Registration Dossier

Administrative data

Description of key information

Eye Irritation: Not irritant
Skin Irritation: Not irritant

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:
From April 14 to April 17, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test conducted according to internationally accepted Guideline and in according to the GLP Principles
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
other: Internal Procedure SOP 118 008 40
Deviations:
no
GLP compliance:
yes
Amount / concentration applied:
23.5 mg (Tissue 1)
23.1 mg (Tissue 2)
23.1 mg (Tissue 3)
Duration of treatment / exposure:
60 ± 5 min. after the first application and the incubation period the inserts were rinsed
Details on study design:
TEST SYSTEM
Specification: Commercially available EpiDermTMKit.
The EpiDermTM tissues are cultured on specially prepared cell cultures inserts.
Origin: EpiDermTMKit from MatTek In Vitro Life Science Laboratories (Batch n. 21654).

CHEMICALS AND MEDIA
MTT Reagent
Contains 1 mg/ml 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide as concentrate in DBPS buffer (5 mg/ml, stored at –20 ± 5 °C).
Origin: prepared by LAUS GmbH
A MTT stock solution of 5 mg/mL in DPBS buffer was prepared and stored in aliquots of 2 ml at – 20 ± 5 °C. 2 ml of the stock solution were thawed and diluted with 8 mL of me-dium (resulting in 1 mg/ml).

DPBS-Buffer
Dulbecco’s Phosphate Buffered Saline (DPBS buffer without CaCl2 and MgCl2).
Origin: MatTek In Vitro Life Science Laboratories and prepared by LAUS GmbH

Composition of the subset from MatTek In Vitro Life Science Laboratories (batch n.: 020315MHA):
Used as negative control and for rinsing the test item from the tissues
KCl 0.2 g
KH2PO4 0.2 g
NaCl 8.0 g
Na2HPO4 * 7H2O 2.16 g
H2O ad 1 l
Composition of the subset from LAUS GmbH (batch: 20150226):
Solvent for MTT concentrate and for rinsing the outside of the inserts at the end of the in-cubation time with MTT.
KCl 0.2 g
KH2PO4 0.2 g
NaCl 8.0 g
Na2HPO4 * 7H2O 2.16 g
H2O ad 1 l

Positive Control
Sodium dodecyl sulphate (SDS): solution in deionised H2O containing 5 % SDS
Origin: MatTek In Vitro Life Science Laboratories (batch no.: 020615TMH).

MEM Medium with Phenol Red for Pre-Test
Serum-free MEM (Minimum Essential Medium).
Origin: Gibco/Invitrogen (Batch no.: 1613667)

Assay Medium
Serum-free DMEM (Dulbecco’s Modified Eagle’s Medium).
Origin: MatTek In Vitro Life Science Laboratories (batch no.: 040915TMA)

Isopropanol:
Purity 99.9 %,
Batch no.: 310060213050,
used as extracting solvent for formazan.

TEST VESSELS
Vessels materials: glass or sterilisable plastic.
Sterilised before use by rinsing with ethanol 70% or autoclaving.
Vessels used: 6, 24 and 96 well-plates

INSTRUMENT AND DEVICES used during the test:
Autoclave, 3870 ELV-B
Stop-watch
96-well-plate photometer, Anthos Reader
Precision scales, Mettler Toledo PB 5001-SO2 Labostyle
Analytical scales, Mettler Toledo XS 205 DU
Incubation chamber Binder
Table water bath, neoLabAdjustable pipettes with sterile tips
Clean bench, category 2 (Axo Safe, MARS 12000)Orbital shaker, GFL 3005
Bulb-headed pipettes, sterile
Single-use pipettes
Nylon mesh circles 8 mm diameter (200 µm pore (EPI-MESH))
Pipetting device, Accujet
Refrigerator and freezer
Weighing funnels
Microscope
Standard Laboratory material (glassware)
Irritation / corrosion parameter:
other:
Value:
2.089
Remarks on result:
other:
Remarks:
Basis: mean of the three tissues (%). Remarks: relative standard deviation of the three tissues = 2.1 %. (migrated information)
Irritation / corrosion parameter:
other:
Value:
112.2
Remarks on result:
other:
Remarks:
Basis: mean. Remarks: percentage values of formazan production calculated in comparison to the negative control. (migrated information)

The photometric absorbance of the negative controls is considered as 100 %. For each replicate of test item and positive control, formazan production is calculated as % photometric absorbance compared with the mean of the negative controls:

% Formazan production = [OD mean of negative controls/ODreplicate test item resp. positive control]*100%

where OD is the Optical Density

Absorbance values blank isopropanol (OD at 570 nm)

