dialkyl-methyldihydro-heteropolycycle

Eye irritation

Administrative data

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

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:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

22 July 2015

Test guideline 3

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)

Test material

Test material information

Constituent 1

Test material form:

liquid

Test animals / tissue source

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.

Test system

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.

Results and discussion

In vitro

Results

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

Any other information on results incl. tables

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

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 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).