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

Endpoint summary

Description of key information

Skin Corrosion: not corrosive (OECD 431, EpiSkin)

Skin Irritation: irritating (OECD 439, EpiDerm)

Eye Irritation: not conclusion (OECD 437, BCOP)

Eye Irritation: irritating (OECD 492, EpiOcular)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-04-14 to 2021-04-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

18 June 2019, corrected 26 June 2020

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No. 2019/1390, L-247, Part B.46. “In vitro Skin Irritation: Reconstructed Human Epidermis Test Method”

Version / remarks:

31-Jul-2019

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 02-Oct-2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Specific details on test material used for the study:

Name: cyclic Glucamide C12-C14
Chemical Name: anhydro –D-glucitol, 1-deoxy-1-(methylamino)-, N-[C12-14(even numbered) acryl] derivs.; Amides, C12-C14, N-(1-deoxy-D-glucitol-1-yl)-N-methyl, dehydrated
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:

human skin model

Source species:

human

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.

Details on test system:

This in vitro method is designed to predict and classify the skin irritation potential of a chemical by assessment of its effect on EpiDermTM, a reconstituted three-dimensional human epidermis model. 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 25 mg + 25 µL Dulbecco’s phosphate buffered saline

Duration of treatment / exposure:

60 ± 1 min

Duration of post-treatment incubation (if applicable):

24 ± 2 h + 18 ± 2 h (42 ± 2 h)

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 and extracted with isopropanol for at least 2h. The extracts are measured photometrically at 570 nm.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

2.1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

Test Acceptance Criteria
The test meets acceptance criteria if:
- mean absolute OD570 nm of the three negative control tissues is ≥ 0.8 and ≤ 2.8
- mean relative tissue viability of the three positive control tissues is < 20%
- standard deviation (SD) of relative tissue viability obtained from each three concurrently tested tissues is ≤ 18%.

Pre-Experiments:

The mixture of 25 mg test item per 1 mL MTT medium showed reduction of MTT compared to the solvent. The mixture turned blue/purple. The coloured test material or MTT reducing substance was classified as “Irritant” i.e. mean tissue viability is < 50%. Therefore, no correction procedures were necessary.

The mixture of 25 mg of the test item per 300 μl aqua dest. and/or per 300 μL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, NSC equalled 0%.

Experiment:

Name	Negative Control			Positive Control			Test Item
Replicate Tissue	1	2	3	1	2	3	1	2	3
Absolute OD570	2.400	2.075	2.298	0.111	0.105	0.115	0.086	0.098	0.094
	2.546	2.085	2.267	0.112	0.101	0.109	0.088	0.098	0.092
Mean Absolute OD570	2.279****			0.109			0.092
OD570(Blank Corrected)	2.354	2.029	2.252	0.065	0.059	0.069	0.040	0.052	0.048
	2.500	2.040	2.222	0.066	0.055	0.063	0.042	0.052	0.046
Mean OD570of the Duplicates (Blank Corrected)	2.427	2.034	2.237	0.065	0.057	0.066	0.041	0.052	0.047
Total Mean OD570of the 3 Replicate Tissues (Blank Corrected)	2.233*			0.063			0.047
SD of Mean OD570of the 3 Replicate Tissues (Blank Corrected)	0.196			0.005			0.006
Relative Tissue Viability [%]	108.7	91.1	100.2	2.9	2.6	2.9	1.8	2.3	2.1
Mean Relative Tissue Viability [%]	100.0			2.8**			2.1
SD of Relative Tissue Viability [%]***	8.8			0.2			0.2
CV [% Viabilities]	8.8			7.8			11.8

^*          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%

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

In this study under the given conditions the test item showed irritant effects. The test item is therefore classified as “irritant” in accordance with UN GHS “Category 2”.

Executive summary:

In the present study the skin irritant potential of cyclic Glucamide C12-C14 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 25 mg test item per 1 mL MTT medium showed reduction of MTT compared to the solvent. The mixture turned blue/purple. The coloured test material or MTT reducing substance was classified as “Irritant” i.e. mean tissue viability is < 50%. Therefore, no correction procedures were necessary.

The mixture of 25 mg of the test item per 300 μl aqua dest. and/or per 300 μL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, NSC equalled 0%.

