dialkyl-methyldihydro-heteropolycycle

Endpoint summary

Description of key information

Skin Irritation

REACH_not irritating | EpiDerm | OECD 439 | #key study#

Eye Irritation

REACH_not irritating | EpiOcular | OECD 492 | #key study#

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

28 July 2015

Deviations:

no

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

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 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.

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.

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)

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

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.

.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-01-29 to 2019-01-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for the Testing of Chemicals No. 492: Reconstructed human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage.

Version / remarks:

25 June 2018

Qualifier:

according to guideline

Guideline:

other: EURL ECVAM DB-ALM Method Summary No. 164: EpiOcular™ Eye Irritation Test - Summary

Version / remarks:

22 July 2015

Qualifier:

according to guideline

Guideline:

other: EpiOcular™ Eye Irritation Test (OCL-200-EIT) For the prediction of acute ocular irritation of chemicals For use with MatTek Corporation’s Reconstructed Human EpiOcular™ Model

Version / remarks:

29 June 2015

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany)

Species:

human

Details on test animals or tissues and environmental conditions:

The test was carried out with the EpiOcular™ reconstructed human cornea-line epithelium (RhCE) model (MatTek). The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified, highly differentiated squamous epithelium morphologically similar to that found in a human cornea. The EpiOcular™ RhCE tissue construct consists of at least 3 viable layers of cells and a non-keratinized surface, showing a cornea-like structure analogous to that found in vivo.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

1. Negative Control 50 μL Aqua dest.
2. Positive Control 50 μL methyl acetate
3. Test Item 50 μL (undiluted)

Duration of treatment / exposure:

Incubation: 30 +/- 2 min.

Duration of post- treatment incubation (in vitro):

Post soak incubation: 12 +/- 2 min
Post-treatment incubation: 120 +/- 15 min
MTT-incubation: 3h

Number of animals or in vitro replicates:

2

Details on study design:

The test was performed on EpiOcular, a reconstituted three-dimensional human corneal epithelium model. 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 30 min exposure period and 120 min post-treatment period and compared to those of the concurrent negative controls.

Irritation parameter:

other: mean relative tissue viability

Remarks:

NSMTT, NSC living, NSC killed corrected

Run / experiment:

1

Value:

94.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: value in %

Other effects / acceptance of results:

Value Cut off pass/fail
Mean Absolute OD570 nm NK 1.624 0.8 < NK < 2.5 pass
Mean Relative Viability PC [%] 39.9 < 50% pass
Max. Difference of % Viability [%] 6.7 < 20% pass

The mixture of 50 μL test item per 1 mL MTT medium showed reduction of MTT as compared to the solvent. The mixture turned blue/purple. Since the mean relative tissue viability of the test item treated tissues (TM) was above the 60% threshold value killed tissue controls were performed for quantitative correction of results.

NSMTT [%] = [(ODKT - ODKU)/ODNC] * 100 = [(0.026 – 0.027)/1.580]*100 = - 0.06% ≈ -0.1%

Difference of NSMTT of the two duplicate tissues must be < 20%, otherwise not accepted.

NSMTT1 [%] = [meanODKT1 - ODKU)/ODNC] * 100 = [(0.026 – 0.027)/1.580]*100 = - 0.06%

NSMTT2 [%] = [meanODKT2 - ODKU)/ODNC] * 100 = [(0.026 – 0.027)/1.580]*100 = - 0.06%

NSMTT1 - NSMTT2 = ± 0%

NSMTT was ≤ 60% relative to the negative control of living epidermis and could therefore be used for determination of the killed control corrected viability (KCCV) according to the following formula:

KCCV [%] = viabilityTM – NSMTT = 94.8 – (-0.1) = 94.9%

The mixture of 50 μL test item per 2 mL isopropanol showed no colouring but per 1 mL Aqua dest colouring was observed as compared to the solvent. Since the mean relative tissue viability of the test item treated tissues (TM) was above the 60% threshold value coloured tissue controls were performed for quantitative correction of results.

NSCliving [%] = [ODTVT/ODNC] * 100 = [0.003/1.580] = 0.19 % ≈ 0.2%

Difference of NSCliving of the two duplicate tissues must be < 20%, otherwise not accepted.

