Benzyldimethyl(octadecyl)ammonium chloride

Endpoint summary

Description of key information

Based on the results of the in vitro studies and taking a conservative approach, the test substance, C18 ADBAC, is considered to be non-irritant to skin, and corrosive to eyes.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From November 08, 2017 to November 10, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identity: Benzyldimethyl(octadecyl)ammonium chloride
Batch no: 15817
Composition: 100%
Cationic activity: 96.2%
Appearance: White solid
Dilution: Not diluted
Expiry date: 09/2019
Storage conditions: Room temperature, protect from humidity and water

Test system:

other: SkinEthic Reconstructed Human Epidermis

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Not specified

Justification for test system used:

The SkinEthicTM skin irritation test method is a validated alternative test procedure to the Draize Rabbit Skin irritation Test and endorsed by OECD Guideline 439.

Vehicle:

unchanged (no vehicle)

Details on test system:

SkinEthicTM Reconstructed Human Epidermis:

SkinEthicTM Reconstructed Human Epidermis is a three dimensional reconstructed human skin model comprising freshly isolated normal epidermal keratinocytes (NHEK: the major cellular component of skin), which are cultured at the air liquid interface on specialised culture inserts to develop a stratified differentiated epidermis characteristic of skin in vivo. The model consist of multiple layers of viable epithelial cells and a functional multi-layered stratum corneum with robust barrier function.

The SkinEthic skin irritation assay predicts and classifies the skin irritation potential of a chemical by assessment of its cytotoxic effect following its topical application directly to the skin surface and its subsequent penetration through the stratum corneum

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

16±2 mg

Duration of treatment / exposure:

42 ± 1 minutes

Duration of post-treatment incubation (if applicable):

42 ± 1 h

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

42 minutes exposure

Value:

96.1

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Interpretation of results:

other: CLP criteria not met

Remarks:

(not irritating)

Conclusions:

Under study conditions, the test substance was determined to be non-irritant to skin

Executive summary:

An in vitro study was conducted to determine the skin irritation potential of the test substance, C18 ADBAC (active: 96.2%), using Reconstructed Human Epidermis (RHE) Test Method, according to OECD Test Guideline 439, in compliance with GLP. On Day 18, SkinEhtic^TM tissues were pre-incubated overnight in the growth culture medium. The tissues were then placed in maintenance culture medium prior to application of the test substance. 10 µL distilled water followed by 16±2 mg of powder was applied to each of the 3 RHE tissue for 42±1 minutes, then rinsed and further incubated, post exposure, for 42±1 h at 37˚C, 5% CO2±1% and 95% humidified atmosphere. Cell viability was quantitatively measured (based on dehydrogenase conversion of the MTT dye (Methylthiazolyldiphenyl-tetrazolium bromide into a blue formazan salt) by determining the absorbance at 570 nm. Sterile Dulbecco’s Phosphate Buffered Saline (DPBS) and sodium dodecyl sulphate (5% in water) were used as negative and positive controls respectively. A test substance is considered to be an irritant to skin if the skin model viability after exposure and post-treatment incubation is ≤50%. The final percentage of viability obtained with the test substance was 96.1%, which was well above the 50% irritant threshold. Optical Density (OD) results obtained with negative and positive controls were within the acceptance limits; hence the study met all the acceptance/validity criteria. Under study conditions, the test substance was determined to be non-irritant to skin (Catoire, 2017).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

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:

From January 30, 2018 to Febraury 02, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 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:

Adopted october 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

human

Strain:

other: human-derived keratinocytes

Details on test animals or tissues and environmental conditions:

The EpiOcular tissue consists of normal, human-derived keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells. These cells are not transformed or transfected with genes to induce an extended life span. The EpiOcularTM tissues are cultured in specially prepared cell culture inserts with a porous membrane through which nutrients can pass to the cells. The tissue surface is 0.6 cm2.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 mg

Duration of treatment / exposure:

6 h

Duration of post- treatment incubation (in vitro):

18 h

Number of animals or in vitro replicates:

Duplicates

Irritation parameter:

other: % relative tissue viability

Run / experiment:

6 h

Value:

5.7

Vehicle controls validity:

not valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

% Viability positive control and test substance:

Treatment	Acceptance criterion	OD570nm	Viability %	Difference of viability	% Mean viability
Water (NC)	0.8≤OD≤2.5	1.653	100.3	0.5	100
	Difference of viability <20%	1.644	99.7
Methyl acetate	%mean viability < 50%	0.173	10.5	13.2	17.1
	Difference of viability <20%	0.391	23.7
Test substance	Difference of viability <20%	0.136	8.2	5.2	5.7
		0.051	3.1

Interpretation of results:

other: CLP criteria not met (Inconclusive)

Conclusions:

Under the conditions of the study, the test substance can be considered to be potentially irritating, however no prediction for classification (i.e., GHS class 1 and GHS class 2) can be made

Executive summary:

