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Description of key information

Skin Irritation

REACH_not irritating | Human skin model | OECD Draft Guideline | #key study#

Eye Irritation

REACH_not irritating | OECD 492 EpiOcular | #key study#

REACH_not irritating | HET-CAM | #key study#

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:
June 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: ECVAM Skin Irritation Validation Study
Principles of method if other than guideline:
This study was conducted using a reconstruced human epidermis model and was in accordance with the following references:
- ECVAM international validation study on in vitro tests for acute skin irritation: Report on the validity of the EPISKIN and EpiDerm assays and on the Integrity Funktion Test (Altern Lab Anim. 2007 Dec; 35 (6): 559-601.
- European Commission: Institute for Health and Consumer Protection and European Centre for the Validation of Alternative Methods (ECVAM): Statement on the validity of in vitro tests for skin irritation (2007)
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Remarks:
EpiSkin Standard Model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
skin obtained from plastic surgery from multiple donors
Source strain:
not specified
Vehicle:
unchanged (no vehicle)
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
15 µL
Duration of treatment / exposure:
15 min
Duration of post-treatment incubation (if applicable):
42 h
Number of replicates:
3
Irritation / corrosion parameter:
other: Optical density (OD)
Run / experiment:
Time point: 15 min
Value:
0.85
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
0.82
Positive controls validity:
valid
Remarks:
0.13
Remarks on result:
other:
Remarks:
Max. score: 1.6
Irritation / corrosion parameter:
other: Rel. Absorbance
Run / experiment:
Time point: 15 min.
Value:
104
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100.0
Positive controls validity:
valid
Remarks:
16.2
Other effects / acceptance of results:
no other effects

Dose group

Treatment Interval

Absorbance

570 nm

Tissue 1*

Absorbance

570 nm

Tissue 2*

Absorbance

570 nm

Tissue 3*

Mean Absorbance of 3 Tissues

Standard

Deviation

of 3

Tissues

Rel. Absorbance [% of Negative Control]**

Standard

Deviation [%]

Negative Control

15 min

0.8909

0.8028

0.7786

0.8241

0.0591

100.0

7.2

Positive Control

15 min

0.1243

0.1320

0.1432

0.1331

0.0095

16.2

1.2

Sa 190

15 min

0.8821

0.7955

0.8939

0.8571

0.0537

104.0

6.5

* Mean of two replicate wells after blank correction /

** relative absorbance [rounded values]:100 (absorbancetest item)/ (absorbancenegative control)

Interpretation of results:
GHS criteria not met
Conclusions:
Classification: not irritating
Executive summary:

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item Sa 190 is not irritant to skin.

This in vitro study was performed to assess the irritation potential of Sa 190 by means of the Human Skin Model Test.

Three tissues of the human skin model EPISKIN were treated with either the test item, the negative or the positive control for 15 minutes.

15 μL of the melted test item were applied to each tissue and spread to match the tissue size.

15 μL of either the negative control (deionised water) or the positive control (5% Sodium lauryl sulfate) were applied to each tissue.

The test item as well as the positive and negative controls were washed off the skin tissues after 15 minutes treatment. After further incubation for 42 hours the tissues were treated with the MTT solution for 3 hours following 72 hours 10 minutes 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 above the required acceptability criterion of mean OD ≥ 0.6 for the 15 minutes treatment interval thus showing the quality of the tissues.

Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the 15 minutes treatment interval thus ensuring the validity of the test system.

The standard deviations between the % variabilities of the test item, the positive and negative controls were below 18% thus ensuring the validity of the study.

The control values are well in the range of our historical data (see chapter 12).

After treatment with the test item Sa 190 the mean relative absorbance value did not decrease (104.0%) compared to the negative control. This value is well above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item Sa 190 is not irritant to skin.

