Quaternary ammonium compounds, C12-14-alkyltrimethyl, Me sulfates

Endpoint summary

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Remarks:

In vitro skin corrosion test with EpiDerm (EPI-200))

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From July 26, 2017 to July 27, 2017

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:

adopted 29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Deviations:

no

GLP compliance:

yes

Test system:

human skin model

Remarks:

EpiDerm Skin Model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts. Recommended test system in international guidelines (OECD and EC)

Vehicle:

unchanged (no vehicle)

Details on test system:

Test system specifications: Three-dimensional human skin model, comprising a reconstructed epidermis with a functional stratum corneum was supplied by MatTek In Vitro Life ScienceLaboratories, Bratislava, Slovak Republic.

System conditions: Human keratinocytes are used to construct the epithelium. Multiple layers of viable epithelial cells are present under a functional stratum corneum. The stratum corneum is multi-layered with the necessary lipid profile to produce a functional barrier. The containment properties of the model prevent the passage of material around the stratum corneum to the viable model tissue. The skin model was supplied free of contamination with bacteria, mycoplasma and fungi.

Assessment of MTT Interacting substances:In order to assess the potential non-specific reduction of the test article, 25 mg of test substance was added to 1 mL of 1.0 mg/mL MTT and the colour change was assessed after incubation for 60 minutes at 37±1°C, 5±1% CO2, 95% RH. There was no change in colour therefore the test article did not interact with MTT.

Assessment for colour interference: 25 mg of test substance was added to both 0.3 mL deionized water and isopropanol before being incubated for 60 minutes at 37±1°C, 5±1% CO2, 95% RH. The solution did not become coloured, therefore the test article was deemed not to have the potential to stain the tissue.

Application of test and control substances: On the day of receipt EpiDerm tissues were transferred to refrigerator at 2 to 8°C and stored overnight. The next day, approximately 1.5 hours before starting the assay, the tissues were prepared for treatment in labelled 6-well plates. The test was performed on a total of four tissues per test substance, negative and positive control, out of which two were used for a 3 minute application and two tissues were used for a 1 hour application. Approximately 25 mg of the test substance was applied to the tissue, which was pre-moistened with 25 μL water to ensure good contact with the tissue surface. Further tissues were concurrently treated with 50 μL distilled water (negative control) and with 50 μL 8M potassium hydroxide (positive control). After the 3 minute or 1-hour contact periods, the tissues were washed with phosphate buffered saline (PBS) to remove residual material. The rinsed tissues were kept in 24-well plates (holding plates) until all tissues were dosed and rinsed. The positive control material was a direct MTT reducer, therefore additional positive controls were performed. The positive control article was applied to two freeze-killed tissues (thawed on the day of treatment) per exposure time. Cell viability measurements: 1 mg/mL MTT-medium and were incubated for 3 hours (37±1°C, 5±1% CO2, 95% RH). After incubation any resultant colour was extracted with 2 mL isopropanol overnight. The optical density of each resultant extract was determined spectrophotometrically at 570 nm and cell viability was calculated for each tissue as a percentage of the mean of the negative control tissue. Skin corrosivity potential of the test substance was classified according to the remaining cell viability obtained after test substance treatment with either of the two treatment times.

Cell viability measurements: Once all tissues had been rinsed, they were transferred to wells containing 300 μL of 1 mg/mL MTT-medium and were incubated for 3 hours (37±1°C, 5±1% CO2, 95% RH). After incubation any resultant colour was extracted with 2 mL isopropanol overnight. The optical density of each resultant extract was determined spectrophotometrically at 570 nm and cell viability was calculated for each tissue as a percentage of the mean of the negative control tissue. Skin corrosivity potential of the test article was classified according to the remaining cell viability obtained after test article treatment with either of the two treatment times.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 mg pre-moistened with 25 μL water to ensure good contact with the tissue surface

Duration of treatment / exposure:

3 minutes and 1 h

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Remarks:

cytotoxicity

Run / experiment:

3 min treatment

Value:

ca. 58

Vehicle controls validity:

valid

Negative controls validity:

not valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Remarks:

cytotoxicity

Run / experiment:

1 h treatment

Value:

ca. 20.4

Vehicle controls validity:

valid

Negative controls validity:

not valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

-The test substance was checked for color interference in aqueous conditions and possible direct MTT reduction by adding the test substance to MTT medium. Because the solutions did not turn blue / purple nor a blue / purple precipitate was observed it was concluded that the test substance did not interfere with the MTT endpoint.

