Cyclohexanaminium, N,N,N-trimethyl-, sulfate (2:1)

Endpoint summary

Description of key information

Skin Irritation (EpiDerm): irritating (aqueous sollution 50 wt%)
Skin Irritation (EpiDerm): not corrosive (pure substance)
Eye Irritation (BCOP + EpiOcular): not irritating (aqueous sollution 50 wt%)
Eye Irritation (EpiOcular): irritating (pure substance)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation / corrosion

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Species:

other: in vitro

Strain:

other: in vitro

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 μL (corrosion test) or 30 μL (irritation test) of the undiluted test substance

Duration of treatment / exposure:

Corrosion test: 3 min and 1 h
Irritation test: 1h followed by a 42-hours post.incubation period

Details on study design:

The EpiDermTM model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi layered, 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 EpiDermTM tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs, 10 mm ∅) and commercially available as kits (EpiDerm™ 200), containing 24 tissues on shipping agarose.

Corrosion test:
From the day of arrival in the laboratory, tissues were kept in the refrigerator. At least 1 hour but not more than 1.5 hours before test-substance application, tissues were transferred to 6- well plates with 0.9 mL assay medium and preconditioned in the incubator at 37°C. The preincubation medium was replaced with fresh medium immediately before application.
Two tissues per exposure time (3 minutes at room temperature or 1 hour in the incubator, as a rule) and test group (test material, negative control and positive control; 12 tissues per test) were used.
Fifty microliter (50 μL) of the undiluted liquid test substance was applied using a pipette. A nylon mesh was placed carefully onto the tissue surface afterwards.
Control tissues were concurrently treated with 50 μL of de-ionized water (negative control, NC) or with 50 μL of 8 N potassium hydroxide (positive control, PC). A nylon mesh was placed carefully onto the tissue surface afterwards (negative control, only).
The tissues were washed with PBS to remove residual test material 3 minutes or 1 hour after start of the application treatment. Rinsed tissues were kept in 24-well plates (holding plates) at room temperature on assay medium until all tissues per application time were dosed and rinsed. The assay medium was then replaced by MTT solution and tissues were incubated for 3 hours. After incubation, the tissues were washed with PBS to stop the MTT-incubation. The formazan that was metabolically produced by the tissues was extracted by incubation of the tissues in isopropanol. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 6 microtiter wells filled with isopropanol for each microtiter plate.

Irritation test:
On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 0.9 mL assay medium and preconditioned in the incubator at 37°C. After 1 hour the preincubation medium was replaced with fresh medium and preconditioning continued for 18 ± 3 hours.
Three tissues were treated with the test substance, the PC and NC, respectively. Thirty microliter (30 μL) of the undiluted liquid test substance was applied using a pipette. A nylon mesh was placed carefully onto the tissue surface afterwards.
Control tissues were concurrently treated with 30 μL of sterile PBS (negative control, NC) or with 30 μL of 5% SDS (positive control, PC). A nylon mesh was placed carefully onto the tissue surface afterwards.
The tissues were kept under the laminar flow hood at room temperature for 25 minutes overall and for 35 minutes in the incubator.
The tissues were washed with sterile PBS to remove residual test material 1 hour after start of application. Rinsed tissues were blotted on sterile absorbent paper and transferred into new 6-well plates, pre-filled with 0.9 mL fresh medium. When all tissues were rinsed, the surface of each tissue was carefully dried with a sterile cotton swab.
Subsequently, the tissues were placed into the incubator at 37°C for 24 ± 2 hours. After 24 ± 2 hours the tissues were transferred into new 6-well plates pre-filled with 0.9 mL of fresh medium and placed into the incubator for additional 18 ± 2 hours post-incubation period.
After the post-incubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours.
After incubation, the tissues were washed with PBS to stop the MTT-incubation. The formazan that was metabolically produced by the tissues was extracted by incubation of the tissues in isopropanol. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 6 microtiter wells filled with isopropanol for each microtiter plate.

Corrosion test: The mean viability of the test-substance treated tissues determined after an exposure period of 3 minutes was 101%, and it was 66% after an exposure period of 1 hour. Irritation test: The mean viability of the test-substance treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 11%.

Based on the observed results and applying the evaluation criteria it was concluded, that Trimethylcyclohexylammonium sulfate , aqueous solution 50 wt.% shows a skin irritation potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy under the test conditions chosen.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

other: OECD (2014a) Draft Proposal for a New Test Guideline: Reconstructed Human Cornealike Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage

GLP compliance:

yes (incl. QA statement)

Species:

other: in vitro

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

a bulk volume of 50 μL (about 24 mg) of the test material was applied covering the whole tissue surface.

