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Administrative data

Description of key information

In an in vitro skin model the test item showed no indication of irritation.

An in vitro test system for mucous membrane irritation (BCOP) gave equivocal results. The in vivo test in rabbits eyes showed no irritating potential.

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:
07 January 2016 until 01 February 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: according to OECD 439 Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
other: UN GHS (2003, last rev. 2009)
Deviations:
no
Principles of method if other than guideline:
Based on a “Statement on the Scientific Validity of In Vitro Tests for Skin Irritation” of the European Commission (November 2008), official acceptance of the test method in the EU was achieved and implemented in EU, 2008a, Council Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to EC Regulation No 1907/2006 of the European Parliament and of the Council on REACH; 1st ATP 2009: EC Regulation No 761/2009 of 23 July 2009 amending, for the purpose of its ATP, EC Regulation No 440/2008 laying down test methods pursuant to EC Regulation No 1907/2006 of the European Parliament and of the Council on REACH, section B46.
GLP compliance:
yes (incl. certificate)
Remarks:
Date of inspection: 13-16 July 2015, Date of Signature: 14 September 2015
Test material information:
Composition 1
Test system:
human skin model
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
Each approximately 25 mg (~ 39 mg/cm2 according to guideline) of the test item were applied to the tissues, wetted with 25 µL of DPBS, and spread to match the surface of the tissue for a complete treatment time of 60 minutes.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight):
DPBS was used as negative control. 30 µL were applied to each of triplicate tissues for 1 hour.

POSITIVE CONTROL
- Amount(s) applied (volume or weight):
A 5% SLS solution in deionised water was used as positive control. 30 µL were applied to each of triplicate tissues for 1 hour.
Duration of treatment / exposure:
60 minutes
Species:
other: reconstituted human epidermis model
Type of coverage:
other: Topical
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
Each approximately 25 mg (~ 39 mg/cm2 according to guideline) of the test item were applied to the tissues, wetted with 25 µL of DPBS, and spread to match the surface of the tissue for a complete treatment time of 60 minutes.
Duration of treatment / exposure:
60 minutes
Details on study design:
Each three tissues of the human skin model EpiDerm™ were treated with the test item, the negative and the positive control for 60 minutes.
Approximately 25 mg of the test item were applied to each tissue, wetted with 25 µL of DPBS, and spread to match the surface of triplicate tissue.
30 µL of either the negative control (DPBS) or the positive control (5% SLS) were applied to triplicate tissue each.
The test item and the positive and negative controls were washed off the skin tissues after 60 minutes treatment. After further incubation for about 43 hours the tissues were treated with the MTT solution for 3 hours following 69.75 hours extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm.
Irritation / corrosion parameter:
% tissue viability
Remarks:
Episkin model, 3 tissues per concentration
Run / experiment:
mean
Value:
80.5
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Other effects / acceptance of results:
No other effects were detected

Results after treatment with Aluminium-Braun 1134 p and the controls (60 minutes exposure interval)


Dose Group Tissue
No.
Absorb.
570 nm
Well 1
Absorb.
570 nm
Well 2
Absorb.
570 nm
Well 3
Mean
Absorbance
of 3 Wells
Mean Absorbance
of 3 Wells
blank
corrected
Mean
Absorbance*
after blank
correction
Rel.
Absorbance
[%] Tissue
1, 2 + 3*
Relative Standard Deviation
[%]
Mean Rel.
Absorbance
[% of Negative Control]**
Mean Absorbance
Blank corrected viable tissue
without MTT
(Step 2)
Mean Rel.
Absorbance
[% of Negative Control]
after colour interference and MTT reduction correction
Blank   0.036 0.037 0.036 0.036 0            
Negative
Control
1 1.736 1.703 1.695 1.711 1.675 1.673 100.1 1.1 100    
2 1.679 1.688 1.699 1.689 1.653 98.8
3 1.725 1.713 1.74 1.726 1.69 101
Positive
Control
1 0.116 0.116 0.118 0.117 0.081 0.075 4.8 6.6 4.5    
2 0.107 0.111 0.109 0.109 0.073 4.4
3 0.108 0.105 0.11 0.107 0.071 4.3
Test
 Item
1 1.322 1.323 1.34 1.328 1.292 1.36 77.2 6.4 81.3 0.013 80.5
2 1.483 1.484 1.515 1.494 1.458 87.2
3 1.363 1.334 1.402 1.366 1.33 79.5

*          relative absorbance per tissue [rounded values]

**        relative absorbance per treatment group [rounded values]


Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item is not irritant to skin according to UN GHS and EU CLP regulation.
Executive summary:

This in vitro study was performed to assess the irritation potential of the test item by means of the Human Skin Model Test.

The test item passed the MTT interference pre-test. Due to its intensive colour, an additional test with one viable tissue (without MTT addition) was necessary to correct the result in the main experiment.

Approximately 25 mg of the test item were applied to each tissue, wetted with 25 µL of DPBS, and spread to match the surface of triplicate tissue.

30 µL of either the negative control (DPBS) or the positive control (5% SLS) were applied to triplicate tissue each.

The test item and the positive and negative controls were washed off the skin tissues after 60 minutes treatment. After further incubation for about 42 hours the tissues were treated with the MTT solution for 3 hours following 69 hours extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm.

After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD³ 0.8 and ≤ 2.8 for the 60 minutes treatment interval thus showing the quality of the tissues.

Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 4.5% thus ensuring the validity of the test system.

