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Diss Factsheets

Toxicological information

Eye irritation

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
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.

Test material

Constituent 1
Chemical structure
Reference substance name:
3-(D-gluconoylamino)propyl(2-hydroxyethyl)dimethylammonium chloride
EC Number:
257-440-3
EC Name:
3-(D-gluconoylamino)propyl(2-hydroxyethyl)dimethylammonium chloride
Cas Number:
51812-80-7
Molecular formula:
C13H29N2O7.Cl
IUPAC Name:
1-Propanaminium, 3-(D-gluconoylamino)-N-(2-hydroxyethyl)-N,N-dimethyl-, chloride
Test material form:
liquid
Details on test material:
Batch 0002042127
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 animals / tissue source

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).

Test system

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.

Results and discussion

In vitro

Results
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

Any other information on results incl. tables

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

 

Applicant's summary and conclusion

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.