The test item showed irritant effects. The mean relative tissue viability (% negative control) was ≤ 50% (2.1%) after 60 min treatment and 42 h post-incubation.

.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 December 2020 to 22 April 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

18 June, 2019

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Council Regulation 440/2008, Method B.40 BIS: “In Vitro Skin Corrosion: Human Skin Model Test”

Version / remarks:

May 30, 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: European Centre for the Validation of Alternative Methods (ECVAM). ECVAM INVITTOX Protocol No 118: ”EpiSkinTM Skin Corrosivity Test“

Version / remarks:

December 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Name: cyclic Glucamide C12-C14
Chemical Name: anhydro –D-glucitol, 1-deoxy-1-(methylamino)-, N-[C12-14(even numbered) acryl] derivs.; Amides, C12-C14, N-(1-deoxy-D-glucitol-1-yl)-N-methyl, dehydrated
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:

human skin model

Source species:

human

Justification for test system used:

This test uses the EPISKIN-SM™ reconstructed human epidermis model (SkinEthic) 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:

physiological saline

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
The EPISKIN-SMTM tissues are provided as kits (SkinEthic), consisting of the following components relevant for this study:
1x EPISKIN-SM™ plate containing 12 reconstructed epidermis units (area: 0.38 cm2); each reconstructed epidermis is attached to the base of a tissue culture insert with an O-ring set and maintained on nutritive agar for transport (Lot: 21 EKIN 002 [main exp.], 21 EKIN 008 [viable tissue controls and respective negative control], 21 EKIN 007 [killed tissue controls])
1x 12-well assay plate
1x flask of sterile maintenance medium (basic medium for incubations, Lot: 21 MAIN3 002 [main exp.], 21 MAIN3 008 [controls])
1x flask of sterile assay medium (basic medium for use in MTT assays, Lot: 21 ESSC 002 [main exp.], 21 ESSC 008 [controls])
Validity controls as provided by the supplier (SkinEthic):

Experimental Procedure
Upon receipt of the EPISKIN-SMTM, the tissues were transferred into 12-well plates containing 2 mL pre-warmed maintenance medium per well. The 12-well plates were incubated in a humidified incubator at 37  1 °C, 5.0% CO2 for at least 1 h to 48 h. Then the medium was replaced by 2.2 mL fresh assay medium. The 12-well plate(s) used for the 60 min and 3 min experiment were placed back into the incubator. The other plate(s) were used for the 4 h treatment.
4 h experiment: the tissues were treated with each dose group in duplicate, starting with the negative control. Start time was recorded with dosing of the first tissue. Then the 12-well plate(s) were incubated for 4 h ± 10 min at room temperature.
60 min experiment: the tissues were treated with the test item and the negative control in duplicate, starting with the negative control. Start time was recorded with dosing of the first tissue. Then the 12-well plate(s) were incubated for 60 min ± 5 min at room temperature.
3 min experiment: the tissues were treated with the test item and the negative control in duplicate, starting with the negative control. Start time was recorded with dosing of the first tissue. A constant time interval of e.g. 30 sec was kept between dosing.
3 min, 60 min and 4 h experiment: after 3 min / 60 min / 4 h application, with forceps, the first insert was removed from the 12-well plate. Using e.g. a wash bottle the tissue was gently rinsed about 15 times with 25 mL PBS to remove any residual test item. Excess PBS was removed by gently shaking the insert and blotting bottom with blotting paper. The surface was swept with a cotton bud. The insert was placed in a prepared 12-well “holding plate“ containing 2.2 mL pre-warmed assay medium per well. All inserts were treated in the same manner.
Then the inserts were transferred into a prepared 12-well “MTT assay plate“ containing 2 mL pre-warmed MTT solution. The plate was incubated for 3 h ± 15 min at 37  1 °C, 5.0% CO2 / 95% air.
After the 3 h MTT incubation period the tissues were placed on blotting paper to remove excess liquid. Afterwards a total biopsy of the epidermis by using the biopsy punch was performed and the epidermis was separated from the collagen matrix with the aid of forceps. Both parts (epidermis and collagen matrix) were transferred into suitable tubes and 500 µL of acidic isopropanol were added. Then the tubes were mixed on a vortex mixer. Extraction was carried out at room temperature overnight, protected from light.
At the end of the formazan extraction period the tubes were mixed by vortexing until solution colour became homogeneous.
If any visible cell/tissue fragments were in suspension, the tubes were centrifuged to eliminate the fragments and avoid possible interference with the absorbance readings.
Per each tissue 2 x 200 µL aliquots of the extract were transferred into a 96-well plate and OD was measured at 570 nm using a filter band pass of maximum ± 30 nm in a plate spectrophotometer using acidified isopropanol as the blank.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

