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Diss Factsheets

Administrative data

Description of key information

In-vivo testing before 1980 showed that concentrations of the test article including 100% induced barely perceptible skin irritation. In-vitro testing in human epidermis model confirmed the lack of potential for significant skin irritation. According to an in-vitro EpiOcular™ Eye Irritation test, CERAPHYL™ 65 has potential for inducing serious eye irritation or serious eye damage. An additional in-vitro test exposing the cornea of Bovine eyes from young cattle to CERAPHYL™ 65 confirmed the potential for severe eye irritation of Fatty acids, mink, reaction products with 3-(dimethylamino)propylamine and 2-chloroethanol.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental starting date :November 8, 2017 - Experimental completion date : November 10, 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
GLP compliance:
no
Remarks:
Ashland testing laboratory works with Standard Operating Procedures.
Specific details on test material used for the study:
Chemical name: CERAPHYL™ 65
Code 826812
Batch number 0001954899
Supplier Ashland
Dry matter (%) 59.4%
Physical state Clear amber to dark amber liquid
pH 7.6
Solvent Propylene Glycol
Treatment None
Storage conditions Room Temperature
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from multiple donors
Vehicle:
unchanged (no vehicle)
Details on test system:
- Ashland 0.5 cm2 reconstructed epidermis from normal human keratinocytes.
- Batch N°: 1710EPID02
- Origin: Foreskin
- Quality Controls: Certified by Ashland Tissue Engineering laboratories, 06/11/2017
- Cells were grown on inert polycarbonate filter on chemically defined medium, airlifted for 17 days
- 3 tissues were used for each chemical.
- The RHE tissues were 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:
Neat
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:
3 per group: Negative control, Quaternium-26, Positive control
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Main
Value:
120.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Interpretation of results:
GHS criteria not met
Executive summary:

According to ASHLAND EQUIVALENT EPIDERMISES SKIN IRRITATION test, CERAPHYL™ 65 is classified as Non irritant.

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The experimental start date was 20 Nov 2017, and the experimental completion date was 27 Nov 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
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
Amended by EC No. 640/2012 OJ No. L193, 20 July 2012.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Identification: Quaternium-26
Appearance: Clear amber to dark amber liquid
Batch: 0001954899
Purity/Composition UVCB
Test item storage At room temperature
Stable under storage conditions until 22 December 2017 (retest date)
Test item handling No specific handling conditions required
Chemical name (IUPAC), synonym or trade name: Ceraphyl 65 (60% Quaternium 26)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from multiple donors
Vehicle:
unchanged (no vehicle)
Details on test system:
EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 17-EKIN-047).
This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen.
The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
Source: SkinEthic Laboratories, Lyon, France.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
Neat
Duration of treatment / exposure:
15 minutes
Procedure:
The test was performed on a total of 3 tissues per test item together with negative and positive controls. Twenty five µl of the undiluted test item was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 µl PBS (negative control) and 3 tissues with 25 µl 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time.
After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item. After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 ml pre-warmed maintenance medium until all tissues were dosed and rinsed.
Duration of post-treatment incubation (if applicable):
42 hours and MTT incubation for 3 hours.
Procedure:
After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 ml MTT-solution (0.3 mg/ml in PBS). The tissues were incubated for 3 h at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in pre-labeled microtubes and extracted with 500 µl isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for approximately 70 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Main
Value:
100
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Interpretation of results:
GHS criteria not met
Executive summary:

In conclusion, Quaternium-26 is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report andshould not be classified.