NSC1 [%] = [ODTVT1 /ODNC] * 100 = [0.003/1.580] = 0.19 % ≈ 0.2%

NSC2 [%] = [ODTVT2 /ODNC] * 100 = [0.003/1.580] = 0.19 % ≈ 0.2%

NSC1 – NSC2 = ± 0.0%

NSCliving was ≤ 60% (0.2%) relative to the negative control of living epidermis and could therefore be used for determination of the NSC-corrected mean relative tissue viability (NSCCV) according to the following formula:

NSCCV [%] = viabilityTM [%] – NSCliving [%] = 94.8% - 0.2% = 94.6%

Since the test item showed non-specific MTT-reduction and 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.

The non-specific colour of additional killed tissues (NSCkilled) was calculated according to the following formula:

NSCkilled [%] = [ODTKT/ODNC]*100 = [0.002/1.580] = ≈ 0.1%

The true tissue viability was then calculated as the percent tissue viability obtained with living tissues minus NSMTT minus NSCliving plus NSCkilled.

True Tissue Viability = [%] mean Tissue viability – NSMTT - NSCliving + NSCkilled = (94.8 + 0.1 – 0.2 + 0.1) = 94.8%

Result of the Test Item

Name	Negative Control		Positive Control		Test Item
Replicate Tissue	1	2	1	2	1	2
Absolute OD570	1.574	1.684	0.667	0.676	1.487	1.569
	1.569	1.669	0.663	0.689	1.516	1.593
Mean Absolute OD570	1.624****		0.674		1.541
OD570(Blank Corrected)	1.531	1.640	0.624	0.632	1.443	1.525
	1.525	1.626	0.619	0.646	1.473	1.550
Mean OD570of the Duplicates (Blank Corrected)	1.528	1.633	0.621	0.639	1.458	1.537
Total Mean OD570of the 2 Replicate Tissues (Blank Corrected)	1.580*		0.630		1.498
TODTT- NSMTT	-		-		1.499
TODTTNSMTT and NSCliving	-		-		1.496
SD of Mean OD570of the Duplicates (Blank Corrected)	0.074		0.012		0.056
Relative Tissue Viability [%]	96.7	103.3	39.3	40.4	92.3	97.3
Relative Tissue Viability Difference [%]***	6.7		1.1		5.0
Mean Relative Tissue Viability [%]	100.0		39.9**		94.8
Mean Tissue Viability [%] - NSMTT corrected	-		-		94.9,
Mean Tissue Viability [%] - NSMTT and NSClivingcorrected	-		-		94.7
True Tissue Viability [%]	-		-		94.8

^*              Corrected mean OD₅₇₀of the negative control corresponds to 100% absolute tissue viability

^**             Mean relative tissue viability of the positive control is < 50%

^***            Relative tissue viability difference of replicate tissues is < 20%

^****        Mean absolute OD₅₇₀of the negative control is ≥ 0.8 and ≤ 2.5      

Result of the NSMTT control 

NSMTT	KU		KT		Negative Control
Replicate Tissue	1	2	1	2	1	2
Absolute OD570	0.070	0.070	0.069	0.070	1.574	1.684
	0.072	0.072	0.070	0.070	1.569	1.669
OD570(Blank Corrected)	0.026	0.027	0.025	0.026	1.531	1.640
	0.028	0.028	0.026	0.026	1.525	1.626
Mean OD570of the Duplicates (Blank Corrected)	0.027	0.027	0.026	0.026	1.528	1.633
Total Mean OD570 of the 2 Replicate Tissues (Blank Corrected)	0.027		0.026		1.580
SD of Mean OD570of the Duplicates (Blank Corrected)	0.000		0.000		0.074
NSMTT [%]	-0.1				-
Relative Tissue Viability [%]	-				96.7	103.3
Relative Tissue Viability Difference [%]	-				6.7
Mean Relative Tissue Viability [%]	-				100.0

Result of the NSC_living control

NSCliving	TVT		Negative Control
Replicate Tissue	1	2	1	2
Absolute OD570	0.045	0.045	1.574	1.684
	0.048	0.048	1.569	1.669
OD570(Blank Corrected)	0.001	0.002	1.531	1.640
	0.005	0.004	1.525	1.626
Mean OD570of the Duplicates (Blank Corrected)	0.003	0.003	1.528	1.633
Total Mean OD570 of the 2 Replicate Tissues (Blank Corrected)	0.003		1.580
SD of Mean OD570of the Duplicates (Blank Corrected)	0.000		0.074
NSCliving[%]	0.2		-
Relative Tissue Viability [%]	-		96.7	103.3
Relative Tissue Viability Difference [%]	-		6.7
Mean Relative Tissue Viability [%]	-		100.0