1. Negative control 50 µL 0.9% NaCl solution
2. Positive control 50 µL glacial acetic acid
3. Test Item 20 ± 2 mg + 100 ± 5 µL 0.9% NaCl

Duration of treatment / exposure:

The test was performed on a total of 6 tissues for each test item and for the negative control, 2 replicates for each treatment period (3 min, 60 min and 4 h exposure time). For the positive control the test was performed with 2 replicates for the 4 h exposure time

Number of replicates:

6 tissues for each test item
negative control: 2 replicates
positive control: 2 replicates

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

4h

Value:

>= 35

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

6 min

Value:

92.9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min

Value:

104.4

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The test item showed non-specific MTT-reducing potential. Therefore, additional killed tissue controls were treated with the test item to determine the non-specific reduction of MTT (NSMTT) and the results were corrected to the true MTT metabolic conversion (TODTT). Moreover, the test item showed water-colouring potential in aqua dest., but not in isopropanol, in the range of 570 ± 30 nm . Therefore, additional viable tissue controls were treated with the test item to determine the non-specific colour (NSCliving). As NSCliving was ≤ 5%, correction with NSCliving and NSCkilled was obsolete.
The controls confirmed the validity of the study. The mean OD570 of the two negative control tissues was between 0.6 and 1.5 for each exposure period. The mean relative tissue viability (% negative control) of the positive control was  20% (4.5%) after 4 h treatment. The maximum inter tissue viability difference of replicate tissues of all dose groups was  30% (7.7%).

 Experiment

Table 2:   Blank values (main experiment)

Name	Blank
absolute OD570 - values (raw data)	0.043	0.045
	0.045	0.045
mean OD570 (mean of 2 aliquots)	0.045

Table 3:   Blank values (controls)

Name	Blank
absolute OD570 - values (raw data)	0.043	0.044
	0.045	0.045
mean OD570 (mean of 2 aliquots)	0.044

 

Table 4:   3 min Experiment

Name	Negative Control		Test Item
Replicate Tissue	1	2	1	2
Absolute OD570	0.748	0.804	0.799	0.815
	0.791	0.872	0.853	0.881
Mean Absolute OD570	0.804****		0.837
OD570 - Blank Corrected	0.704	0.759	0.754	0.770
	0.747	0.828	0.808	0.836
Mean OD570 of 2 Aliquots (Blank Corrected)	0.725	0.794	0.781	0.803
SD OD570  of 2 Aliquots	0.030	0.048	0.038	0.047
Total Mean OD570 of 2 Replicate Tissues (Blank Corrected)	0.759*		0.792
TODTT NSMTT	-		0.793
TODTT NSMTT und NSCliving	-		no correction necessary
SD OD570  of 2 Replicate Tissues	0.048		0.016
Mean Relative Tissue Viability [%]	100.0		104.3
Mean Relative Tissue Viability [%] - NSMTT Corrected	-		104.4
Mean Relative Tissue Viability [%] - NSMTT and NSCliving Corrected	-		no correction necessary
True Tissue Viability [%]	-		double correction not necessary
Coefficient Of Variation [%]^***	6.3		2.0

*                      corrected mean OD₅₇₀ of the negative control corresponds to 100% absolute tissue viability.

***                difference between each two replicates is £ 30% (in the range of 20 – 100% viability and for ODs > 0.3).

