cyclic C12/14-Glucamide;cyclic N-methyl-N-dodecanoyl/tetradecanoyl-glucamine;anhydro-N-dodecanoyl/tetradecanoyl-N-methylglucamine;Amides, C12-C14, N-(1-deoxy-D-glucitol-1-yl)-N-methyl, dehydrated

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 October 2021 to 02 December 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guidline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

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:

18 June 2019

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

Reference substance name:

D-Glucitol, anhydro-1-deoxy-1-(methylamino)-, N-[C12-14(even numbered) acyl] derivs.

Molecular formula:

C19H37NO5 C21H41NO5

IUPAC Name:

D-Glucitol, anhydro-1-deoxy-1-(methylamino)-, N-[C12-14(even numbered) acyl] derivs.

Test material form:

semi-solid (amorphous): gel

Specific details on test material used for the study:

Batch No.: S148866
Aggregate State at RT: slightly turbid solid mass
Colour: brown
Storage Conditions: room temperature
Expiry Date: 30 September 2022
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.

Test system

Amount / concentration applied:

1. Negative Control 50 µL Aqua dest.
2. Positive Control 50 µL methyl acetate
3. Test Item 50 mg

Duration of treatment / exposure:

incubation: 30 +/- 2 min.

Observation period (in vivo):

post soak: 12 +/- 2 min.
post treatment: 120 +/- 15 min.

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

Run / experiment:

1

Value:

999 999.999

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Value Cut off pass/fail
Mean Absolute OD570 nm NC: 1.359 0.8 < NC < 2.8 pass
Mean Relative Viability PC [%]: 34.1 < 50% pass
Max. Difference of % Viability [%] 18.1 < 20% pass

Any other information on results incl. tables

Pre-Experiments

The mixture of 50 mg 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 below the 60% threshold value no killed tissue controls were performed, since the test item has to be classified as irritant in any case.

 

The mixture of 50 mg test item per 1 mL Aqua dest. and per 2 mL isopropanol showed no colouring as compared to the solvent. Therefore, NSC_living equalled 0%.

 

The test item showed non-specific reduction of MTT, but 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.

 

Result of the Test Item cyclic Glucamide C12-C14

 

Name	Negative Control		Positive Control		Test Item
Replicate Tissue	1	2	1	2	1	2
Absolute OD570	1.461	1.240	0.384	0.581	0.079	0.079
	1.496	1.240	0.378	0.631	0.082	0.082
Mean Absolute OD570	1.359****		0.494		0.080
OD570 (Blank Corrected)	1.415	1.195	0.339	0.536	0.033	0.033
	1.450	1.194	0.333	0.585	0.036	0.037
Mean OD570 of the Duplicates (Blank Corrected)	1.433	1.195	0.336	0.560	0.035	0.035
Total Mean OD570 of the 2 Replicate Tissues (Blank Corrected)	1.314*		0.448		0.035
SD of Mean OD570 of the Duplicates (Blank Corrected)	0.168		0.159		0.000
Relative Tissue Viability [%]	109.1	90.9	25.6	42.7	2.6	2.7
Relative Tissue Viability Difference [%]^***	18.1		17.1		0.0
Mean Relative Tissue Viability [%]	100.0		34.1**		2.6

^*              Corrected mean OD₅₇₀ of 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 OD₅₇₀ of the negative control is > 0.8 and < 2.8                                                                              

 

 

 

Test Acceptance Criteria

 

	Value	Cut off	pass/fail
Mean Absolute OD570 nm NC	1.359	0.8 < NC < 2.8	pass
Mean Relative Viability PC [%]	34.1	< 50%	pass
Max. Difference of % Viability [%]	18.1	< 20%	pass

 

Historical Data

 

	Mean Absolute OD570±30nm NC	Mean Relative Viability [%] PC	SD Viability [%] NC, PC, TI
Mean	1.690	29.1	6.6
SD	0.291	12.2	6.5
Range of LCL – UCL	1.109 – 2.271	4.6 – 53.6	0.0 – 19.6
n	85	85	383

LCL:       Lower control limit (95%, mean – 2*SD)

UCL:      Upper control limit (95%, mean + 2*SD)

n:            number of control values

Applicant's summary and conclusion

Conclusions:

In this study under the given conditions the test item showed irritant effects. No prediction of the ocular irritation potential of the test item can be made, thus additional testing will be required.

Executive summary:

In the present study the eye irritating potential of cyclic Glucamide C12-C14 was analysed. Since irritant substances are cytotoxic to the corneal epithelium after a short time exposure the cytotoxic effects of the test item on EpiOcularÔ, a reconstituted three-dimensional human corneal epithelium model, were determined. Hereby, the test item was applied topically to the EpiOcularÔ tissue. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid (MTT) after a 6 h exposure period and 18 h post-treatment period and compared to those of the concurrent negative controls.

 

The test item showed non-specific MTT-reducing potential, but was classified as irritant. Therefore no additional controls were necessary. The test item showed no water-colouring potential.

 

The test item showed irritant effects. The mean relative tissue viability (% negative control) was ≤ 60% (2.6%).