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:
2017-12-19 to 2018-01-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD Guideline for the Testing of Chemicals No. 492: Reconstructed human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labelling for eye irritation ornea-like Epithelium (RhCE) test method)
Version / remarks:
28 July 2015
Qualifier:
according to guideline
Guideline:
other: EURL ECVAM DB-ALM Method Summary No. 164: EpiOcular™ Eye Irritation Test - Summary
Version / remarks:
22 July 2015
Qualifier:
according to guideline
Guideline:
other: EpiOcular™ Eye Irritation Test (OCL-200-EIT) For the prediction of acute ocular irritation of chemicals For use with MatTek Corporation’s Reconstructed Human EpiOcular™ Model irritation of chemicals
Version / remarks:
29 June 2015
GLP compliance:
yes (incl. QA statement)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany)
Species:
other: Reconstructed human cornea-like epithelium (RhCE) EpiOcular
Details on test animals or tissues and environmental conditions:
The test was carried out with the EpiOcular™ reconstructed human cornea-line epithelium (RhCE) model (MatTek). The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified, highly differentiated squamous epithelium morphologically similar to that found in a human cornea. The EpiOcular™ RhCE tissue construct consists of at least 3 viable layers of cells and a non-keratinised surface, showing a cornea-like structure analogous to that found in vivo.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
1. Negative Control 50 μL Aqua dest.
2. Positive Control 50 μL methyl acetate
3. Test Item 50 μL (undiluted)
Duration of treatment / exposure:
30 +/- 2 min.
Duration of post- treatment incubation (in vitro):
- post soak incubation: 12 +/- 2 min.
- post treatment incubation: 120 +/- 15min
- MTT incubation: 3h
Number of animals or in vitro replicates:
The test was performed on a total of 2 tissues per dose group.
Details on study design:
The test was performed on EpiOcular, a reconstituted three-dimensional human corneal epithelium model. Hereby, the test item was applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 30 min exposure period and 120 min post-treatment period and compared to those of the concurrent negative controls.
Irritation parameter:
other: Mean relative tissue viability [%]
Run / experiment:
Mean tissues 1 and 2
Value:
97.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100
Positive controls validity:
valid
Remarks:
20.5
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
TEST ACCEPTANCE CRITERIA

Value Cut off pass/fail
Mean Absolute OD570 nm NK 1.814 0.8 < NK < 2.5 pass
Mean Relative Viability PC [%] 20.5 < 50% pass
Max. Difference of % Viability [%] 1.4 < 20% pass

The mixture of 50 µl test item per 1 mL MTT medium showed reduction of MTT as compared to the solvent. The mixture turned blue/purple. Since the mean relative tissue viability of the test item treated tissues (TM) was above the 60% threshold value killed tissue controls were performed for quantitative correction of results

NSMTT [%] = [(ODKT- ODKU)/ODNK] * 100 = 0.99%

Difference of NSMTT of the two duplicate tissues must be < 20%, otherwise not accepted.

NSMTT1 [%] = [meanODKT1 - ODKU)/ODNK] * 100 = -0.34%

NSMTT2 [%] = [meanODKT2 - ODKU)/ODNK] * 100 = 2.32%

NSMTT1 - NSMTT2 =± 2.66%

NSMTT was ≤ 60% (0.99%) relative to the negative control of living epidermis and could therefore be used for determination of the killed control corrected viability (KCCV) according to the following formula:

KCCV [%] = viabilityTM- NSMTT = 97.0%

 

The mixture of 50 µL test item per 1 mL A. dest showed colouring as compared to the solvent. Since the mean relative tissue viability of the test item treated tissues (TM) was above the 60% threshold value coloured tissue controls were performed for quantitative correction of results.

NSCliving [%] = [ODTVT/ODNK]*100 = 0.47%

Difference of NSCliving of the two duplicate tissues must be < 20%, otherwise not accepted.

NSC1 [%] = [ODTVT1/ODNK] * 100 = 0.39%

NSC2 [%] = [ODTVT2/ODNK] * 100 = 0.54%

NSC1 – NSC2 = ±0.15%

NSClivingwas ≤ 60% (0.47%) relative to the negative control of living epidermis and could therefore be used for determination of theNSC-corrected mean relative tissue viability (NSCCV) according to the following formula:

NSCCV [%] = viabilityTM[%] – NSCliving[%] = 96.4%

 

Since the test item showed non-specific MTT-reduction and non-specific colouring of living tissues, a third control for non-specific colour in killed tissues (NSCkilled) was performed to avoid a possible double-correction for colour interference.

The non-specificcolour of additional killed tissues (NSCkilled) was calculated according to the following formula:

NSCkilled [%] = [ODTKT/ODNK]*100 = 1.45%

The true tissue viability was then calculated as the percent tissue viability obtained with living tissues minus NSMTT minus NSClivingplus NSCkilled.