****              The mean absolute OD₅₇₀ of the negative control is ≥ 0.6 and ≤ 1.5.

 

 

 

Table 5:   60 min Experiment

Name	Negative Control		Test Item
Replicate Tissue	1	2	1	2
Absolute OD570	0.813	0.853	0.816	0.810
	0.880	0.920	0.844	0.798
Mean Absolute OD570	0.867****		0.817
OD570 - Blank Corrected	0.769	0.809	0.771	0.765
	0.835	0.876	0.799	0.754
Mean OD570 of 2 Aliquots (Blank Corrected)	0.802	0.842	0.785	0.759
SD OD570  of 2 Aliquots	0.047	0.047	0.020	0.008
Total Mean OD570 of 2 Replicate Tissues (Blank Corrected)	0.822*		0.772
TODTT NSMTT	-		0.764
TODTT NSMTT und NSCliving	-		no correction necessary
SD OD570  of 2 Replicate Tissues	0.028		0.018
Mean Relative Tissue Viability [%]	100.0		93.9
Mean Relative Tissue Viability [%] - NSMTT Corrected	-		92.9
Mean Relative Tissue Viability [%] - NSMTT and NSCliving Corrected	-		no correction necessary
True Tissue Viability [%]	-		double correction not necessary
Coefficient Of Variation [%]^***	3.4		2.4

*                      corrected mean OD₅₇₀ of the negative control corresponds to 100% absolute tissue viability.

***                difference between each two replicates is £ 30% (in the range of 20 – 100% viability and for ODs > 0.3).

****              The mean absolute OD₅₇₀ of the negative control is ≥ 0.6 and ≤ 1.5.

 

 

Table 6:   4 h Experiment

Name	Negative Control		Positive Control		Test Item
Replicate Tissue	1	2	1	2	1	2
Absolute OD570	0.807	0.794	0.074	0.084	0.782	0.864
	0.883	0.880	0.076	0.086	0.847	0.941
Mean Absolute OD570	0.841****		0.080		0.859
OD570 - Blank Corrected	0.762	0.750	0.029	0.040	0.737	0.820
	0.839	0.836	0.032	0.042	0.803	0.896
Mean OD570 of 2 Aliquots (Blank Corrected)	0.801	0.793	0.031	0.041	0.770	0.858
SD OD570  of 2 Aliquots	0.054	0.061	0.002	0.001	0.047	0.054
Total Mean OD570 of 2 Replicate Tissues (Blank Corrected)	0.797*		0.036		0.814
TODTT NSMTT	-		-		0.819
TODTT NSMTT und NSCliving	-		-		no correction necessary
SD OD570  of 2 Replicate Tissues	0.005		0.007		0.062
Mean Relative Tissue Viability [%]	100.0		4.5**		102.2
Mean Relative Tissue Viability [%] - NSMTT Corrected	-		-		102.7
Mean Relative Tissue Viability [%] - NSMTT and NSCliving Corrected	-		-		no correction necessary
True Tissue Viability [%]	-		-		double correction not necessary
Coefficient Of Variation [%]^***	0.7		20.3		7.7

*                      corrected mean OD₅₇₀ of the negative control corresponds to 100% absolute tissue viability.

**                  mean relative tissue viability of the two positive control tissues of the 4 h treatment period is £ 20%.

***                difference between each two replicates is £ 30% (in the range of 20 – 100% viability and for ODs > 0.3).

****              The mean absolute OD₅₇₀ of the negative control is ≥ 0.6 and ≤ 1.5.

 

 

Table 7:   Result of the NSMTT control 3 min

NSMTT	KU		KT		Negative Control
Replicate Tissue	1	2	1	2	1	2
absolute OD570 - values	0.097	0.092	0.088	0.089	0.821	0.808
	0.105	0.097	0.094	0.118	0.896	0.869
OD570 (Blank Corrected)	0.053	0.048	0.044	0.044	0.777	0.764
	0.061	0.053	0.049	0.073	0.852	0.825
mean OD570 (mean of 2 aliquots)	0.057	0.050	0.047	0.059	0.814	0.794
total mean OD570 (mean of the replicate tissues)	0.054		0.053		0.804
SD OD570 (of the replicate tissues)	0.005		0.009		0.014
NSMTT [%]	-0.1				-
Relative Tissue Viability [%]	-				101.2	98.8
Mean Relative Tissue Viability [%]	-				100.0
SD Tissue Viability [%]	-				1.7
CV [% Viabilities]	-				1.7

 