Replicate

1

2

3

4

5

6

7

8

Mean

Absorbance

0.041

0.038

0.038

0.038

0.037

0.037

0.038

0.037

0.038

Absorbance Values of Negative control, Positive Control and Test item

Designation

Measurement

Negative Control

AP 1300 S

Positive Control

Tissue 1 

1

2.023

2.148

0.097

2

1.919

2.206

0.089

Tissue 2 

1

1.701

2.131

0.088

2

1.726

2.069

0.085

Tissue 3 

1

2.002

2.129

0.089

2

2.025

2.081

0.087

Mean absorbance Values

Designation

Negative Control

AP 1300 S

Positive Control

Mean – blank (tissue 1)

1.933

2.139

0.055

Mean – blank (tissue 2)

1.676

2.062

0.049

Mean – blank (tissue 3) 

1.976

2.067

0.050

Mean of the three tissues

1.862

2.089

0.051

Relative standard deviation
of the three tissues

8.7 %

2.1 %

6.3 %

% Formazan Production

Designation

AP 1300 S

Positive Control

% Formazan production (tissue 1)

114.9 %

3.0 %

% Formazan production (tissue 2)

110.7 %

2.6 %

% Formazan production (tissue 3)

111.0 %

2.7 %

% Formazan production (mean)

112.2 %

2.8 %

Interpretation of results:
not classified
Remarks:
Migrated information According to the CLP Regulation Criteria used for interpretation of results: EU
Conclusions:
112.2 % of the relative absorbance
Executive summary:

Method

The substance has been tested in order to evaluate the skin irritation potential according to the EU method B.46 and OECD 439.

During the test three tissues of the human skin model EpiDermTM has been treated with on average 25 mg of test item for 60 minutes. Both positive and negative control have been prepared.

Observations

After the treatment with the test item, the relative absorbance values were increased, compared to the negative control, to 112.2 %.

The Optical Density (OD) of the negative control (1.9) was well within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8. The positive control induced a decrease in the relative absorbance as compared to the negative control to 2.8 % (required: ≤ 20 %). For these reasons the validity criteria of the test system has been fulfilled.

Conclusion

The 112.2 % value of the relative absorbance is above the threshold for irritation potential set out in the OECD guideline, therefore the substance is considered as not irritant in the Human Skin Model Test.

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
Study period:
May 5th, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test conducted according to internationally accepted Guideline and in according to the GLP Principles
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Principles of method if other than guideline:
This study design is in agreement with the procedures described in the OECD series on testing and assessment no. 160 – Guidance Document on the bovine corneal opacity and permeability (BCOP) and isolated chicken eye (ICE) test methods: collection of tissues for histological evaluation and collection of data on non-severe irritants.
GLP compliance:
yes (incl. certificate)
Controls:
yes
Amount / concentration applied:
A suspension of the test item at 20 % w/v was prepared in physiological saline, with correction for the displacement due to the volume of the test item. Concentrations were expressed in terms of material as received. The suspension was sonicated and maintained in magnetic agitation until aliquots preparation. Three syringes were filled in with the suspension and maintained at room temperature until use.
Duration of treatment / exposure:
4-hour exposure period
Observation period (in vivo):
There was no post-exposure period.
Number of animals or in vitro replicates:
n.a.
Details on study design:
REPLICANTS: three corneas per Test Item, Negative and Positive control respectively

CONTROLS: positive and negative control
Positive control: 20 % (w/v) Imidazole (Sigma, batch no. 0001422794), in physiological saline (0.9 % NaCl) (Baxter, batch no. 13D0406).
Negative control: Physiological saline (0.9 % NaCl) (Baxter, batch no. 13D0406).

REMOVAL OF TEST SUBSTANCE
- Washing and time after start of exposure:
After exposure, corneas were rinsed thoroughly with complete EMEM with phenol red. For the test item treated group, additional washings were performed and the glass windows of the anterior chambers were removed to allow a more effective removal of the test item from the corneas. A final wash with prewarmed complete EMEM without phenol red was carried out. Finally, the anterior chamber was re-filled with prewarmed complete EMEM without phenol red.

SCORING SYSTEM: in vitro irritacy score (IVIS)

TOOL USED TO ASSESS SCORE: opacitometer
Irritation parameter:
other: IVIS (in vitroirritation score)
Basis:
mean
Time point:
other: 4 hours
Score:
ca. 0
Reversibility:
other: No effects observed under test condition
Remarks on result:
other: in vitro test
Irritant / corrosive response data:
No relevant increases in opacity or permeability values were recorded after treatment with the test item, when compared to those of negative control.
The calculated in vitro irritancy score (IVIS ) was 0.0.

Negative and positive controls gave the expected results. The test is therefore considered as valid.

Assessment of ocular corrosion/irritation bovine corneal opacity/permeability (BCOP) assay.