Details on study design:

Direct MTT reduction
To assess the ability of the test material to directly reduce MTT a pretest was performed. The test substance was added to 0.9 mL of the MTT solution. The mixture was incubated in the dark at about 37 °C for 3 hours. A negative control (de-ionized water) was tested concurrently. If the MTT solution color or, in case of water-insoluble test substances the border to the water-phase, turned blue / purple, the test substance was presumed to directly reduce MTT.
The direct reduction of MTT by a test substance interferes with the color density produced by metabolic capacity of the tissue and would falsify the test results.
In case where direct MTT reduction occurred, two freeze-killed control tissues each were treated with the test article and the negative control, in the same way as described in the following section.
Basic procedure
Several test substances were tested in parallel within the present test (test no. 59) using the same control tissues (NC and PC). Two tissues were treated with each, the test substance, the PC and the NC. There are two separate protocols for liquids and solids, differing in exposure time and postincubation
period. Due to the physical condition of the test substance the protocol for solids was applied.
Pre-incubation of the tissues
On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 1 mL assay medium and preconditioned in the incubator at 37°C. After 1 hour the preincubation medium was replaced with fresh medium and preconditioning continued in the incubator at standard culture conditions for 16 – 24 hours.
Pretreatment of the tissues
After the pre-incubation the tissues were pre-treated with 20 μL of PBS in order to wet the tissue surface. The tissues were incubated at standard culture conditions for 30 minutes.
Application of the test substance
Using a sharp spoon, a bulk volume of 50 μL of the test material was applied covering the whole tissue surface. Control tissues were concurrently applied with 50 μL of sterile de-ionized water (NC) or with 50 μL of methyl acetate (PC). After application, the tissues were placed into the incubator until the total exposure time of 6 hours was completed.
Removal of the test substance and postincubation period
To remove the test substance, the tissues were washed with sterile PBS. For this purpose the tissues were immersed and swiveled three times in each of three beakers filled with PBS. Washed tissues were immediately immersed into 12-well plates, pre-filled with 5 mL/well prewarmed medium (post-soak immersion) in order to remove residual test substance. After 25 minutes of post-soak immersion, each tissue was dried on absorbent paper and transferred to fresh 6-well plates filled with 1 mL/well pre-warmed medium. Subsequently, the tissues were incubated at standard culture conditions for 18 hours (postincubation period).
MTT incubation
After the post-incubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours. After incubation, the tissues were washed with PBS to stop the MTT-incubation. The formazan that was metabolically produced by the tissues was extracted by incubation of the tissues in isopropanol at room temperature overnight or for at least 2 hours on a plate shaker. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 4 microtiter wells filled with isopropanol for each microtiter plate.

Test substance		tissue 1	tissue 2	mean	inter-tissue variability [%]
NC	mean OD570	1.709	1.691	1.700
	viability [% of NC]	100.5	99.5	100.0	1.1
14/0711-1	mean OD570	0.378	0.268	0.323
	viability [% of NC]	22.3	15.8	19.0	6.5
PC	mean OD570	0.427	0.550	0.489
	viability [% of NC]	25.1	32.4	28.7	7.3

Based on the observed results and applying the evaluation criteria, it was concluded, that Trimethylcyclohexylammonium sulfate shows an eye irritation potential in the EpiOcular™ eye irritation test under the test conditions chosen.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Additional information

There are studies available for Trimethylcyclohexylammonium sulfate as aqueous solution 50 wt.% and for the pure substance.

Skin irritation (in vitro):

EpiDerm

The potential of Trimethylcyclohexylammonium sulfate to cause dermal corrosion was assessed by a single topical application of 25

μL bulk volume (about 17 mg) of the undiluted test substance to a reconstructed three dimensional human epidermis model

(EpiDerm™). Two EpiDerm™ tissue samples were incubated with the test substance for 3 minutes and 1 hour, each. Viability of the

test-substance treated tissues determined after an exposure period of 3 minutes was 105.4%. Viability of the test-substance treated

tissues determined after an exposure period of 1 hour was 90.5%. Based on the observed results and applying the evaluation criteria

it was concluded, that Trimethylcyclohexylammonium sulfate does not show a corrosion potential in the EpiDerm™ skin corrosion

test under the test conditions chosen.