The mean absorption at 570 nm measured after treatment with the test substance and controls were as follows:
1) Negative control 1.103 (3 minute application) and 1.115 (1 h application)
2) Test substance 0.634 (3 minute application) and 0.228 (1 h application)
3) Positive control 0.312 (3 minute application) and 0.186 (1 h application)

- The mean tissue viability obtained after 3 minute and 1 h treatments with the test substance compared to the negative control tissues. Skin corrosion is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after the 3 minute and 1 h treatments with the test substance compared to the negative control tissues was 58% and 20.4% respectively. Because the mean relative tissue viability for the test substance was not below 50% after 3 minutes treatment and not below 15% after 1 h treatment the test substance is considered to be not corrosive.

- The OD values for the negative controls were between 0.8 and 2.8, and were within observed historical ranges. The mean relative tissue viability following the 1 h exposure to the positive control was -0.4%.


- In the range of 20 - 100% viability the coefficient of variation between tissue replicates was <30%, indicating that the test system functioned properly. The coefficient of variation between tissue replicates treated with the test substance was 29.97% for the 3-minute treatment, which is above acceptance criteria. Since all individual viabilities were >50%, the test outcome was considered valid.

Interpretation of results:

other: CLP criteria not met

Conclusions:

Under the study conditions, the test substance was determined to be non corrosive to skin based on human three dimensional epidermal model (EpiDerm (EPI-200)).

Executive summary:

A study was conducted to determine the in vitro skin corrosion potential of the test substance, Quaternary ammonium compounds, C12-14 alkyltrimethyl, Me sulfates, according to OECD Guideline 431 and EU Method B.40 bis, in compliance with GLP. The test substance was checked for colour interference in aqueous conditions and possible interference with MTT reduction by adding the test substance to MTT medium. Because the solutions did not turn blue/purple nor a blue/purple precipitate was observed, it was concluded that the test substance did not interfere with the MTT. Duplicate tissue samples were exposed to approximately 25 mg of the test substance, which was pre-moistened with 25 μL water to ensure good contact with the tissue surface. Post treatment, a determination of the cytotoxic effect was performed. Cytotoxicity was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Skin corrosion was expressed as the remaining cell viability after exposure to the test substance. The positive control had a mean relative tissue viability of -0.4% after the 1 h exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD Guideline 431 and the laboratory historical control data range. In the range of 20 - 100% viability the coefficient of variation between tissue replicates was <30%, indicating that the test system functioned properly. The relative mean tissue viability obtained after 3 min and 1 h treatment periods with the test substance compared to the negative control tissues were 58% and 20.4%, respectively. Because the mean relative tissue viability for the test substance was above 50 and 15% after 3 min and 1 h respectively, so the test substance is considered to be non-corrosive. Under the study conditions, the test substance was determined to be non-corrosive to skin based on human three dimensional epidermal model (Payne, 2017). 

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From December 04, 2017 to December 08, 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

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Not specified

Justification for test system used:

Three-dimensional human skin model, comprising a reconstructed epidermis with a functional stratum corneum Human keratinocytes are used to construct the epithelium. Multiple layers of viable epithelial cells are present under a functional stratum corneum. The stratum corneum is multi-layered with the necessary lipid profile to produce a functional barrier. The containment properties of the model prevent the passage of material around the stratum corneum to the viable model tissue. The skin model is supplied free of contamination with bacteria, mycoplasma and fungi. Recommended test system in international guidelines (OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

Specification: EpiDermTM SIT (EPI-200) three-dimensional human skin model, comprising a reconstructed epidermis with a functional stratum corneum, supplied by MatTek In Vitro Life Science Laboratories, Bratislava, Slovak Republic.

General model conditions: Human keratinocytes are used to construct the epithelium. Multiple layers of viable epithelial cells are present under a functional stratum corneum. The stratum corneum is multi-layered with the necessary lipid profile to produce a functional barrier. The containment properties of the model prevent the passage of material around the stratum corneum to the viable model tissue. The skin model is supplied free of contamination with bacteria, mycoplasma and fungi.