Table 8:   Result of the NSMTT control 60 min

NSMTT	KU		KT		Negative Control
Replicate Tissue	1	2	1	2	1	2
absolute OD570 - values	0.094	0.107	0.098	0.103	0.787	0.778
	0.099	0.101	0.099	0.133	0.850	0.836
OD570 (Blank Corrected)	0.050	0.062	0.053	0.059	0.743	0.734
	0.054	0.057	0.055	0.089	0.806	0.791
mean OD570 (mean of 2 aliquots)	0.052	0.060	0.054	0.074	0.774	0.763
total mean OD570 (mean of the replicate tissues)	0.056		0.064		0.768
SD OD570 (of the replicate tissues)	0.005		0.014		0.008
NSMTT [%]	1.1				-
Relative Tissue Viability [%]	-				100.8	99.2
Mean Relative Tissue Viability [%]	-				100.0
SD Tissue Viability [%]	-				1.1
CV [% Viabilities]	-				1.1

 

 

 

Table 9:   Result of the NSMTT control 4 h

NSMTT	KU		KT		Negative Control
Replicate Tissue	1	2	1	2	1	2
absolute OD570 - values	0.109	0.096	0.108	0.087	0.963	0.952
	0.107	0.099	0.102	0.097	0.963	1.030
OD570 (Blank Corrected)	0.065	0.052	0.063	0.043	0.919	0.907
	0.063	0.055	0.057	0.053	0.918	0.985
mean OD570 (mean of 2 aliquots)	0.064	0.053	0.060	0.048	0.919	0.946
total mean OD570 (mean of the replicate tissues)	0.059		0.054		0.932
SD OD570 (of the replicate tissues)	0.007		0.009		0.020
NSMTT [%]	-0.5				-
Relative Tissue Viability [%]	-				98.5	101.5
Mean Relative Tissue Viability [%]	-				100.0
SD Tissue Viability [%]	-				2.1
CV [% Viabilities]	-				2.1

 

Table 10: Result of the NSC_living control 3 min

NSCliving	TVT		Negative Control
Replicate Tissue	1	2	1	2
absolute OD570 - values	0.063	0.063	0.821	0.808
	0.064	0.066	0.896	0.869
absolute OD570 - Blank corrected values	0.019	0.018	0.777	0.764
	0.020	0.021	0.852	0.825
mean OD570 (mean of 2 aliquots)	0.019	0.020	0.814	0.794
total mean OD570 (mean of replicate tissues)	0.020		0.804
SD OD570 (of the 2 replicate tissues)	0.000		0.014
NSCliving [%]	2.4		-
Relative Tissue Viability [%]	-		101.2	98.8
Mean Relative Tissue Viability [%]	-		100.0
SD Tissue Viability [%]	-		1.7
CV [% Viabilities]	-		1.7

 

 

 

Table 11: Result of the NSC_living control 60 min

NSCliving	TVT		Negative Control
Replicate Tissue	1	2	1	2
absolute OD570 - values	0.059	0.073	0.787	0.778
	0.060	0.071	0.850	0.836
absolute OD570 - Blank corrected values	0.015	0.029	0.743	0.734
	0.016	0.026	0.806	0.791
mean OD570 (mean of 2 aliquots)	0.015	0.028	0.774	0.763
total mean OD570 (mean of replicate tissues)	0.021		0.768
SD OD570 (of the 2 replicate tissues)	0.009		0.008
NSCliving [%]	2.8		-
Relative Tissue Viability [%]	-		100.8	99.2
Mean Relative Tissue Viability [%]	-		100.0
SD Tissue Viability [%]	-		1.1
CV [% Viabilities]	-		1.1

 

Table 12: Result of the NSC_living control 4 h

NSCliving	TVT		Negative Control
Replicate Tissue	1	2	1	2
absolute OD570 - values	0.074	0.064	0.963	0.952
	0.076	0.062	0.963	1.030
absolute OD570 - Blank corrected values	0.030	0.019	0.919	0.907
	0.031	0.017	0.918	0.985
mean OD570 (mean of 2 aliquots)	0.031	0.018	0.919	0.946
total mean OD570 (mean of replicate tissues)	0.024		0.932
SD OD570 (of the 2 replicate tissues)	0.009		0.020
NSCliving [%]	2.6		-
Relative Tissue Viability [%]	-		98.5	101.5
Mean Relative Tissue Viability [%]	-		100.0
SD Tissue Viability [%]	-		2.1
CV [% Viabilities]	-		2.1

 

 

 