The objective was to assess the potential for corrosive activity and skin irritation of Trimethylcyclohexylammonium sulfate , aqueous

solution 50 wt.%. Two in vitro assays were part of this in vitro skin irritation and corrosion test strategy: The Skin Corrosion Test

(SCT) and Skin Irritation Test (SIT). The potential of Trimethylcyclohexylammonium sulfate , aqueous solution 50 wt.% to cause

dermal corrosion/irritation was assessed by a single topical application of 50 μL (corrosion test) or 30 μL (irritation test) of the

undiluted test substance to a reconstructed three dimensional human epidermis model (EpiDerm™). For the corrosion test two

EpiDerm™ tissue samples were incubated with the test substance for 3 minutes and 1 hour, respectively. The irritation test was

performed with three EpiDerm™ tissue samples, which were incubated with the test substance for 1 hour followed by a 42-hours

post-incubation period. Corrosion test: The mean viability of the test-substance treated tissues determined after an exposure period

of 3 minutes was 101%, and it was 66% after an exposure period of 1 hour. Irritation test: The mean viability of the test-substance

treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 11%.

Based on the observed results and applying the evaluation criteria it was concluded, that Trimethylcyclohexylammonium sulfate ,

aqueous solution 50 wt.% shows a skin irritation potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy under the

test conditions chosen.

Eye irritation (in vitro):

EpiOcular

The potential of Trimethylcyclohexylammonium sulfate to cause ocular irritation was assessed by a single topical application of 50 μL bulk volume (about 24 mg) of the undiluted test substance to a reconstructed three dimensional human cornea model (EpiOcular™).

Two EpiOcular™ tissue samples were incubated with the test substance for 6 hours followed by a 18-hour post-incubation period.

Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the testsubstance treated epidermal tissues is compared to that of negative control tissues. The ratio of the values indicates the relative tissue viability. The test substance is not able to reduce MTT directly. The mean viability of the test-substance treated tissues was 19%. Based on the observed results and applying the evaluation criteria it was concluded, that Trimethylcyclohexylammonium sulfate shows an eye irritation potential in the EpiOcular™ eye irritation test under the test conditions chosen.

BCOP

The potential of Trimethylcyclohexylammonium sulfate , aqueous solution 50 wt.% to cause ocular irritation or serious damage to the

eyes was assessed by a single topical application of 750 μL of the undiluted test substance to the epithelial surface of isolated bovine

corneas. Three corneas were treated with the test substance for 10 minutes followed by a 2-hours post-incubation period.

In addition to the test substance a negative control (NC; de-ionized water) and two positive controls (PC1 and PC2; 100% ethanol

and 100% dimethylformamide) were applied to three corneas each. Corneal opacity was measured quantitatively as the amount of

light transmitted through the cornea. Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes

across the full thickness of the cornea. Both measurements were used to calculate an In Vitro Irritancy Score of the test substance. In

addition H&E-stained cross sections of the corneas were evaluated in addition to the assessment of opacity and permeability.

The mean IVIS (In Vitro Irritancy Score) was 5.5. Histological evaluation revealed changes indicating minimal eye damage in all

corneas treated with the test substance.

EpiOcular

The potential of Trimethylcyclohexylammonium sulfate , aqueous solution 50 wt.% to cause ocular irritation was assessed by a single topical application of 50 μL of the undiluted test substance to a reconstructed three dimensional human cornea model (EpiOcular™). Two EpiOcular™ tissue samples were incubated with the test substance for 30 minutes followed by a 2-hours post-incubation period. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the testsubstance treated epidermal tissues is compared to that of negative control tissues. The ratio of the values indicates the relative tissue viability. The EpiOcular™ eye irritation test showed the following results:

The test substance is not able to reduce MTT directly. The mean viability of the test-substance treated tissues was 81%. Based on the results for BCOP and EpiOcular™ Test and applying the evaluation criteria, Trimethylcyclohexylammonium sulfate , aqueous solution 50 wt.% does not show an eye irritation potential in the in vitro eye irritation test strategy under the test conditions chosen.

Effects on skin irritation/corrosion: irritating

Effects on eye irritation: irritating

Justification for classification or non-classification

Based on the available studies data on skin and eye irritating properties the test item is classified as Xi, R36/28 (according to Directive 67/548/EEC (DSD) and as eye and skin irritating (H315/H319) according to Regulation (EC) No 1272/2008 (CLP).