3-(D-gluconoylamino)propyl(2-hydroxyethyl)dimethylammonium chloride

Endpoint summary

Description of key information

Key information of skin irritation is based on the in-vitro Ashland Epidermis skin irritation test with CERAPHYL® 60.

Key information of eye irritation is based on the in-vitro EpiOcular Eye Irrittation Test (EIT) with CERAPHYL® 60.

CERAPHYL® 60 is the marketed product and contains 60% 1-Propanaminium, 3-(D-gluconoylamino)-N- (2-hydroxyethyl)-N, N-dimethyl-, chloride.

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.

Specific details on test material used for the study:

Solid fraction: 60.2%
Physical state: Clear yellow to light amber liquid
pH: 4.9
Storage at room temperature

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 60% solids in water

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:

24-well plates

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Main Ashland Equivalent Epidermis Skin Irritation Test

Value:

91.12

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™ 60	91.12	Non irritant

 

Interpretation of results:

GHS criteria not met

Conclusions:

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

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar 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:

A draft test guideline on RhCE test method pf July 27, 2015

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.

Specific details on test material used for the study:

Solid fraction: 60.2%
Physical state: Clear yellow to light amber liquid
pH: 4.9
Storage at room temperature

Species:

human

Details on test animals or tissues and environmental conditions:

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).

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

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.

Duration of treatment / exposure:

30 minutes

Duration of post- treatment incubation (in vitro):

For Liquids (except colorant):
· 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.

Details on study design:

ACCEPTANCE CRITERIA
· Negative control: The negative control data meet the acceptance criteria if
o The mean OD value of tissues is > 0.8 and < 2.5
o The variability between two tissue replicates should be less than 20%.
· Positive control: The positive control data meet the acceptance criteria if the mean relative viability, expressed as % of the NC, is:
o ≤ 50% for 30-minute exposure for liquid substances
o ≤ 50% for 6-hrs exposure for solid substances
· Tested Substance: A test meets the acceptance criterion if :
o The variability between two tissue replicates should be less than 20%.
o This applies also to the killed controls and the colorant controls which are calculated as percent values related to the viability of the relating negative control.

Irritation parameter:

in vitro irritation score

Remarks:

% tissue viability

Run / experiment:

EpiOcular™ Eye Irritation test (EIT)

Value:

91.72

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Cell viability results :

Group	Mean Viability (%)	Classification
Negative Control / Water	100	No Category
Positive Control / Methyl acetate	33.16	Cat1/Cat2
Tested Product / Neat CERAPHYL™ 60	91.72	No Category

 

Interpretation of results:

GHS criteria not met

Conclusions:

According to EpiOcular™ Eye Irritation test, CERAPHYL™ 60 is not classified for eye irritation or serious eye damage and defined as UN GHS: No category.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Justification for classification or non-classification

According to the in-vitro Ashland Epidermis skin irritation test, CERAPHYL® 60 containing 60% 1-Propanaminium, 3-(D-gluconoylamino)-N-(2-hydroxyethyl)-N,N-dimethyl-, chloride is classified as non-irritant.

According to the in-vitro EpiOcular Eye Irrittation Test (EIT), CERAPHYL® 60 containing 60% 1-Propanaminium, 3-(D-gluconoylamino)-N-(2-hydroxyethyl)-N,N-dimethyl-, chloride is classified as non-irritant.