anhydro-D-glucitol, 1-deoxy-1-(methylamino)-, N-[C8-10(even numbered) acyl] derivs.

Endpoint summary

Description of key information

OECD 439: irritating

OECD 431: corrosive

OECD 492: irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019

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

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Remarks:

EPISKIN SM™

Source species:

human

Details on animal used as source of test system:

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: 19-EKIN-046 for main experiment, 19-EKIN-041 for killed tissue controls)
1x 12-well assay plate
1x flask of sterile maintenance medium (basic medium for incubations, Lot: 19MAIN3052)
1x flask of sterile assay medium (basic medium for use in MTT assays, Lot: 19ESSC048)
Validity controls as provided by the supplier (SkinEthic):

Vehicle:

unchanged (no vehicle)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Duration of treatment / exposure:

3min, 60min, 4h

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3min

Value:

81.2

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60min

Value:

75.4

Negative controls validity:

other: not relevant

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

4h

Value:

30.6

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive 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). The test item showed no water-colouring potential.
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, excepted for the 60 minute time point (1,651). This do not influence the outcome of the test because the corrosivity was shown with the 4 hours’ time point and the outcome of the test would not change. The mean relative tissue viability (% negative control) of the positive control was  20% (3.2%) after 4 h treatment. The maximum inter tissue viability difference of replicate tissues of all dose groups was  30% (0.1% - 25.3%).

Interpretation of results:

Category 1B (corrosive) based on GHS criteria

Conclusions:

In this study under the given conditions the test item showed corrosive effects. The relative mean tissue viability after 4 h treatment was decreased below 35%. Additionally, the relative mean tissue viability was decreased to not more than 35% after 60 min treatment. The test item is therefore classified as “corrosive“ in accordance with a combination of optional sub-categories 1B and 1C.

Executive summary:

In the present study the skin corrosivity potential of cyclic Glucamide C8-C10 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 minand 4 hexposure 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). The test item showed no water-colouring potential.

The test item showed corrosive effects. The mean relative tissue viability (% negative control) was reduced below 35% (30.6%, NSMTT-corrected) after 4 h treatment and to not more than 35% (75.4%, NSMTT-corrected) after 60 min treatment. Relative mean tissue viability was reduced to 81.2%, NSMTT-correctedafter 3 min treatment.

The controls confirmed the validity of the study. The mean OD₅₇₀of the two negative control tissues was between 0.6 and 1.5 for each exposure period, excepted for the 60 minute time point (1,651). This do not influence the outcome of the test because the corrosivity was shown with the 4 hours’ time point and the outcome of the test would not change. The mean relative tissue viability (% negative control) of the positive control was£ 20% (3.2%) after 4 h treatment. The maximum inter tissue viability difference of replicate tissues of all dose groups was£ 30% (0.1% - 25.3%).

In this study under the given conditions the test item showed corrosive effects. The relative mean tissue viability after 4 h treatment was decreased below 35%. Additionally, the relative mean tissue viability was decreased to not more than35%after 60 min treatment. The test item is therefore classified as “corrosive“ in accordance witha combination of optional sub-categories 1B and 1C.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (corrosive)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-06-28 to 2019-07-17

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:

Name: cyclic Glucamide C8-C10
Chemical Name: Amides, C8-C10, N-(1-deoxy-D-glucitol-1-yl)-N-methyl, dehydrated (CAS name)
Batch No.: BP 1c (reactor mix)
CAS No 2290526-77-9
Compounds: UVCB
main compounds: C15H29NO5 (C8-acyl-derivative)
C17H33NO5 (C10-acyl-derivative)
8.5% unidentified components
Aggregate State at RT: highly viscous mass (20°C)
Colour: brown
Storage Conditions: room temperature, dry
Expiry Date: 28 March 2021
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.

Amount / concentration applied:

50 µL

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:

4.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

Value Cut off pass/fail
Mean Absolute OD570 nm NK 1.924 0.8 < NK < 2.5 pass
Mean Relative Viability PC [%] 47.9 < 50% pass
Max. Difference of % Viability [%] 1.4 < 20% pass

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. The killed tissue controls were performed for quantitative correction of results.