Table 13: Result of the NSC_killed control 3 min

NSCkilled	TKT		Negative Control
Replicate Tissue	1	2	1	2
absolute OD570 - values	0.073	0.064	0.821	0.808
	0.096	0.079	0.896	0.869
absolute OD570 - Blank corrected values	0.028	0.019	0.777	0.764
	0.052	0.035	0.852	0.825
mean OD570 (mean of 2 aliquots)	0.040	0.027	0.814	0.794
total mean OD570 (mean of replicate tissues)	0.034		0.804
SD OD570 (of the 2 replicate tissues)	0.009		0.014
NSCkilled [%]	4.2		-
Relative Tissue Viability [%]	-		101.2	98.8
Mean Relative Tissue Viability [%]	-		100.0
SD Tissue Viability [%]	-		1.7
CV [% Viabilities]	-		1.7

 

Table 14: Result of the NSC_killed control 60 min

NSCkilled	TKT		Negative Control
Replicate Tissue	1	2	1	2
absolute OD570 - values	0.071	0.078	0.787	0.778
	0.065	0.064	0.850	0.836
absolute OD570 - Blank corrected values	0.027	0.034	0.743	0.734
	0.021	0.019	0.806	0.791
mean OD570 (mean of 2 aliquots)	0.024	0.026	0.774	0.763
total mean OD570 (mean of replicate tissues)	0.025		0.768
SD OD570 (of the 2 replicate tissues)	0.002		0.008
NSCkilled [%]	3.3		-
Relative Tissue Viability [%]	-		100.8	99.2
Mean Relative Tissue Viability [%]	-		100.0
SD Tissue Viability [%]	-		1.1
CV [% Viabilities]	-		1.1

 

 

 

Table 15: Result of the NSC_killed control 4 h

NSCkilled	TKT		Negative Control
Replicate Tissue	1	2	1	2
absolute OD570 - values	0.084	0.104	0.963	0.952
	0.071	0.123	0.963	1.030
absolute OD570 - Blank corrected values	0.040	0.059	0.919	0.907
	0.027	0.078	0.918	0.985
mean OD570 (mean of 2 aliquots)	0.033	0.069	0.919	0.946
total mean OD570 (mean of replicate tissues)	0.051		0.932
SD OD570 (of the 2 replicate tissues)	0.025		0.020
NSCkilled [%]	5.5		-
Relative Tissue Viability [%]	-		98.5	101.5
Mean Relative Tissue Viability [%]	-		100.0
SD Tissue Viability [%]	-		2.1
CV [% Viabilities]	-		2.1

 

 

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

In this study under the given conditions the test item showed no corrosive effects. The relative mean tissue viability after 4 h treatment was not decreased to less than 35% of the corresponding negative control tissues. The test item is therefore classified as “non-corrosive“.

Executive summary:

In the present study the skin corrosivity potential of cyclic Glucamide C12-C14 was analysed. Since corrosive chemicals are cytotoxic after a short time exposure to the stratum corneum of the epidermis the cytotoxic effects of the test item on EPISKIN-SM™, a reconstituted three-dimensional human epidermis 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 3 min, 60 min and 4 h exposure period and compared to those of the concurrent negative controls.

The test item showed non-specific MTT-reducing potential. Therefore, additional killed tissue controls were treated with the test item to determine the non-specific reduction of MTT (NSMTT) and the results were corrected to the true MTT metabolic conversion (TOD_TT). Moreover, the test item showed water-colouring potential in aqua dest., but not in isopropanol, in the range of 570 ± 30 nm (see Figure 1 and Figure 2). Therefore, additional viable tissue controls were treated with the test item to determine the non-specific colour (NSC_living). As NSC_living was ≤ 5%, correction with NSC_living and NSC_killed was obsolete.

The test item showed no corrosive effects. The mean relative tissue viability (% negative control) was ³ 35% (102.7%, NSMTT-corrected) after 4 h treatment.

Relative mean tissue viability was 92.9% (NSMTT-corrected) after 60 min treatment and 104.4% (NSMTT-corrected) after 3 min treatment.

The controls confirmed the validity of the study. The mean OD₅₇₀ of the two negative control tissues was between 0.6 and 1.5 for each exposure period. The mean relative tissue viability (% negative control) of the positive control was £ 20% (4.5%) after 4 h treatment. The maximum inter tissue viability difference of replicate tissues of all dose groups was £ 30% (7.7%).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

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

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

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)

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.

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.

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

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

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%).