True Tissue Viability = [%] mean tissue viability – NSMTT –NSCliving+ NSCkilled= 97.9%

Result of the Test Item Sa 190

Name

Negative Control

Positive Control

Test item

Tissue

1

2

1

2

1

2

OD570values

1.798

1.775

0.400

0.405

1.763

1.755

1.847

1.837

0.401

0.424

1.817

1.777

OD570values
(blank-corrected)

1.754

1.731

0.356

0.361

1.719

1.711

1.803

1.794

0.357

0.380

1.774

1.733

mean of the duplicates

1.778

1.762

0.356

0.371

1.746

1.722

mean OD

1.770*

0.364

1.734

TODTT- NSMTT

 -

 -

1.717

TODTTNSMTT und NSCliving

 -

 -

1.707

mean sd OD

0.011

0.010

0.017

tissue viability [%]

100.5

99.5

20.1

20.9

98.6

97.3

relative tissue viability difference [%]***

0.9

0.8

1.4

mean tissue viability [%]

100.0

20.5**

98.0

mean tissue viability [%]
- NSMTT corrected

 -

 -

97.0

mean tissue viability [%]
- NSMTT and NSClivingcorrected

 -

 -

96.4

True Tissue Viability

 -

 -

97.9

 

*              Corrected mean OD570 of the negative control corresponds to 100% absolute tissue viability

**             Mean relative tissue viability of the positive control is < 50%

***            Relative tissue viability difference of replicate tissues is < 20%

Result of the NSMTT Control

NSMTT

KU

KT

Negative Control

Tissue

1

2

1

2

1

2

absolute OD570 -values

0.074

0.075

0.067

0.115

1.798

1.775

0.075

0.076

0.071

0.117

1.847

1.837

OD570(Blank Corrected)

0.030

0.031

0.023

0.071

1.754

1.731

0.031

0.032

0.027

0.073

1.803

1.794

mean OD570
(mean of 2 aliquots)

0.031

0.031

0.025

0.072

1.778

1.762

total mean OD570
(mean of the replicate tissues)

0.031

0.049

1.770

SD OD570(of the replicate tissues)

0.000

0.033

0.011

NSMTT [%]

0.99

 -

Relative Tissue Viability [%]

 -

100.5

99.5

Mean Relative Tissue Viability [%]

 -

100.0

SD Tissue Viability [%]

 -

0.6

CV [% Viabilities]

 -

0.6

Result of the NSCliving Control

NSCliving

TVT

Negative Control

Tissue

1

2

1

2

absolute OD570 -values

0.050

0.053

2.098

2.048

0.051

0.055

2.111

2.021

absolute OD570 -
Blank corrected values

0.007

0.010

2.055

2.005

0.008

0.012

2.068

1.978

mean OD570
(mean of 2 aliquots)

0.008

0.011

2.062

1.991

total mean OD570
(mean of replicate tissues)

0.010

2.027

SD OD570
(of the 2 replicate tissues)

0.002

0.050

NSCliving[%]

0.47

 -

Relative Tissue Viability [%]

 -

101.7

98.3

Mean Relative Tissue Viability [%]

 -

100.0

SD Tissue Viability [%]***

 -

2.5

CV [% Viabilities]

 -

2.5

 

Result of the NSCkilled Control 

NSCkilled

TKT

Negative Control

Tissue

1

2

1

2

Absolute OD570 -values

0.065

0.078

2.098

2.048

0.064

0.082

2.111

2.021

Absolute OD570 -
Blank corrected values

0.022

0.035

2.055

2.005

0.021

0.039

2.068

1.978

Mean OD570
(mean of 3 aliquots)

0.022

0.037

2.062

1.991

Total Mean OD570
(Mean of Replicate Tissues)

0.029

2.027

SD OD570
(of the 2 replicate tissues)

0.011

0.050

NSCkilled[%]

1.45

 -

Relative Tissue Viability [%]

 -

101.7

98.3

Mean Relative Tissue Viability [%]

 -

100.0

SD Tissue Viability [%]

 -

2.5

CV [% Viabilities]

 -

2.5

Interpretation of results:
GHS criteria not met
Conclusions:
In this study under the given conditions the test item showed no irritant effects. The test item is classified as “non-irritant“ in accordance with UN GHS “No Category”
Executive summary:

In the present study the eye irritating potential of Sa 190 was analysed. Since irritant substances are cytotoxic to the corneal epithelium after a short time exposure the cytotoxic effects of the test item on EpiOcular, a reconstituted three-dimensional human corneal epithelium model, were determined. Hereby, the test item was applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 30 min exposure period and 120 min post-treatment period and compared to those of the concurrent negative controls.