Treatment code Pre-test corna number Basal opacity Mean group basal opacity
N1 10 0 0.00
N2 6 0
N3 9 0
P1 2 1 0.00
P2 5 0
P3 7 0
A1 1 1 0.00
A2 4 0
A3 8 0

Mean of basal opacity: 0.00; the mean refers to the total number of selected pre-test corn

The IVIS cut-off values for identifying test chemicals as inducing serious eye damage (UN GHS Category 1) and test chemicals not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are:

- IVIS ≤ 3 –No Classification(UN GHS)

- IVIS > 3; ≤ 55 – No prediction can be made

- IVIS > 55 –Category 1(UN GHS)

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Calculated in vitro irritancy score (IVIS ) = 0.0.
No relevant increases in opacity or permeability values were recorded after treatment with the test item, when compared to those of negative control.
Executive summary:

Method

The potential of the test item to cause corrosion/severe irritation by using the Bovine Corneal Opacity and Permeability (BCOP) assay was examined, in agreement with OECD Guideline no. 437 and the Guidance Document OECD series on testing and assessment no. 160.

The test item, a non surfactant powder, was dissolved at 20 % (w/v) in physiological saline and tested on the epithelial surface of three idoneous bovine corneas, for an exposure period of 4 hours.

Observations

The opacity of two of the treated corneas, detected with an opacimeter at the end of the test item exposure period, was 0.0; residual test item was observed on the surface of the third replicate thus the opacity value obtained (18) was not considered reliable. This was confirmed by macroscopic observation, where no alterations of corneas surfaces were noted. After the determination of opacity, the epithelial surface was treated with a 0.5 % solution of sodium fluorescein in DPBS for approximately 90 minutes to investigate alteration in cornea permeability. The mean value of permeability (OD490) of the corneas treated with the test item was 0.0022. The calculated in vitro irritancy score (IVIS) of the test item was 0.0.

Positive and negative controls [a 20 % (w/v) Imidazole solution in physiological saline and physiological saline alone, respectively] were concurrently tested in similar conditions and gave the expected results.

Result

According to the OECD Guideline no. 437, the test item can be classified as Not irritant.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin Irritation

The test substance was evaluated for its skin irritation potential according to the EU method B.46 and OECD 439.

During the test three tissues of the human skin model EpiDermTM has been treated with on average 25 mg of test item for 60 minutes. Both positive and negative control have been prepared. The observed value of the relative absorbance is above the threshold for irritation potential set out in the OECD guideline, therefore the substance is considered as not irritant in the Human Skin Model Test.

 

Eye Irritation

The test substance was evaluated for its eye irritation potential according to the OECD Guideline no. 437 and the Guidance Document OECD series on testing and assessment no. 160.

The test item, a non surfactant powder, was dissolved at 20 % (w/v) in physiological saline and tested on the epithelial surface of three idoneous bovine corneas, for an exposure period of 4 hours.

Under the test conditions, the test item does not show any eye irritation potential.


Justification for selection of skin irritation / corrosion endpoint:
Test conducted according to internationally accepted Guideline and in according to the GLP Principles

Justification for selection of eye irritation endpoint:
Test conducted according to internationally accepted Guideline and in according to the GLP Principles

Justification for classification or non-classification

Skin Irritation

The ECHA Guidance on the application of the CLP Criteria set out that normally, therecommendations for classification according to GHS criteria based on the results of anin vitro test are mentioned in the corresponding OECD test guideline. According to the OECD Guideline 439 the cut-off values for the prediction of irritation are:

-  The test chemical is considered to be irritant to skin in accordance with UN GHS Category 2 if the tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50 %.

- The test chemical may be considered as non-irritant to skin in accordance with UN GHS No Category if the tissue viability after exposure and post-treatment incubation is more than (>) 50 %.

 

The test item showed a value of the relative absorbance (112.2 %) above the threshold for irritation potential set out in the OECD guideline, therefore the substance is considered as NOT IRRITANT

 

Eye Irritation

The ECHA Guidance on the application of the CLP Criteria set out that asubstance can be considered as causing serious eye damage (Category 1) based on positive results in the BCOP test, and a negative results from the BCOP test methods can be used for classification purposes.

According to the OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants),the IVIS cut-off values for identifying test chemicals as inducing serious eye damage (UN GHS Category 1) and test chemicals not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are:

- IVIS ≤ 3 – No Classification (UN GHS)

- IVIS > 3; ≤ 55 – No prediction can be made

- IVIS > 55 – Category 1 (UN GHS)

The test item showed a value ofin vitro irritancy score (IVIS ) of 0.0 therefore, according to the OECD Guideline no. 437, the test item can be classified as “No Category” and considered as NOT IRRITANT.