Functional Model Conditions: The magnitude of viability is quantified using MTT and measuring the optical density (OD) of the extracted (solubilised) dye from each tissue. The negative control tissue has been shown to be stable in culture for the duration of the test exposure period. The stratum corneum is sufficiently robust to resist the rapid penetration of certain cytotoxic marker chemicals (e.g. 5% SDS). The tissue has been shown to demonstrate reproducibility over time.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 mg

Duration of treatment / exposure:

1 h

Duration of post-treatment incubation (if applicable):

41 h 50 minutes

Number of replicates:

Triplicate

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 h

Value:

3.3

Vehicle controls validity:

not valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Cell viability measurement:

Substance	Tissue Replicate	OD570			Corrected Mean	% Relative Survival		Standard Deviation	Coefficient of Variance
		Aliquot 1	Aliquot 2	Mean		Tissue	Mean
Negative control	A	1.172	1.160	1.166	1.166	102.1	100	2.17	2.17
	B	1.090	1.143	1.116	1.116	97.8
	C	1.127	1.157	1.142	1.142	100.1
Test substance	A	0.037	0.037	0.037	0.037	3.2	3.3	0.31	9.59
	B	0.041	0.041	0.041	0.041	3.6
	C	0.034	0.041	0.034	0.034	3.0
Positive control	A	0.035	0.043	0.039	0.039	3.4	3.2	0.26	8.17
	B	0.033	0.033	0.033	0.033	2.9
	C	0.037	0.039	0.038	0.038	3.3
Blank		0	-0.00

Interpretation of results:

other: Category 2 (irritant) based on CLP criteria

Conclusions:

Under the study conditions, based on the percentage of viability of 3.3%, the test substance was concluded to be irritant to the skin.

Executive summary:

A study was conducted to determine the skin irritation potential of the test substance, Quaternary ammonium compounds, C12-14 alkyltrimethyl, Me sulfates, using Reconstructed Human Epidermis (RhE) Test Method, according to OECD Guideline 439, in compliance with GLP. The test substance was checked for colour interference in aqueous conditions and possible interference with MTT reduction by adding the test substance to MTT medium. Because the solutions did not turn blue/purple nor a blue/purple precipitate was observed, it was concluded that the test substance did not interfere with the MTT. Three tissues of the human skin model EpiDermTM were treated with the test substance, positive or negative control by application of 25 mg (tissues wetted with 25 µLwith PBS) for 60 min (25 min at room temperature and 35 min at 37°C, 5% CO2) and 42 h post incubation period. PBS was used as negative control and 5% of sodium dodecyl sulphate solution in PBS was used as positive control. Subsequently, viability of the tissues was assessed and compared to the negative control. The absolute mean of the positive and negative control tissues was within the acceptance limits of the guideline and the laboratory historical control data range indicated that the test system functioned properly. Under the study conditions, based on the percentage of viability of 3.3%, the test substance was concluded to be irritant to the skin. (Dreher, 2018). 

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:

From October 16, 2017 to October 19, 2017

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)

Deviations:

no

Principles of method if other than guideline:

This study was performed in order to evaluate the potential of test substance in a Reconstructed human Cornea-like Epithelium (RhCE) model in an in vitro study. The EpiOcularTM Eye Irritation Test (EIT) predicts the acute ocular irritation potential of chemicals by measurement of irreversible tissue damage caused by cytotoxic effects in the human cornea model. Within a testing strategy, the EpiOcularTM EIT is used as a replacement of the Draize Eye Irritation Test. It is utilized for the classification and labelling of chemicals concerning their eye irritation potential. The EpiOcularTM EIT is intended to differentiate substances that are “not eye irritant” from those that require labelling as either GHS category 1 or 2 for serious eye damage or eye irritation potential. Eye irritant materials are identified by their ability to produce a decrease in cell viability as determined. The cell viability is measured by dehydrogenase conversion of MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide), present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percentage reduction of cell viability in comparison with untreated negative controls is used to predict the eye irritation potential.

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:

1) Tissue 51.7 mg
2) Tissue 49.8 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:

1.1

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:

Designation	Positive Control	Test substance
% Viability (Tissue 1)	39.90%	1.3%
% Viability (Tissue 2)	43.00%	0.9%
% Viability Mean	41.40%	1.1%

Interpretation of results:

other: CLP criteria not met (Inconclusive)

Conclusions:

Under the study conditions, based on the percentage viability of 1.1%, the test substance was considered either eye irritant or inducing serious eye damage to the human eye.

