Registration Dossier

Administrative data

Description of key information

The substance was found to be not eye irritating nor skin irritating.

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:
08.08.2016 - 15.08.2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2009
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium fuer Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Source strain:
other: not applicable
Justification for test system used:
Dermal irritation is generally defined as "the production of reversible inflammatory changes in the skin". The potential for chemical induced skin irritation is usually determined in vivo in the Draize rabbit skin irritation test as described in OECD guideline 404. However, because systemic reactions play a minor role in modulating local skin toxicity potential of chemicals, skin irritation potential may be predicted by in vitro systems, provided they are sufficiently complex to mimic human skin barrier and cell reactivity. In an international prevalidation study performed by ECVAM, the in vitro skin irritation test using the human skin model EpiDermTM and EpiSkinTM and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for skin irritancy potential.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi-200-SIT Kit
- Tissue batch number(s): 23349

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 35 minutes at 37 °C and 25 minutes at room temperature
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: at least 15 times
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 42 hours
- Spectrophotometer: Versamax® Molecular Devices, Softmax Pro, version 4.7.1
- Filter: 570 nm

NUMBER OF REPLICATE TISSUES: 3

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The substance is considered skin irritant category 2 according to UN GHS (published 2003, last (6th) revision 2015) if the mean relative tissue viability of three individual tissues is reduced ≤ 50% of the negative control.

ACCEPTANCE CRITERIA
- Negative control: The absolute OD 570 nm of the negative control tissues in the MTT test is an indicator of tissue viability obtained after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is meeting the acceptance criterion if the mean OD570 of the negative control tissues is ≥ 0.8 and ≤ 2.8.
- Positive control: An assay is meeting the acceptance criterion if mean relative tissue viability of the positive control is ≤ 20%.
- Standard deviation: The rel. SD of 3 identical replicates should be < 18%. OD values should not be below historically established boundaries.
- Historical data and the quality certificate of the supplier of the test kit demonstrated the robustness of the test system / test kit.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 µL
- Concentration (if solution): undiluted; 47 µL/cm2

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): undiluted

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5%
Duration of treatment / exposure:
60 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
99.8
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not lead to a change in colour.
Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.
The acceptance criteria were met.

Results

* Relative absorbance per tissue [rounded values]: 100 × (absorbance tissue) / (mean absorbance negative control)

** Relative absorbance per treatment group [rounded values]: 100 × (mean absorbance test item/positive control) / (mean absorbance negative control )

 

Dose Group

Tissue No.

Absorbance 570 nm Well 1

Absorbance 570 nm Well 2

Absorbance 570 nm Well 3

Mean Absorbance of 3 Wells

Mean Absorbance
 of 3 wells blank corrected

Mean Absorbance
of 3 tissues after blank correction

Rel. Absorbance [%] Tissue 1, 2 + 3*

Relative Standard Deviation [%]

Mean Rel. Absorbance [% of Negative Control]**

Blank

 

0.038

0.039

0.039

0.038

0.000

 

 

 

 

Negative Control

1

1.658

1.633

1.664

1.652

1.613

1.690

95.5

4.0

100.0

2

1.792

1.783

1.755

1.777

1.738

102.9

3

1.782

1.766

1.721

1.756

1.718

101.7

Positive Control

1

0.104

0.100

0.101

0.101

0.063

0.060

3.7

5.3

3.5

2

0.094

0.096

0.096

0.095

0.057

3.4

3

0.097

0.098

0.097

0.097

0.059

3.5

Test Item

1

1.698

1.650

1.668

1.672

1.634

1.687

96.7

3.4

99.8

2

1.776

1.705

1.673

1.718

1.679

99.4

3

1.811

1.749

1.798

1.786

1.748

103.4

 

 

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008
Conclusions:
In this study and under the experimental conditions reported, the test item is non-irritant to skin.
Executive summary:

In the current study the skin irritation potential of the test item was assessed by means of the Human Skin Model Test according to OECD 439 and GLP.

In a pretest the colourless test item did not reduce MTT (test for direct MTT reduction) or did not change the colour when it was mixed with deionised water (test for colour interference). Consequently, additional tests with freeze-killed or viable tissues were not necessary.

The main study consisted of topical exposure of the test item to the human reconstructed epidermis model followed by a cell viability test. The cell viability was measured by dehydrogenase conversion of MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazoliumbromide], in cell mitochondria, into a blue formazan salt that was quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison of untreated negative controls was used to predict the skin irritation potential of the substance and used for the purpose of classification as irritating or non-irritating. The test chemical is considered to be irritant to skin in accordance with UN GHS and EU CLP Category 2 if the tissue viability after exposure and post-treatment incubation is ≤ 50%.

