Dimethyl[3-[(1-oxooctadecyl)amino]propyl][2-oxo-2-(tetradecyloxy)ethyl]ammonium chloride

Endpoint summary

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:

Experimental starting date :October 4, 2017 - Experimental completion date : October 6, 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)

Version / remarks:

July 22, 2010

Deviations:

no

Remarks:

Only minor modifications.

GLP compliance:

no

Remarks:

Laboratory works with standard operating procedures and according to GLP procedures with the exception of audits by a separate Quality Assurance Unit.

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Justification for test system used:

Certified by Ashland Tissue Engineering laboratories, October 2, 2017.

Vehicle:

unchanged (no vehicle)

Remarks:

Product is 50% solids in propylene glycol

Details on test system:

- Ashland 0.5 cm2 reconstructed epidermis from normal human keratinocytes.
- Cells are grown on inert polycarbonate filter on chemically defined medium, airlifted for 17 days
- 3 tissues will be used for each chemical.
- The RHE tissues are incubated at 37°C, 5% CO2 until test substance application (usually 24 hours).

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Undiluted

Duration of treatment / exposure:

42 minutes
Procedure:
. Fill a 24-well plate with 300μl by well of pre-warmed maintenance culture medium.
· Transfer tissues.
· Dispense 16 μL ± 0.5 μL (or 16 μg) of products and controls on the top of epidermis.
· Carefully apply a nylon mesh (Ø = 7.5mm) on the whole surface with forceps.
· Keep the plates containing the treated epidermis for 42 minutes at room temperature.

Duration of post-treatment incubation (if applicable):

42 hours and MTT incubation for 3 hours.
Procedure:
· Remove the nylon mesh with fine forceps from the epidermal surface of a treated tissue.
· Remove the treated units using forceps, and rinse thoroughly 25 times with 1 ml DPBS.
· Transfer the washed tissue on 2 mL growth culture medium (6-well plate).
· Incubate the treated and rinsed epidermis at 37°C, 5% CO2 for 42 hours (± 60 minutes).
Assessment of non-specific MTT reduction (NSMTT)
· To identify direct MTT reducers, each test chemical should be added to freshly prepared MTT solution. If the MTT mixture containing the test chemical turns blue/purple, the test chemical is presumed to directly reduce MTT and a further functional check on non-viable RhE tissues should be performed.
· Each MTT reducing test chemical is applied on at least two killed tissue replicates which undergo the entire testing procedure to generate a non-specific MTT reduction (NSMTT)

Number of replicates:

9 replicate wells

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Main Ashland Equivalent Epidermis Skin Irritation Test

Value:

90.65

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

viability: 100%

Positive controls validity:

valid

Remarks:

viability: 3.08%

Remarks on result:

no indication of irritation

Cell viability results :

Group	Mean Viability (%)	Classification
Negative Control / Water	100	Non irritant
Positive Control / SDS 5%	3.08	Irritant
Tested Product / Neat CERAPHYL™ 70	90.65	Non irritant

 

Interpretation of results:

GHS criteria not met

Conclusions:

According to the in-vitro Ashland Epidermis skin irritation test, CERAPHYL® 70 is classified as non-irritant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not 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:

weight of evidence

Study period:

Experimental starting date : October 3, 2017 - Experimental completion date : October 6, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: CD 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 9 October 2017

Deviations:

no

GLP compliance:

no

Remarks:

Performed according to a Standard Operating Procedure

Vehicle:

water

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

Neat

Duration of treatment / exposure:

30 minutes

Duration of post- treatment incubation (in vitro):

Post-exposure immersion 12 minutes
Post-exposure incubation 120 minutes
Duration of MTT incubation 3 hours

Number of animals or in vitro replicates:

4 replicates

Details on study design:

Assessment of Direct Test Article Reduction by MTT
It is necessary to assess this ability for each test article prior to conducting any assays with viable tissues.
1. 50 μl ( for liquid test articles) or approximately 50 mg (for solid test articles) are added to 1 ml of a 1.0 mg/mL MTT solution in a 6-well plate and the mixture is incubated in the dark at 37°C in a humidified atmosphere of 5 % CO2 for three hours.
2. A negative control (50 μl of sterile deionized water) should be run concurrently.
3. If the MTT solution colour turns blue/purple, the test article is presumed to have reduced the MTT.
4. In cases where the test article is shown to reduce MTT, a functional check using freeze-killed tissue controls (killed controls = KC) should be performed.