Executive summary:

A study was conducted to determine the eye irritation potential of the test substance, Quaternary ammonium compounds, C12-14 alkyltrimethyl, Me sulfates, using Reconstructed human Cornea-like Epithelium (RhCE) test method, according to OECD Guideline 492, in compliance with GLP. Two tissues of the EpiOcularTM tissue model were treated with the test substance, positive or negative control. Tissues were pre-wetted with 20 μL of PBS (Sterile Phosphate Buffered Saline) prior to topical application of approximately 50±2 mg of neat test substance. Sterile demineralised water was used as negative control and methyl acetate as positive control. After 6 h exposure on the surface of EpiOcularTM reconstructed ocular epithelium and 18 h post-incubation time, the viability of the tissues was assessed and compared to a negative control. The MTT viability test readings were conducted at 570 nm without reference filter. Under the study conditions, based on the percentage viability of 1.1%, the test substance was concluded to be either eye irritant or inducing serious eye damage to the human eye (Andres, 2018).

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Remarks:

The Bovine Corneal Opacity and Permeability Assay (BCOP)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

(adopted 26 July 2013)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes

Species:

other: Bovine

Details on test animals or tissues and environmental conditions:

Test System: Bovine eyes were used as soon as possible after slaughter.
Rationale: In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing (1-6). As a consequence a validated and accepted in vitro test for eye irritation should be performed before in vivo tests are conducted. One of the proposed validated in vitro eye irritation tests is the Bovine Corneal Opacity and Permeability (BCOP) test.

Source: Bovine eyes from young cattle were obtained from the slaughterhouse, where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
Transport: Eyes were collected after slaughter, completely immersed in Hanks’ Balanced Salt Solution (containing penicillin at 100 IU/mL and streptomycin at 100 μg/mL) in a suitably sized container and transported on the same day to the testing facility.

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

750 µL (20%w/v test substance in 0.9% sodium chloride solution) at 32°C

Duration of treatment / exposure:

4 h

Observation period (in vivo):

-

Duration of post- treatment incubation (in vitro):

Permeability endpoint, 1 mL of sodium fluorescein (5 mg/mL solution) was added into the anterior chamber and the corneas were incubated in the vertical position for 1 h and 25 minutes.

Number of animals or in vitro replicates:

3

Details on study design:

Preparation of Corneas:
On arrival at the test facility the eyes were carefully examined for defects including increased opacity, scratches and neovascularisation. Only corneas free from such defects were used.

Cornea Selection and Opacity Reading:
Upon arrival at the test facility, the corneas were excised from the eyes and loaded onto the specifically designed holders. Both chambers of each holder were filled with pre-warmed Minimal Essential Medium (MEM), with the posterior chamber filled first, ensuring that no bubbles were formed. The holders were incubated at 32±1°C for 1 hour. After the incubation, the media was removed from both the anterior and posterior chambers. Fresh media was added to the posterior chamber first and then the anterior chamber (this media replacement order ensured the cornea retained its natural curvature as much as possible). The opacity of each cornea was measured using an opacitometer. Any corneas found to have scratches or increased neovascularization or an opacity of >7 opacity units when examined prior to treatment were discarded.

Treatment of Corneas and Opacity Measurements:
A volume of 750 μL of the test susbtance formulation was applied to each of three corneas followed by a four hour incubation at 32°C. After this incubation, each cornea was washed with media containing phenol red (as a pH indicator) until this indicator
showed no pH effect occurring (and demonstrating that the test article had been removed successfully). The corneas were then washed once in media without phenol red, corneal opacity was measured and the anterior chamber emptied. For the permeability endpoint, 1 mL of sodium fluorescein (5 mg/mL solution) was added into the anterior chamber and the corneas were incubated in the vertical position for 1 hour and 25 minutes. Following this period, the media in the posterior chamber was
removed and held in a labelled tube. Three 350 μL aliqots of this media (per cornea) were analysed for optical density at 490 nanometers (OD490).

A volume of 750 μL of the negative or positive control was similarly applied to further groups of three corneas. These groups were subject to the procedures detailed above. It was noted during the permeability assessment that a small gap was present
between the anterior and posterior chambers, for one replicate of the negative control. This replicate was subsequently excluded from all analysis, with the mean value from the remaining two negative control replicates being used.


Test substance formulation: The test substance was ground to a fine powder, and tested as a 20% w/v solution in 0.9% sodium chloride solution.

