(Blank Corrected)	0.001	0.001	1.646	1.751	1.683
	0.001	0.002	1.642	1.801	1.730
Mean OD570of the Duplicates (Blank Corrected)	0.001	0.002	1.644	1.776	1.707
Total Mean OD570of the 2 or 3 Replicate Tissues (Blank Corrected)	0.001		1.709
SD of Mean OD570of the Duplicates (Blank Corrected)	0.001		0.066
NSCkilled[%]	0.1		-
Relative Tissue Viability [%]	-		96.2	103.9	99.9
Mean Relative Tissue Viability [%]	-		100.0
SD of Relative Tissue Viability [%]	-		3.9
CV [% Viabilities]	-		3.9

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 the skin irritant potential of dialkyl-methyldihydro-heteropolycycle

was analysed. The EpiDerm™-Standard Model (EPI-200™), a reconstituted three-dimensional human epidermis model, was used as a replacement for the Draize Skin Irritation Test (OECD TG 404, [7]) to distinguish between UN GHS “Category 2” skin irritating test substances and not categorized test substances (“No Category”) which may be considered as non-irritant. Hereby, the test item was applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 60 min exposure and 42 h post-incubation period and compared to those of the concurrent negative controls.

The mixture of 30 μL test item per 1 mL MTT medium showed reduction of MTT compared to the solvent. The test item turned blue/purple.

For quantitative correction of results, two killed tissues were treated with 30 μL of the test item (KT) and two killed tissues were left untreated as a control (KU), respectively. NSMTT was calculated relative to the negative control of living tissues (NC) according to the following formula:

NSMTT [%] = [(ODKT - ODKU)/ODNC] * 100 = [(0.049-0.045)/1.709] * 100 = 0.2%

Mean ODKT = 0.049

Mean ODKU = 0.045

Mean ODNC = 1.709

NSMTT was ≤ 30% (0.2%) relative to the negative control of living epidermis, the true MTT metabolic conversion (TODTT) of the test item treated living tissues TM was therefore corrected according to the following formula:

TODTT = ODTM – (ODKT – ODKU) = 1.648 – (0.049-0.045) = 1.644

The mixture of 30 μL of the test item per 300 μl aqua dest. showed colouring detectable by unaided eye-assessment (no colouring was detected with 300 μl isopropanol). Therefore, the absorption of the chemical in water was measured in the range of 570 ± 30 nm.

The test item in water absorbed light in the relevant range (see Figure 1). For quantitative correction of results, the non-specific colour of additional viable tissues (NSCliving) was determined by using additional viable tissues without MTT-staining and calculated according to the following formula:

NSCliving [%] = [ODTVT/ODNC]*100 = [0.002/1.709]*100 = 0.1%

Mean ODTVT = 0.002

NSCliving was ≤ 5% relative to the negative control of living epidermis, therefore no correction of the results was necessary.

For test items which act as non-specific MTT-reducers and show non-specific colouring of living tissues, a third control for non-specific colour in killed tissues (NSCkilled) was performed to avoid a possible double-correction for colour interference. Therefore, two killed tissues were treated with 30 μL of the test item (TKT). The non-specific colour of additional viable tissues (NSCkilled) was then calculated according to the following formula:

NSCkilled [%] = [ODTKT/ODNC]*100 = [0.001/1.709]*100 = 0.1%

Mean ODTKT = 0.001

As correction with the NSCliving control was not necessary, correction with the NSCkilled control was also not required.

The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 50% (96.2%, NSMTT-corrected) after 60 min treatment and 42 h post-incubation.

.