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

Skin irritation / corrosion

Administrative data

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

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:

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

Version / remarks:

18 June 2019, corrected 26 June 2020

Deviations:

no

Test guideline 2

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

Test guideline 3

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

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:

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

In vitro test system

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.

Test system

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.

Results and discussion

In vitro

Results

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%.

Any other information on results incl. tables

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%

Applicant's summary and conclusion

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.

.