Pre-incubation step
· For a given chemical a minimum of 2 tissues were used.
· An appropriate numbers of 6-well plates were filled with 1 ml of EpiOcular™ Assay Medium. EpiOcular™ EIT test kit was open and tissues were transferred into Assay medium filled wells, using sterile forceps.
· Place the tissues at 37°C, 5% CO2 until test substance application (usually 16 – 24 hours).

Treatment of liquid test article
· After the overnight incubation, the tissues are pre-wetted with 20 μL of Ca2+Mg2+-Free-DPBS. The tissues are incubated at standard culture conditions for 30 ± 2 minutes.
· Each liquid test and control article is tested by applying 50 μl topically on the EpiOcular™ tissues. The tissues are incubated at standard culture conditions for 30 ± 2 minutes.
· At the end of the treatment time, the test articles are removed by extensively rinsing the tissues with Ca2+Mg2+-free D-PBS, and any remaining liquid should be decanted onto the absorbent material.
· Epitheliums were immersed in a new 12-well plate containing 5 mL of fresh Assay Medium, for 12 ± 2 minutes at room temperature.

Viability measurement
· 24-well plates were filled with 300 μL MTT solution (1 mg/ml).
· Treated tissues were transferred in the pre-filled MTT 24-well plates and Incubated for 3 hours (± 10 minutes) at 37°C and 5% CO2.
· Treated tissue insert bottom was dried on sterile absorbent paper and transferred in new 24-well plate containing 2 mL of isopropanol so that isopropanol is flowing into the insert on the tissue surface.
· Plate was protected from evaporation by stretching 3 parafilm layers over the plate and adding the lid on the plate and incubated in the dark for 2 hours (± 5 minutes) at room temperature with gentle agitation (about 150 rpm) or overnight at 2-8°C for formazan extraction.
· Tissue and polycarbonate filter were pierced with a tip in order to get the whole extraction solution in the corresponding well.
· Extraction solution was homogenized by pipetting 3 times up and down to complete Formazan crystals solubilization.
· 2 x 200 μL extraction solution per well (= 2 wells per tissue i.e. 2 replicates per tissue) were transferred into a 96-well plate.
· Optical Densities (OD) was read using a 96-well plate spectrophotometer: The concentration of Formazan was measured by determining the OD at 570 nm.

Irritation parameter:

in vitro irritation score

Remarks:

% cell viability

Value:

53.46

Negative controls validity:

valid

Remarks:

100 % viability

Positive controls validity:

valid

Remarks:

33.16 % viability

Remarks on result:

positive indication of irritation

Interpretation of results:

other: Cat1/Cat2

Conclusions:

According to EpiOcular™ Eye Irritation test, CERAPHYL™ 70 is classified for eye irritation or serious eye damage and defined as UN GHS Cat1/Cat2.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The experimental start date was 13 Nov 2017, the experimental completion date was 17 Nov 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)

Version / remarks:

Adopted 09 October 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Details on test animals or tissues and environmental conditions:

EpiOcular™ (OCL-200-EIT MatTek Corporation, Lot: 27406 Kit Q.
Source: MatTek Corporation, Ashland MA, U.S.A.
The EpiOcular tissue construct is a non-keratinized epithelium (0.6 cm2) prepared from normal human keratinocytes (MatTek). It models the cornea epithelium with progressively stratified, but not cornified cells. These cells are not transformed or transfected with genes to induce an extended life span in culture.
The “tissue” is prepared in inserts with a porous membrane through which the nutrients pass to the cells. A cell suspension is seeded into the insert in specialized medium. After an initial period of submerged culture, the medium is removed from the top of the tissue so that the epithelial surface is in direct contact with the air. This allows the test material to be directly applied to the epithelial surface in a fashion similar to how the corneal epithelium would be exposed in vivo.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Neat, the test item (an excessive amount per tissue) was applied directly on top of the skin tissue and spread to match the size of the tissue. Since the test item induced color interference in aqueous conditions, two tissues were treated with test item and incubated with DMEM instead of MTT solution.

Duration of treatment / exposure:

6 hours ± 15 minutes at 37.0 ± 1.0°C

Duration of post- treatment incubation (in vitro):

Post-Soak: 25 ± 2 minute immersion incubation at room temperature.
After the Post-Soak period cell culture inserts were each dried carefully and transferred to the 6-well plate containing 1.0 ml of warm Assay Medium and were incubated for 18 hours ± 15 minutes at 37°C.
After incubation, cell culture inserts were dried carefully to remove excess medium and subsequently transferred into a 24-wells plate prefilled with 0.3 ml MTT-medium (1.0 mg/ml). The tissues were incubated for 180 ± 10 minutes at 37°C.

Number of animals or in vitro replicates:

2 tissues per test item together with a negative control and positive control

Details on study design:

Quaternium-70 was checked for possible color interference and direct MTT reduction before the study was started.
On the day of receipt the tissues were equilibrated (in its 24-well shipping container) to room temperature. Subsequently, tissues were transferred to 6-well plates and incubated for
20 ± 4 hours at 37°C in 1.0 ml fresh pre-warmed Assay Medium, which was refreshed after approximately 1 hour.
DMEM (Dulbecco’s Modified Eagle’s Medium): Supplemented DMEM medium, serum-free supplied by MatTek Corporation.
MTT medium: MTT concentrate (5 mg/ml) diluted (1:5) with MTT diluent.
The tissues were pre-incubated at standard culture conditions for 30 ± 2 minutes.
After the exposure period, tissues were thoroughly rinsed with Ca2+Mg2+-free D-PBS to remove residual test item.
Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Eye hazard potential of the test item was classified according to remaining cell viability following exposure of the test item.

Irritation parameter:

in vitro irritation score

Remarks:

mean % tissue viability

Run / experiment:

Main

Value:

12

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Milli-Q water

Positive controls validity:

valid

Remarks:

Methyl Acetate

Remarks on result:

positive indication of irritation

The relative mean tissue viability obtained after 6 hours±15 minutes treatment with Quaternium-70 compared to the negative control tissues was 12%. Since the mean relative tissue viability for Quaternium-70 was below 60%, it is considered to be potentially irritant or corrosive to the eye. The positive control had a mean cell viability after 6 hours ±15 minutes exposure of 31%. The absolute mean OD₅₇₀ of the negative control tissues was within the laboratory historical control data range . The difference between the percentage of viability of two tissues treated identically was less than 17%, indicating that the test system functioned properly.

Interpretation of results:

other: Category 2 or Category 1

Conclusions:

Quaternium-70 is potentially irritant or corrosive in the EpiOcular™ test under the experimental conditions described in this report. The test item is identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1). Assuming a worst case scenario, the ultimate classification is set at cat. 1.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Justification for classification or non-classification

According to the in-vitro Ashland Epidermis skin irritation test, CERAPHYL® 70 containing 50-60 % Quaternium-70 is classified as non-irritant.

Based on two separate tests, Quaternium-70 is potentially irritant or corrosive in the EpiOcular™ test under the experimental conditions described in this report.  The test item is identified as potentially requiring classification and labelling according to UN GHS: Eye irritant Category 2 or Category 1. Assuming a worst case scenario, the ultimate classification is set at eye irritant cat. 1.