The relative standard deviations between the % variabilities of the test item, the positive and negative controls in the main test were below 7% (threshold of the "OECD Guideline for the Testing of Chemicals 439:In vitroSkin Irritation: Reconstructed Human Epidermis Test Method”: < 18%), thus ensuring the validity of the study.

Compared to the relative absorbance value of the negative control the mean relative absorbance value was reduced to 80.5% (corrected value) after exposure of the skin tissues to the test item. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

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:
supporting study
Study period:
The test was performed on 05 February 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.
Reference:
Composition 1
Qualifier:
according to
Guideline:
other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997.
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD Guideline for Testing of Chemicals 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 (July, 2013).
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Date of inspection: 25 April 2012, 23 ,25,Date of inspection: 13-16 July 2015, Date of Signature: 14 September 2015
Test material information:
Composition 1
Species:
other: Freshly isolated bovine cornea (at least 9 month old donor cattle)
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Source: Schlachthof Aschaffenburg, 63739 Aschaffenburg, Germany
Vehicle:
physiological saline
Controls:
other: 10% (w/v) Benzalkonium chloride in 0.9% (w/v) NaCl (saline); negative control: Saline
Amount / concentration applied:
The test item was tested as a 20% suspension (w/v) in saline. The test item could not be suspended or solved homogeneously, therefore,
each 0.75 mL of the prepared stock was distributed to each cornea.
Duration of treatment / exposure:
240 minutes
Number of animals or in vitro replicates:
3 corneae per group (test item, negative control, positive control)
Details on study design:
Three corneas were exposed to each 0.75 mL of a 20% (w/v) suspension of the stest item in physiological saline for 240 minutes.
After treatment the test item suspension was rinsed off the corneas and the corneas' opacity was determined. In a second step the permeability of the corneas was determined photometrically after 90 minutes treatment with fluorescein solution.

SCORING SYSTEM:
Opacity measurement
The opacitometer determines changes in the light transmission passing through the corneae, and displays a numerical opacity value. This value was recorded in a table. The opacitometer OP_KiT opacitometer (Electro Design, 63-Riom France) was calibrated as described in the manual and the opacity of each of the corneae was determined by reading each holder placed in the photoreceptor compartment for treated cornea.

For equilibration and prior to application of the test item or controls, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath. At the end of the incubation period, the basal opacity was determined (t0). After exposure of the corneae to the test groups, after rinsing and further incubation of the corneae for two hours, the opacity value was determined again (t240).

Permeability Determination
Following to the opacity readings, the permeability was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the incubation medium will be removed from the anterior compartment and replaced by 1 mL of a 0.5% (w/v) sodium fluorescein solution in HBSS. Corneae were incubated again in a horizontal position for 90 ± 10 minutes in a water-bath at 32 ± 1 °C. Incubation medium from the posterior compartment were removed, well mixed and transferred into a 96 well plate and the optical density at 490 nm (OD490) was determined with a spectrophotometer (Versamax® Molecular Devices). The absorbance values will be determined using the software SoftMax Pro Enterprise (version 4.7.1).

DATA EVALUATION
Opacity
The change of opacity value of each treated cornea or positive and negative control corneae is calculated by subtracting the initial basal opacity from the post treatment opacity reading (t240 – t0), for each individual cornea.
The average change in opacity of the negative control corneae is calculated and this value is subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.

Permeability
The corrected OD490 value of each cornea treated with positive control and test item is calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.

IVIS Calculation
The following formula is used to determine the IVIS of the negative control:
IVIS = opacity value + (15 x OD490 value)
The following formula is used to determine the IVIS of the positive control and the test item:
IVIS = (opacity value – opacity value mean negative control) + (15 x corrected OD490 value)
The mean IVIS value of each treated group is calculated from the IVIS values.
Depending on the score obtained, the test item is classified into the following category according to OECD guideline 437:

IVIS: In vitro Irritancy Score (according to OECD 437):

≤ 3 No Category (according to GHS)
> 3; ≤ 55 No prediction can be made
> 55 Serious eye damaging according to CLP/EPA/GHS (Cat 1)


Criteria for Determination of a Valid Test

The test will be acceptable if
• the positive control gives an IVIS that falls within two standard deviations of the current historical mean (updated every three months), and if
• the negative control responses result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.
Irritant / corrosive response data:
moderate increase of the corneal opacity and permeability
no prediction for the damage hazard of the test item to the eye can be made
Other effects:
none

Results after 240 Minutes Incubation Time


Test Group

Opacity value = Difference (t240-t0) of Opacity

Permeability at 490 nm (OD490)

IVIS

Mean IVIS

Proposedin vitroIrritancy Score

 

 

Mean

 

Mean

 

 

 

Negative Control

1

0.33

0.080

0.074

2.20

1.44

Not categorized

0

0.062

0.093

0

0.080

1.20

Positive Control

152.67*

0.087*

153.97

151.43

Category 1

149.67*

0.150*

151.92

146.67*

0.155*

148.39

Aluminium-Braun

1134 p

11.67*

0.124*

13.53

15.98

No prediction can be made

15.67*

0.060*

16.57

16.67*

0.078*

17.84

*corrected values

Interpretation of results:
study cannot be used for classification
Remarks:
Migrated information
Conclusions:
In conclusion, according to the current study and under the experimental conditions reported, the test substance is not serious eye damaging (CLP/EPA/GHS (Cat 1) but a prediction for the damage hazard cannot be made (GHS).
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

No classification

Neither in a skin model nor in an in vivo eye irritation study any irritant effects were deteced.