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 June 2021 to 28 July 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for the Testing of Chemicals, no. 437 (adopted: 26 June 2020)

Deviations:

yes

Remarks:

According to the guideline, due to the relatively high viscosity of the test item preparation, 750 µL thereof was applied directly onto the cornea by removing the window-locking ring and glass window prior to treatment.

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Munich, Germany

Specific details on test material used for the study:

Name: cyclic Glucamide C12-C14
Product
(Common Name/Code): cyclic N-dodecanoyl/tetradecanoyl-N-methylglucamine / cyclic Glucamide C12-C14
Lot No.: S148866
Storage Conditions: room temperature
Physical State at Room
Temperature: slightly turbid solid mass
Color: brown
Density: 1.07 g/cm³
Molecular Weight: main compounds: 359,5 g/mol (C12-derivative); 387,5 g/mol (C14-derivative)
pH at Room Temperature: approx. 6-8 (not tested)
Date of Analysis: 30 September 2020
Expiry Date: 30 September 2022
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.

Vehicle:

physiological saline

Amount / concentration applied:

The test item was suspended with physiological saline 0.9% NaCl (B. Braun Melsungen, lot no. 20192410, expiry date: 04/2023) to give a 20% concentration.

Duration of treatment / exposure:

After 4 hours ± 5 minutes incubation at 32 ± 1 °C either the test substance or the control substance was removed and the epithelium washed at least three times with MEM (containing phenol red).

Number of animals or in vitro replicates:

3 corneas for the test item
3 corneas as negative controls treated with physiological saline 0.9% NaCl
3 corneas as positive controls treated with imidazole 20% in physiological saline 0.9% NaCl

Details on study design:

Test System
Preparation of the Corneas
The assay uses isolated corneas obtained as a by-product from animals freshly slaughtered at the abattoir Vion Beef B.V., Buchloe, Germany.
On the test day, fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories. Immediately after arrival of the eyes, cornea preparation was initiated.
The eyes were carefully examined for defects and any defective eyes were discarded.
The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing HBSS. Before the corneas were mounted in corneal holders (Duratec GmbH) with the endothelial side against the O-ring of the posterior chamber, they had been visually examined for defects and any defective cornea had been discarded. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with RPMI 1640 medium (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI 1640 medium). The posterior chamber was always filled first. The corneas were incubated for one hour at 32 ± 1 °C.


Calibration of the Opacitometer
The opacitometer (BASF-OP3.0, Duratec GmbH) was switched on at least 15 min before starting the calibration procedure. The filter holder was placed into the opacitometer and the readout was adjusted to 1000 ± 10 lux using the “Calibrate”-turning knob. For calibration the glass filter F2 was introduced into the filter holder. The readout lay in the range between 540-560 lux. To test the linearity of the measurement, two additional calibration filters, glass filter F3 and glass filter F4, were measured. For these glass filters, the opacitometer displayed values between 300-310 lux and between 90-100 lux. The calibration procedure was performed before the test and is documented in the raw data.

Treatment of the Corneas
After the equilibration period, the medium was removed from both chambers and replaced with fresh complete RPMI 1640 medium. An initial measurement was performed on each of the corneas using the opacitometer. Three corneas with illuminance readings approximately equivalent to the median illuminance of all corneas were selected as negative-control corneas. The illuminance of each cornea was read and recorded. Only corneas that had an initial illuminance reading I > I0/1.1651 lux were used for the assay. The medium was removed from the anterior chamber and replaced with the test item or control.
750 µL of the test item preparation (open-chamber method) or the control substance (closed-chamber method) was introduced into the anterior chamber. After 4 hours ± 5 minutes incubation at 32 ± 1 °C either the test substance or the control substance was removed and the epithelium washed at least three times with MEM (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI 1640 medium (without phenol red). The anterior chamber was refilled with complete RPMI 1640 medium and an illuminance measurement was performed. Also, each cornea was observed visually and pertinent observations were recorded.
After the illuminance measurement was performed, the medium was removed from both chambers of the holder. The posterior chamber was refilled with fresh complete RPMI 1640 medium. 1 mL of a 5 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes at 32 ± 1 °C. Then the medium from the posterior chamber was removed and its optical density at 490 nm (OD490) was determined, using a spectrophotometer (Jenway 6405 UV/VIS).