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
June 1979
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
yes
Remarks:
Species: guinea pig instead of rabbit. Exposure period: 16 hours.
GLP compliance:
yes
Species:
guinea pig
Strain:
Hartley
Details on test animals or test system and environmental conditions:
Supplier: The animals were obtained through a suitably licensed breeding farm, and were carefully checked upon receipt and prior to test initiation for evidence of poor health.
Housing: They were housed in galvanized or stainless steel cages and were identified through individual markings as well as cage labels.
Diet: consisted of a growth and maintenance ration from a commercial producer
Water: ad libitum.
Type of coverage:
occlusive
Preparation of test site:
clipped
Vehicle:
unchanged (no vehicle)
Controls:
not required
Amount / concentration applied:
0.5 ml of 100% and of 50%, 25% and 10% of the test article in water.
Duration of treatment / exposure:
16 h
Observation period:
after removal of the occlusive wrap and at 24 h.
Number of animals:
1 per concentration
Details on study design:
The animals were prepared by close-clipping the mid-dorsal area of the trunk with a small animal clipper equipped with a 1/4-0 (surgical) head. Decreasing concentrations of the test article, suspended or dissolved in an appropriate, non-irritating vehicle, were applied to guinea pigs under occluded patch for 16 hours. At the end of 16 hours, the occlusive wrap was removed and the test site scored then and at 24- hours for erythema and edema. (Refer to the scoring scale appended.) The highest concentration tested which produced no irritation was chosen for the induction phase.
Irritation parameter:
erythema score
Basis:
animal #1
Remarks:
100%
Time point:
24 h
Score:
1
Max. score:
4
Reversibility:
not specified
Remarks on result:
probability of weak irritation
Irritation parameter:
edema score
Basis:
animal #1
Remarks:
100%
Time point:
24 h
Score:
0
Max. score:
4
Reversibility:
not specified
Remarks on result:
no indication of irritation
Irritation parameter:
erythema score
Basis:
animal #2
Remarks:
50%
Time point:
24 h
Score:
1
Max. score:
4
Reversibility:
not specified
Remarks on result:
probability of weak irritation
Irritation parameter:
edema score
Basis:
animal #2
Remarks:
50%
Time point:
24 h
Score:
0
Max. score:
4
Reversibility:
not specified
Remarks on result:
no indication of irritation
Irritation parameter:
erythema score
Basis:
animal #3
Remarks:
25%
Time point:
24 h
Score:
0
Max. score:
4
Reversibility:
not specified
Remarks on result:
no indication of irritation
Irritation parameter:
edema score
Basis:
animal #3
Remarks:
25%
Time point:
24 h
Score:
0
Max. score:
4
Reversibility:
not specified
Remarks on result:
no indication of irritation
Irritation parameter:
erythema score
Basis:
animal #4
Remarks:
10%
Time point:
24 h
Score:
0
Max. score:
4
Reversibility:
not specified
Remarks on result:
no indication of irritation
Irritation parameter:
edema score
Basis:
animal #4
Remarks:
10%
Time point:
24 h
Score:
0
Max. score:
4
Reversibility:
not specified
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
Very slight erythema (barely perceptible) and no edema at concentrations including 100%.
Interpretation of results:
GHS criteria not met
Conclusions:
Concentrations of the test article including 100% induced barely perceptible skin irritation.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The experimental start date was 21 Nov 2017, and the experimental completion date was 21 Nov 2017.
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 July 26, 2013
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Identification: Quaternium-26
Appearance: Clear amber to dark amber liquid
Batch: 0001954899
Purity/Composition: UVCB
Test item storage: At room temperature
Stable under storage conditions until 22 December 2017 (retest date)
Test item handling No specific handling conditions required
Chemical name (IUPAC), synonym or trade name: Ceraphyl 65
pH 7.6 (as is)
Species:
cattle
Details on test animals or tissues and environmental conditions:
Source: Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
Transport: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
Preparation of Corneas: The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded. The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1°C. The corneas were incubated for the minimum of 1 hour at 32 ± 1°C.

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
Neat
Duration of treatment / exposure:
Corneas were incubated in a horizontal position with 750 ul of either the negative control, positive control (Ethanol) or test item for 10 ± 1 minutes at 32 ± 1°C.
Duration of post- treatment incubation (in vitro):
After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Earle’s Minimum Essential Medium, Life Technologies) and thereafter with cMEM. Possible pH effects of the test item on the corneas were recorded. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. Subsequently the corneas were incubated for 120 ± 10 minutes at 32 ± 1°C. After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns.
Number of animals or in vitro replicates:
3
Details on study design:
Opacity Measurement:
The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter.
The opacity value (measured with the device OP-KIT) was calculated according to:
Opacity=(I0/I - 0.9894)/0.0251
With I0 the empirically determined illuminance through a cornea holder but with windows and medium, and I the measured illuminance through a holder with cornea.
The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each treated cornea with the test item or positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test item or positive control treated cornea. The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.
Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Sigma-Aldrich, Germany) was evaluated.
Interpretation:
The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score: In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)
Additionally the opacity and permeability values were evaluated independently to determine whether the test item induced irritation through only one of the two endpoints.
In vitro score range UN GHS
≤ 3 No Category
> 3; ≤ 55 No prediction can be made
>55 Category 1
Irritation parameter:
in vitro irritation score
Run / experiment:
Main
Value:
>= 57 - <= 105
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was 47 and 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.
Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
Quaternium-26 induced serious eye damage through both endpoints, resulting in a mean in vitro irritancy score of 81 after 10 minutes of treatment.
Executive summary:

The objective of this study was to evaluate the eye hazard potential of Quaternium-26 as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test). This report describes the potency of chemicals to induce serious eye damage using isolated bovine corneas. The eye damage of Quaternium-26 was tested through topical application for 10 minutes. The study procedures described in this report were based on the most recent OECD guideline.

Batch 0001954899 of Quaternium-26 was aclear amber to dark amber liquid. The test item was applied as it is (750 µl) directly on top of the corneas.

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical rangeindicating that the negative control did not induce irritancy on the corneas. The meanin vitroirritancy score of the positive control (Ethanol) was 47 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. 

Quaternium-26 induced serious eye damage through both endpoints, resulting in a mean in vitro irritancy score of 81 after 10 minutes of treatment.

In conclusion, since Quaternium-26 induced an IVIS ≥ 55, it is concluded that Quaternium-26 inducesserious eye damagein the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report andshould be classified category 1 according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.
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:
0
Vehicle controls validity:
not applicable
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™ 65 is classified for eye irritation or serious eye damage and defined as UN GHS Cat1/Cat2.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Quaternium 26 applied as such induced severe eye damage in in-vitro studies. Results of in-vivo studies with concentrations including 4% of the product showed mild and reversible eye irritation. Hence, although Quaternium 26 is classified as potentially inducing irreversible effects on the eye Category 1, formulations containing up to and including 4% of Quaternium 26 do not induce irreversible damage to the eyes.

Justification for classification or non-classification

Since Quaternium-26 induced an IVIS ≥ 55 in the Bovine Corneal Opacity and Permeability test, it should be classified category 1 according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.