The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 60% (97.9%; NSMTT-,NSCliving and NSCkilled-corrected).

The controls confirmed the validity of the study. The mean absolute OD570of the two negative control tissues was > 0.8 and < 2.5 (1.814). The mean relative tissue viability (% negative control) of the positive control was < 50% (20.5%). The maximum inter tissue difference of replicate tissues of all dose groups was < 20% (1.4%).

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test procedure according to validation study and GLP
Qualifier:
according to guideline
Guideline:
other: HET-CAM Test
Principles of method if other than guideline:
This study was conducted according to the procedures indicated by the folowing internationally accepted guideline and recomendation:
INVITTOX Protocol No. 47: HET-CAM Test. http://ecvam-sis.jrc.it
GLP compliance:
yes (incl. QA statement)
Species:
other: HET-CAM
Strain:
not specified
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
For each replicate about 300 µl of the test item were applied directly to the chorioallantoic membrane in order to cover at least 50 % of the membrane. 300 µl of each of the positive controls ( 1% solution in deion. water) and the negative control (physiological sodium chloride solution, 0.9% w/w) were applied to three eggs each.
Duration of treatment / exposure:
300 s
Observation period (in vivo):
300 s
Number of animals or in vitro replicates:
- test item: 6
- controls: 3 each
Details on study design:
CAM Preparation
On day 9, the day of the performance of the experiment, the eggs were candled once again in order to determine the position of the egg's oxygen bubble. The position was marked on the eggshell. Along the marking the eggshell was sawed with an electric saw. Afterwards, the shell above the oxygen bubble was removed. The egg skin beneath was covered with 0.9% (w/v) NaCl in deion. water (saline, produced in-house) in order to separate the egg skin from the chorioallantoic membrane beneath the skin. After 3 minutes incubation at 37.5 ± 0.5 °C the egg skin was removed from the membrane using forceps.

Treatment
In this HET-CAM assay the test item was tested pure. Six eggs were treated with the test item.
For each replicate about 300 μL of the test item were applied directly to the membrane in order to cover at least 50% of the membrane. 300 μL of each of the positive controls and the negative control were applied to three eggs each. During the observation period of 300 seconds any lesions in close proximity to the covered membrane were monitored and recorded.

Observations
The membranes of the eggs were observed for 300 seconds. Lesions of the underlying blood vessels were monitored and noted. In particular three endpoints were observed and the time point at which an effect occurs was recorded. The three endpoints were:
• haemorrhage
• coagulation
• lysis of the blood vesse
Irritation parameter:
in vitro irritation score
Run / experiment:
HET-CAM 300 s
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
0
Positive controls validity:
valid
Remarks:
9.92 (DDS); 19.12 (NaOH)
Other effects / acceptance of results:
no other effects

Mean irritancy index of 6 eggs: Test Item = 0.00

Mean irritancy index of 3 eggs:

Positive control (1% Sodium dodecyl sulphate) = 9.92

Positive control (0.1 N Sodium hydroxide solution) = 19.12

Negative control (Physiological sodium chloride solution (0.9%)) = 0.00

Interpretation of results:
GHS criteria not met
Remarks:
Criteria used for interpretation of results: INVITTOX
Conclusions:
Classification: not irritating
Executive summary:

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item Sa 190 does not possess any irritating potential.

This in vitro study was performed to assess the irritating potential of SAT 080004 by detection of damages in blood vessels under the chorioallantoic membrane of chicken eggs incubated for 9 days (Hen's Egg Test - Chorioallantoic Membrane Test, HET-CAM.

The test item was tested pure.

The observation time was 5 minutes at room temperature.

Physiological sodium chloride solution (0.9 % (w/v)) was used as negative control.

The negative control showed no irritating effect on the blood vessels under the membrane (mean irritancy index 0.00).

1 % solution of sodium dodecyl sulphate (SDS) and 0.1 N sodium hydroxid (NaOH) were used as positive controls.

The positive controls induced severe irritation on the blood vessels (mean irritancy indices of 9.92 for SDS and 19.12 for NaOH).

The mean irritancy indices of the controls are well comparable with the historical control values and are within the acceptance criteria. Therefore, it was concluded that the test was valid.

No irritating effects were observed during 5 min incubation with the test substance Sa 190. The calculated mean irritancy index is 0.00.

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

The test substance did not produce skin irritation and eye irritation. The substance does not need to be classified for irritation according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.