Irritation parameter:

in vitro irritation score

Run / experiment:

4 h exposure

Value:

23

Vehicle controls validity:

valid

Negative controls validity:

not valid

Positive controls validity:

valid

Remarks on result:

other: Inconcluisve

Other effects / acceptance of results:

The mean corrected opacity reading for the test substance was 23.0. The mean corrected opacity reading for the negative control was 0.0. This result was calculated using 2 negative control replicates. The mean corrected opacity reading for the positive control was 62.0. The corneas treated with the test substance were noted to be slightly cloudy and wrinkled and the corneas treated with the positive control were completely clouded over following treatment.

The mean group corrected optical density for the test substance was 0.013. The mean group corrected optical density for the negative control was 0.000. This result was calculated using 2 negative control replicates. The mean group corrected optical density for the positive control was 0.744.

Corneal opacity:

Test chemical	Cornea number	Initial opacity	Post incubation opacity	Change in opacity	Mean change in opacity	Corrected opacity	Mean corrected opacity
Negative control	9	0	1	1	2.00	-1.0	0
Negative control	10	0	3	3		1
Negative control	11*	-	2*	2*		0.0
Test substance	4	0	29	29	NA	27.0	23.0
Test substance	6	0	20	20		18.0
Test substance	8	0	26	26		24.0
Positive control	1	0	58	58	NA	56.0	62.0
Positive control	25	-1	68	69		67.0
Positive control	26	-1	64	65		63

*Cornea 11 excluded from analysis. Opacity values shown are the mean of corneas 9 and 10.

Corneal permeability:

Test chemical	Cornea number	Mean blank OD490	OD490	Corrected OD490	Mean corrected OD490	Final corrected OD490	Mean Group Corrected OD490
Negative control	9	0.038	0.044	0.01	0.010	-0.004	0
Negative control	10		0.052	0.014		0.004
Negative control	11*		0.048	0.01		0.000
Test substance	4	NA	0.062	0.025	NA	0.014	0.013
Test substance	6		0.058	0.020		0.010
Test substance	8		0.062	0.025		0.014
Positive control	1	NA	0.511	0.473	NA	0.463	0.744
Positive control	25		0.846	0.809		0.799
Positive control	26		1.019	0.981		0.971

*Cornea 11 excluded from analysis. Values shown are the mean of corneas 9 and 10.

Calculated IVIS:

Test chemical	Mean Opacity	Mean Permeability	IVIS (Mean Opacity + (15 x Mean Permeability))
Negative control	0	0	0
Test substance	23	0.013	23.19
Positive control	62.0	0.744	73.16

NA: Not applicable

Interpretation of results:

other: Inconclusive

Conclusions:

Under the study conditions, eye irritation potential of the test substance was determined to be inconclusive based on bovine corneal opacity and permeability test (IVIS score - 23.0).

Executive summary:

A study was conducted to determine the in vitro eye damage potential of the test substance, Quaternary ammonium compounds, C12-14 alkyltrimethyl, Me sulfates, according to OECD Guideline 437, in compliance with GLP. The eye damage potential of the test substance was tested through topical application for 4 h. The test substance was applied as fomulation 20% w/v solution in 0.9% sodium chloride solution (750 µL) directly on top of the freshly isolated bovine cornea sample. The negative control responses for opacity and permeability was less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean corrected opacity reading for the test substance was 23, and the mean corrected opacity reading for the positive control was 62.0. The mean group corrected optical density for the test substance was 0.013 and the mean group corrected optical density for the positive control was 0.744. The mean in vitro irritancy score of the positive control (Imidazole) was 62.0 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. The test substance produced IVIS score of 23 after 4 h of treatment. Under the study conditions, eye irritation potential of the test substance was determined to be inconclusive based on bovine corneal opacity and permeability test (Payne, 2017).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

Study 1:

A study was conducted to determine the in vitro skin corrosion potential of the test substance, Quaternary ammonium compounds, C12-14 alkyltrimethyl, Me sulfates, according to OECD Guideline 431 and EU Method B.40 bis, in compliance with GLP. The test substance was checked for colour interference in aqueous conditions and possible interference with MTT reduction by adding the test substance to MTT medium. Because the solutions did not turn blue/purple nor a blue/purple precipitate was observed, it was concluded that the test substance did not interfere with the MTT. Duplicate tissue samples were exposed to approximately 25 mg of the test substance, which was pre-moistened with 25 μL water to ensure good contact with the tissue surface. Post treatment, a determination of the cytotoxic effect was performed. Cytotoxicity was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Skin corrosion was expressed as the remaining cell viability after exposure to the test substance. The positive control had a mean relative tissue viability of -0.4% after the 1 h exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD Guideline 431 and the laboratory historical control data range. In the range of 20 - 100% viability the coefficient of variation between tissue replicates was <30%, indicating that the test system functioned properly. The relative mean tissue viability obtained after 3 min and 1 h treatment periods with the test substance compared to the negative control tissues were 58% and 20.4%, respectively. Because the mean relative tissue viability for the test substance was above 50 and 15% after 3 min and 1 h respectively, so the test substance is considered to be non-corrosive. Under the study conditions, the test substance was determined to be non-corrosive to skin based on human three dimensional epidermal model (Payne, 2017). 