Result of the NSC_killedcontrol

NSCkilled	TKT		Negative Control
Replicate Tissue	1	2	1	2
Absolute OD570	0.045	0.046	1.574	1.684
	0.046	0.047	1.569	1.669
OD570(Blank Corrected)	0.001	0.002	1.531	1.640
	0.003	0.004	1.525	1.626
Mean OD570of the Duplicates (Blank Corrected)	0.002	0.003	1.528	1.633
Total Mean OD570 of the 2 Replicate Tissues (Blank Corrected)	0.002		1.580
SD of Mean OD570of the Duplicates (Blank Corrected)	0.001		0.074
NSCkilled[%]	0.1		-
Relative Tissue Viability [%]	-		96.7	103.3
Relative Tissue Viability Difference [%]	-		6.7
Mean Relative Tissue Viability [%]	-		100.0

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 classified as “non-irritant“ in accordance with UN GHS “No Category”.

Executive summary:

In the present study the eye irritating potential of dialkyl-methyldihydro-heteropolycycle was analysed. Since irritant substances are cytotoxic to the corneal epithelium after a short time exposure the cytotoxic effects of the test item on EpiOcularTM, a reconstituted three-dimensional human corneal epithelium model, were determined.

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 30 min exposure period and 120 min post-treatment period and compared to those of the concurrent negative controls.

The mixture of 50 μL test item per 1 mL MTT medium showed reduction of MTT as compared to the solvent. The mixture turned blue/purple. Since the mean relative tissue viability of the test item treated tissues (TM) was above the 60% threshold value killed tissue controls were performed for quantitative correction of results.

NSMTT [%] = [(ODKT - ODKU)/ODNC] * 100 = [(0.026 – 0.027)/1.580]*100 = - 0.06% ≈ -0.1%

Difference of NSMTT of the two duplicate tissues must be < 20%, otherwise not accepted.

NSMTT1 [%] = [meanODKT1 - ODKU)/ODNC] * 100 = [(0.026 – 0.027)/1.580]*100 = - 0.06%

NSMTT2 [%] = [meanODKT2 - ODKU)/ODNC] * 100 = [(0.026 – 0.027)/1.580]*100 = - 0.06%

NSMTT1 - NSMTT2 = ± 0%

NSMTT was ≤ 60% relative to the negative control of living epidermis and could therefore be used for determination of the killed control corrected viability (KCCV) according to the following formula:

KCCV [%] = viabilityTM – NSMTT = 94.8 – (-0.1) = 94.9%

The mixture of 50 μL test item per 2 mL isopropanol showed no colouring but per 1 mL Aqua dest colouring was observed as compared to the solvent. Since the mean relative tissue viability of the test item treated tissues (TM) was above the 60% threshold value coloured tissue controls were performed for quantitative correction of results.

NSCliving [%] = [ODTVT/ODNC] * 100 = [0.003/1.580] = 0.19 % ≈ 0.2%

Difference of NSCliving of the two duplicate tissues must be < 20%, otherwise not accepted.

NSC1 [%] = [ODTVT1 /ODNC] * 100 = [0.003/1.580] = 0.19 % ≈ 0.2%

NSC2 [%] = [ODTVT2 /ODNC] * 100 = [0.003/1.580] = 0.19 % ≈ 0.2%

NSC1 – NSC2 = ± 0.0%

NSCliving was ≤ 60% (0.2%) relative to the negative control of living epidermis and could therefore be used for determination of the NSC-corrected mean relative tissue viability (NSCCV) according to the following formula:

NSCCV [%] = viabilityTM [%] – NSCliving [%] = 94.8% - 0.2% = 94.6%

Since the test item showed non-specific MTT-reduction and 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.

The non-specific colour of additional killed tissues (NSCkilled) was calculated according to the following formula:

NSCkilled [%] = [ODTKT/ODNC]*100 = [0.002/1.580] = ≈ 0.1%

The true tissue viability was then calculated as the percent tissue viability obtained with living tissues minus NSMTT minus NSCliving plus NSCkilled.

True Tissue Viability = [%] mean Tissue viability – NSMTT - NSCliving + NSCkilled = (94.8 + 0.1 – 0.2 + 0.1) = 94.8%

The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 60% (94.8% NSMTT-, NSCliving-,NSCkilled- corrected).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Skin Irritation

The test substance did not produce skin irritation. The substance does not need to be classified for irritation according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.

Eye Irritation

The test substance did not produce eye irritation. The substance does not need to be classified for irritation according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.