Test Groups
3 corneas for the test item
3 corneas as negative controls treated with physiological saline 0.9% NaCl
3 corneas as positive controls treated with imidazole 20% in physiological saline 0.9% NaCl
The BCOP assay is considered to be valid if the in vitro irritation score obtained with the positive control falls within the two standard deviations of the current historical mean.
The negative control responses should result in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.


Evaluation of Results
The following formula was used to calculate the opacity, whereas the values a and b are equipment-specific variables empirically determined by the manufacturer:

Opacity = ( I0/I-b)/a

with a = 0.025 and b = 0.9894

The value I0 (=I zero) is the illuminance through a holder without cornea, but with windows and liquid. This value is determined by taking the mean for a set of cornea holders and is reevaluated periodically.
The change in opacity for each cornea was calculated by subtracting the initial opacity reading from the final opacity reading. These values were corrected by subtracting from each the average change in opacity observed for the negative-control corneas. The mean opacity value for each treatment was calculated by averaging the corrected opacity values of each cornea for a given treatment.
The mean OD490 for the blank cuvettes was calculated. The mean blank OD490 was subtracted from the OD490 of each cuvette (corrected OD490). Any dilutions that were made to bring the OD490 values into the linear range of the spectrophotometer (OD490 should be less than 1.500), were taken into account by multiplying the OD490 value of the dilution by the dilution factor. The final-corrected OD490 of the test article and the positive control were calculated by subtracting the average-corrected OD490 of the negative-control corneas from the corrected OD490 value of each treated cornea:
Final-corrected OD490 = (OD490 – mean blank OD490) – average-corrected negative control OD490
The mean OD490 value of each treatment group was calculated by averaging the final corrected OD490 values of the treated corneas for that treatment condition.

The following formula was used to determine the in vitro irritation score (IVIS):

IVIS = mean opacity value + (15 x mean permeability OD490 value)

The IVIS cut-off values for identifying test substances as inducing serious eye damage (UN GHS Category 1) and test substances not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given below:

Evaluation of the BCOP Assay

IVIS UN GHS
≤ 3 No Category
> 3; ≤ 55 No stand-alone prediction can be made
> 55 Category 1

An identification of test substances that should be classified as irritating to eyes (UN GHS Category 2 or Category 2A) or test substances that should be classified as mildly irritating to eyes (UN GHS Category 2B) cannot be made.
For this purpose further testing with another suitable method is required.

Irritation parameter:

in vitro irritation score

Remarks:

mean

Value:

14.86

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

study cannot be used for classification

Conclusions:

No stand-alone prediction can be made regarding the classification of the test substance cyclic Glucamide C12-C14 according to the evaluation criteria. Further testing in another suitable method is required.

Executive summary:

Summary Results

The eye irritancy potential of cyclic Glucamide C12-C14 was investigated in the bovine corneal opacity and permeability assay.

Due to the fact that the IVIS of the positive control did not fall within the two standard deviations of the current historical mean in the first experiment, it was not considered to be valid. Therefore the experiment was repeated. The results of both experiments are reported, noting that data from the first experiment is invalid and is not considered for experimental outcome in the present study.

All data in the report refers to the 2^nd experiment.

Preparation of the test item:                         The test item was suspended with physiological saline 0.9% NaCl to give a 20% concentration

Visual observation after treatment:              All 3 corneas treated with cyclic Glucamide C12-C14 showed slight opacity of the tissue.

Mean in vitro irritation score:                        14.86

Classification:

	UN GHS No Category
X	No stand-alone prediction can be made
	UN GHS Category 1

In the second experiment the in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid.

The negative control responses resulted in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

Conclusion

No stand-alone prediction can be made regarding the classification of the test substance cyclic Glucamide C12-C14 according to the evaluation criteria. Further testing in another suitable method is required.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The registered substance caused no corrosive effects in an in-vitro skin corrosive study according to OECD 431. It caused irritating effects in an in-vitro skin irritation study according to OECD 439. Based on the available information, the substance is classfied as “irritant” for skin irritation in accordance with UN GHS/CLP “Category 2”.

In BCOP study according to OECD 431 the mean in-vitro irritation score is 14.86. It caused irritating effects in an in-vitro EpiOcular study according to OECD 492. Based on the available information, the substance is classfied as “irritant" for eye irritation in accordance with UN GHS/CLP “Category 2”.