NSMTT [%] = [(OD_KT- OD_KU)/OD_NC] * 100 = [(0.042-0.093)/1.877] = -2.72%

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

NSMTT1 [%] = [meanOD_KT1- OD_KU)/OD_NC] * 100 = [(0.042-0.093)/1.877] = -2.72%

NSMTT2 [%] = [meanOD_KT2- OD_KU)/OD_NC] * 100 = [(0.042-0.093)/1.877] = -2.72%

NSMTT1 - NSMTT2 = 0%

NSMTT was ≤ 60% (-2.72% NSMTT) 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 = 1.6% - (-2.72%) ≈ 4.2%

The mixture of 50 µL 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 cyclic Glucamide C8-C10

Name	Negative Control		Positive Control		Test Item
Replicate Tissue	1	2	1	2	1	2
Absolute OD570	1.921	1.887	0.939	0.944	0.074	0.075
	1.953	1.933	0.940	0.956	0.077	0.076
Mean Absolute OD570	1.924****		0.945		0.076
OD570(Blank Corrected)	1.874	1.840	0.893	0.898	0.028	0.029
	1.907	1.887	0.894	0.910	0.031	0.029
Mean OD570of the Duplicates (Blank Corrected)	1.891	1.864	0.893	0.904	0.029	0.029
Total Mean OD570of the 2 Replicate Tissues (Blank Corrected)	1.877*		0.899		0.029
TODTT	-		-		0.079
SD of Mean OD570of the Duplicates (Blank Corrected)	0.019		0.008		0.000
Relative Tissue Viability [%]	100.7	99.3	47.6	48.2	1.6	1.5
Relative Tissue Viability Difference [%]^***	1.4		0.6		0.0
Mean Relative Tissue Viability [%]	100.0		47.9**		1.6
Mean Relative Tissue Viability [%] - NSMTT corrected	-		-		4.2

^*              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	0.146	0.128	0.086	0.087	1.921		1.887
	0.151	0.131	0.091	0.090	1.953		1.933
OD570(Blank Corrected)	0.100	0.081	0.040	0.041	1.874		1.840
	0.104	0.085	0.045	0.044	1.907		1.887
Mean OD570of the Duplicates (Blank Corrected)	0.102	0.083	0.042	0.042	1.891		1.864
Total Mean OD570 of the 2 Replicate Tissues (Blank Corrected)	0.093		0.042		1.877
SD of Mean OD570of the Duplicates (Blank Corrected)	0.014		0.000		0.019
NSMTT [%]	-2.7				-
Relative Tissue Viability [%]	-				100.7	99.3
Relative Tissue Viability Difference [%]	-				1.4
Mean Relative Tissue Viability [%]	-				100.0

Conclusions:

In this study under the given conditions the test item showed irritant effects.

Executive summary:

In the present study cyclic Glucamide C8-C10 was applied topically to the EpiOcular ^TM tissue for 30 min followed by 12 min post-soaking incubation after removal of the test item. After a 120 min 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 µL test item per 1 mL MTT medium showed reduction of MTT as compared to the solvent. The mixture turned blue/purple. The killed tissue controls were performed for quantitative correction of results.

NSMTT [%] = [(OD_KT- OD_KU)/OD_NC] * 100 = [(0.042-0.093)/1.877] = -2.72%

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

NSMTT1 [%] = [meanOD_KT1- OD_KU)/OD_NC] * 100 =[(0.042-0.093)/1.877] = -2.72%

NSMTT2 [%] = [meanOD_KT2- OD_KU)/OD_NC] * 100 =[(0.042-0.093)/1.877] = -2.72%

NSMTT1 - NSMTT2 =0%

NSMTT was ≤ 60% (-2.72% NSMTT) 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 = 1.6% - (-2.72%) ≈ 4.2%

The mixture of 50 µL 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 irritant effects. The mean relative tissue viability (% negative control) was ≤ 60% (4.2% NSMTT-corrected).

The controls confirmed the validity of the study. The mean absolute OD₅₇₀ of the two negative control tissues was > 0.8 and < 2.5 (1.924). The mean relative tissue viability (% negative control) of the positive control was < 50% (47.9%). The maximum inter tissue difference of replicate tissues of all dose groups was < 20% (1.4%).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the experimental results, the substance is classified as skin corrosive "Cat. 1B or 1C" accoridng to CLP. The substance also induce irritation effects and classified as causing serious eye damage "Cat. 1" based on skin corrosion property worst-case scenario.