Fatty acids, C12-18 and C18-unsatd., reaction products with N,N-dimethyl-1,3-propanediamine and triethanolamine

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-07-18 to 2019-09-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guidline 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.

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:

Fatty acids, C12-18 and C18-unsatd., reaction products with N,N-dimethyl-1,3-propanediamine and triethanolamine

Cas Number:

91845-03-3

Molecular formula:

Unknown

IUPAC Name:

Fatty acids, C12-18 and C18-unsatd., reaction products with N,N-dimethyl-1,3-propanediamine and triethanolamine

Test material form:

solid: bulk

Specific details on test material used for the study:

Name: 35760/270
Batch No.: 19953859
Aggregate State at RT: solid
Colour: beige
Storage Conditions: room temperature; protected from light
Stability: stable
Expiry Date: 16 January 2020
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.

Test system

Amount / concentration applied:

Dose Groups:
1. Negative Control 50 µL Aqua dest.
2. Positive Control 50 µL methyl acetate
3. Test Item 50 mg
The test was performed on a total of 2 tissues per dose group.

Duration of treatment / exposure:

incubation: 6 h +/- 0.25 h

Observation period (in vivo):

post soak: 25 +/- 2 min
post treatment: 18 +/- 0.25 h

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 6 h exposure period and 18 h 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 - NSMTT corrected

Run / experiment:

1

Value:

93.7

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:

Value Cut off pass/fail
Mean Absolute OD570 nm NK 1.211 0.8 < NK < 2.5 pass
Mean Relative Viability PC [%] 41.1 < 50% pass
Max. Difference of % Viability [%] 16.4 < 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 above the 60% threshold value killed tissue controls were performed for quantitative correction of results.

NSMTT [%] = [(OD_KT- OD_KU)/OD_NC] * 100 = 0.9%

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

NSMTT1 [%] = [meanOD_KT1- OD_KU)/OD_NC] * 100 = 0.8%

NSMTT2 [%] = [meanOD_KT2- OD_KU)/OD_NC] * 100 = 0.9%

NSMTT1 - NSMTT2 = ± -0.1%

NSMTT was ≤ 60% (0.9%) 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 [%] = viability_TM – NSMTT = 93.7%

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%.

Result of the Test Item 35760/270

Name	Negative Control		Positive Control		Test Item
Replicate Tissue	1	2	1	2	1	2
Absolute OD570	1.190	1.207	0.538	0.506	1.035	1.235
	1.207	1.239	0.540	0.517	1.069	1.251
Mean Absolute OD570	1.211****		0.525		1.147
OD570(Blank Corrected)	1.144	1.161	0.491	0.459	0.988	1.188
	1.160	1.193	0.493	0.470	1.022	1.204
Mean OD570of the Duplicates (Blank Corrected)	1.152	1.177	0.492	0.465	1.005	1.196
Total Mean OD570of the 2 Replicate Tissues (Blank Corrected)	1.164*		0.478		1.101
TODTT	-		-		1.091
SD of Mean OD570of the Duplicates (Blank Corrected)	0.017		0.019		0.135
Relative Tissue Viability [%]	98.9	101.1	42.3	39.9	86.3	102.7
Relative Tissue Viability Difference [%]^***	2.1		2.4		16.4
Mean Relative Tissue Viability [%]	100.0		41.1**		94.5
Mean Relative Tissue Viability [%] - NSMTT corrected	-		-		93.7

^*              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 ≤ 2.5       

Result of the NSMTT control

NSMTT	KU		KT		Negative Control
Replicate Tissue	1	2	1	2	1	2
Absolute OD570	0.070	0.076	0.083	0.084	1.190	1.207
	0.075	0.076	0.085	0.085	1.207	1.239
OD570(Blank Corrected)	0.024	0.029	0.036	0.037	1.144	1.161
	0.028	0.029	0.038	0.038	1.160	1.193
Mean OD570of the Duplicates (Blank Corrected)	0.026	0.029	0.037	0.038	1.152	1.177
Total Mean OD570 of the 2 Replicate Tissues (Blank Corrected)	0.028		0.037		1.164
SD of Mean OD570of the Duplicates (Blank Corrected)	0.002		0.000		0.017
NSMTT [%]	0.9				-
Relative Tissue Viability [%]	-				98.9		101.1
Relative Tissue Viability Difference [%]	-				2.1
Mean Relative Tissue Viability [%]	-				100.0

Applicant's summary and conclusion

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 35760/270 was applied topically to the EpiOcular^TM tissue for 6 h followed by 25 min post-soaking incubation after removal of the test item. After a 18 h post-treatment period cytotoxic effects were determined via MTT reduction assay.

Ocular irritation potential of the test item was predicted from the relative mean tissue viabilities compared to the negative control tissues concurrently treated with Aqua dest.

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 above the 60% threshold value killed tissue controls were performed for quantitative correction of results.

NSMTT [%] = [(OD_KT- OD_KU)/OD_NC] * 100 = 0.9%

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

NSMTT1 [%] = [meanOD_KT1- OD_KU)/OD_NC] * 100 = 0.8%

NSMTT2 [%] = [meanOD_KT2- OD_KU)/OD_NC] * 100 = 0.9%

NSMTT1 - NSMTT2 = ± -0.1%

NSMTT was ≤ 60% (0.9%) 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 [%] = viability_TM – NSMTT = 93.7%

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 no irritant effects. The mean relative tissue viability (% negative control) was > 60% (93.7% NSMTT-corrected).