Study 2:

A study was conducted to determine the skin irritation potential of the test substance, Quaternary ammonium compounds, C12-14 alkyltrimethyl, Me sulfates, using Reconstructed Human Epidermis (RhE) Test Method, according to OECD Guideline 439, in compliance with GLP. The test substance was checked for colour interference in aqueous conditions and possible interference with MTT reduction by adding the test substance to MTT medium. Because the solutions did not turn blue/purple nor a blue/purple precipitate was observed, it was concluded that the test substance did not interfere with the MTT. Three tissues of the human skin model EpiDermTM were treated with the test substance, positive or negative control by application of 25 mg (tissues wetted with 25 µLwith PBS) for 60 min (25 min at room temperature and 35 min at 37°C, 5% CO2) and 42 h post incubation period. PBS was used as negative control and 5% of sodium dodecyl sulphate solution in PBS was used as positive control. Subsequently, viability of the tissues was assessed and compared to the negative control. The absolute mean of the positive and negative control tissues was within the acceptance limits of the guideline and the laboratory historical control data range indicated that the test system functioned properly. Under the study conditions, based on the percentage of viability of 3.3%, the test substance was concluded to be irritant to the skin. (Dreher, 2018). 

Eye irritation

Study 1:

A study was conducted to determine the in vitro eye damage potential of the test substance, Quaternary ammonium compounds, C12-14 alkyltrimethyl, Me sulfates, according to OECD Guideline 437, in compliance with GLP. The eye damage potential of the test substance was tested through topical application for 4 h. The test substance was applied as fomulation 20% w/v solution in 0.9% sodium chloride solution (750 µL) directly on top of the freshly isolated bovine cornea sample. The negative control responses for opacity and permeability was less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean corrected opacity reading for the test substance was 23, and the mean corrected opacity reading for the positive control was 62.0. The mean group corrected optical density for the test substance was 0.013 and the mean group corrected optical density for the positive control was 0.744. The mean in vitro irritancy score of the positive control (Imidazole) was 62.0 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. The test substance produced IVIS score of 23 after 4 h of treatment. Under the study conditions, eye irritation potential of the test substance was determined to be inconclusive based on bovine corneal opacity and permeability test (Payne, 2017).

Study 2:

A study was conducted to determine the eye irritation potential of the test substance, Quaternary ammonium compounds, C12-14 alkyltrimethyl, Me sulfates, using Reconstructed human Cornea-like Epithelium (RhCE) test method, according to OECD Guideline 492, in compliance with GLP. Two tissues of the EpiOcularTM tissue model were treated with the test substance, positive or negative control. Tissues were pre-wetted with 20 μL of PBS (Sterile Phosphate Buffered Saline) prior to topical application of approximately 50±2 mg of neat test substance. Sterile demineralised water was used as negative control and methyl acetate as positive control. After 6 h exposure on the surface of EpiOcularTM reconstructed ocular epithelium and 18 h post-incubation time, the viability of the tissues was assessed and compared to a negative control. The MTT viability test readings were conducted at 570 nm without reference filter. Under the study conditions, based on the percentage viability of 1.1%, the test substance was concluded to be either eye irritant or inducing serious eye damage to the human eye (Andres, 2018).

Justification for classification or non-classification

Skin irritation:

Based on the results of the in vitro skin irritation studies, the test substance, Quaternary ammonium compounds, C12-14 alkyltrimethyl, Me sulfates, warrants a classification as Skin Irritant 2 (H315: Causes skin irritation) according to EU CLP (1272/2008/EC) criteria.

 

Eye irritation:

Based on weight of evidence from the results of in vitro assays and as a conservative approach, the test substance, Quaternary ammonium compounds, C12 -14 alkyltrimethyl, Me sulfates is considered to be irritant to the eyes and warrants a classification as Eye irritant 2 (H319 - causes serious eye irritation) according to EU CLP (1272/2008/EC) criteria.