A study was conducted to determine the eye irritation potential of the test substance, C18 ADBAC (active: 96.2%), using the commercially available MatTek® Human Corneal Epithelium (EpiOcular) test method, according to OECD 492 Guideline, in compliance with GLP.EpiOcularTM tissues were placed in 1 mL of fresh maintenance medium (6-well plate). The tissues were incubated overnight at 37°C, 5% CO2, in humidified incubator and after 1 h, the medium was replaced by 1 mL of fresh medium for an overnight incubation at at 37°C, 5% CO2, in humidified incubator. The tissues in duplicate were exposed to single topical application of 50 mg neat test substance or 50 µL of reference substances (negative control: sterile water; positive control: methyl acetate) for 6 h, followed by a 25 minutes post-treatment immersion, and 18 h post-treatment incubation, prior to the MTT endpoint. After different treatment exposure period, tissues were transferred to 24 well plates containing MTT medium (1 mg/mL). After 3 h MTT incubation, the blue formazan salt formed was extracted with 2 mL of isopropanol per tissues for 2 to 3 h at room temperature or overnight at 4°C. The absorbance of the resulting coloured solution was measured at 570 nm. The viability of the tissues (which has been shown to directly correlate with the irritation potential) were assessed and compared to a negative control. The percentage viability obtained with the test substance was determined to be 5.7%, which is well below the threshold (i.e., ≤60%) indicating no prediction can be made. All the assay acceptance criteria were met and the study was considered valid. Under the conditions of the study, the test substance can be considered to be potentially irritating, however no prediction for classification (i.e., GHS class 1 and GHS class 2) can be made (Catoire, 2018).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin:

An in vitro study was conducted to determine the skin irritation potential of the test substance, C18 ADBAC (active: 96.2%), using Reconstructed Human Epidermis (RHE) Test Method, according to OECD Test Guideline 439, in compliance with GLP. On Day 18,SkinEhtic^TMtissues were pre-incubated overnight in the growth culture medium. The tissues were then placed in maintenance culture medium prior to application of the test substance. 10 µL distilled water followed by 16±2 mg of powder was applied to each of the 3 RHE tissue for 42±1 minutes, then rinsed and further incubated, post exposure, for 42±1 h at 37˚C, 5% CO2±1% and 95% humidified atmosphere. Cell viability was quantitatively measured (based on dehydrogenase conversion of the MTT dye (Methylthiazolyldiphenyl-tetrazolium bromide into a blue formazan salt) by determining the absorbance at 570 nm. Sterile Dulbecco’s Phosphate Buffered Saline (DPBS) and sodium dodecyl sulphate (5% in water) were used as negative and positive controls respectively. A test substance is considered to be an irritant to skin if the skin model viability after exposure and post-treatment incubation is ≤50%. The final percentage of viability obtained with the test substance was 96.1%, which was well above the 50% irritant threshold. Optical Density (OD) results obtained with negative and positive controls were within the acceptance limits; hence the study met all the acceptance/validity criteria. Under study conditions, the test substance was determined to be non-irritant to skin (Catoire, 2017).

Eye:

A study was conducted to determine the eye irritation potential of the test substance, C18 ADBAC (active: 96.2%), using the commercially available MatTek® Human Corneal Epithelium (EpiOcular) test method, according to OECD 492 Guideline, in compliance with GLP.EpiOcularTM tissues were placed in 1 mL of fresh maintenance medium (6-well plate). The tissues were incubated overnight at 37°C, 5% CO2, in humidified incubator and after 1 h, the medium was replaced by 1 mL of fresh medium for an overnight incubation at at 37°C, 5% CO2, in humidified incubator. The tissues in duplicate were exposed to single topical application of 50 mg neat test substance or 50 µL of reference substances (negative control: sterile water; positive control: methyl acetate) for 6 h, followed by a 25 minutes post-treatment immersion, and 18 h post-treatment incubation, prior to the MTT endpoint. After different treatment exposure period, tissues were transferred to 24 well plates containing MTT medium (1 mg/mL). After 3 h MTT incubation, the blue formazan salt formed was extracted with 2 mL of isopropanol per tissues for 2 to 3 h at room temperature or overnight at 4°C. The absorbance of the resulting coloured solution was measured at 570 nm. The viability of the tissues (which has been shown to directly correlate with the irritation potential) were assessed and compared to a negative control. The percentage viability obtained with the test substance was determined to be 5.7%, which is well below the threshold (i.e., ≤60%) indicating no prediction can be made. All the assay acceptance criteria were met and the study was considered valid. Under the conditions of the study, the test substance can be considered to be potentially irritating, however no prediction for classification (i.e., GHS class 1 and GHS class 2) can be made (Catoire, 2018).

In absence of further testing with the test substance and considering the very low percentage viability (i.e., 5.7%) observed in the EpiOcular test together with the corrosive classifications of structurally similar ADBACs, indicates that a similar corrosive conclusion for eye can be considered for the test substance, as a conservative approach (worst case).

Justification for classification or non-classification

Skin irritation:

Based on the results of the in vitro skin irritation study, test substance, C18 ADBAC, is concluded not to warrant classification for skin irritation endpoint, according to EU CLP criteria (Regulation 1272/2008/EC).

Eye irritation:

Based on the results of in vitro assay and as a conservative approach, the test substance, C18 ADBAC, is considered to be corrosive to the eyes and warrants a classification as ‘Eye Damage 1: H318- causes serious eye damage’ according to EU CLP criteria (Regulation 1272/2008/EC).