In the main study three tissues of the human skin model EpiDermTM were treated with the test item, the negative control (DPBS) or the positive control (5% SLS) for 60 minutes.

Hereafter the skin tissues were washed and further incubated for about 42 hours, whereafter the tissues were treated with MTT for 3 hours following about 2.7 hours extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm.

The negative control had absorbance values well within the required range of the acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues. The positive control induced a sufficient decrease in the relative absorbance compared to the negative control, thus assuring the validity of the test system.

The relative standard deviations between the % variability values of the test item, the positive and negative controls in the main test were below the threshold of the OECD TG, thus ensuring the validity of the study.

Treatment with the test item resulted in a mean relative absorbance value of 99.8% compared to the negative control. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritating potential.

It can be stated that in this study and under the experimental conditions reported, the test item is non-irritant to skin.

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

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10.-14.10.2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
Feb. 1987
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Version / remarks:
Jan. 1997.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Ministerium fuer Landwirtschaft, Umweltschutz und Raumordnung, Land Brandenburg, Potsdam
Species:
rabbit
Strain:
other: Chbb:IIM(SPF) - Littlerussian
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Weight animals: 2.4 kg bw
- Housing: individually in PPO cages (floor area: 2576 sq.cm) with perforated floor.
- Diet: pelleted complete rabbit diet "Altromin 2123" from Altromin, D-32791 Lage, Lippe; ad libitum.
- Water: free access to domestic quality drinking water acidified with hydrochloric acid to pH 2.5
- Acclimation period: at least one week

ENVIRONMENTAL CONDITIONS
- Temperature: 20°C ± 3°C
- Humidity: 55% ± 15%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): cycle of 12 hours light and 12 hours darkness. Light: 6 a.m. to 6 p.m..

DATES:
The experimental work was carried out between 10.10.2000 and 14.10.2000.
Vehicle:
unchanged (no vehicle)
Controls:
other: yes, the untreated eye of the rabbit served as control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1mL
- Concentration: undiluted
Duration of treatment / exposure:
Single exposure, no rinsing.
Observation period (in vivo):
72 hours
Number of animals or in vitro replicates:
4
Details on study design:
REMOVAL OF TEST SUBSTANCE
- no rinsing

SCORING SYSTEM: See 'Any other information on materials and methods'

TOOL USED TO ASSESS SCORE:
After the first 24h reading, flurescein was instilled. After rinsing with 20 mL 0.9% sodium chloride solution the eyes were examined again using UV-light to detect possible corneal damage.
Irritation parameter:
cornea opacity score
Basis:
animal: #1, #2, #3, #4
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: reversibility: not applicable
Irritation parameter:
iris score
Basis:
animal: #1, #2, #3, #4
Time point:
24/48/72 h
Score:
0
Max. score:
2
Reversibility:
other: reversibility: not applicable
Irritation parameter:
conjunctivae score
Basis:
animal: #2, #3, #4
Time point:
24/48/72 h
Score:
0.33
Max. score:
3
Reversibility:
fully reversible within: 48 h
Irritation parameter:
conjunctivae score
Basis:
animal: #1
Time point:
24/48/72 h
Score:
0
Max. score:
3
Reversibility:
other: reversibility: not applicable
Irritation parameter:
chemosis score
Basis:
animal: #1, #2, #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: reversibility: not applicable
Irritation parameter:
chemosis score
Basis:
animal: #4
Time point:
24/48/72 h
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 48 h
Irritant / corrosive response data:
One hour after application of the test article animals No. 1926 and No. 1928 showed some conjunctival vessels definitely injected and a discharge different from normal. In animal No. 1929 some conjunctival vessels definitely injected were observed and a discharge with moistening of the lids and hairs just adjacent to lids. Animal No. 1930 showed some conjunctival vessels definitely injected, a swelling above normal and a discharge with moistening of the lids and hairs just adjacent to lids.

24 hours after application of the test article animals No. 1928 and No. 1929 had some conjunctival vessels definitely injected. In animal No. 1930 were observed some conjunctival vessels definitely injected and a swelling above normal. The animal No. 1926 was free of any signs of eye irritation.

48 hours and 72 hours after application of the test article all four animals No. 1926, No. 1928, No. 1929 and No. 1930 were free of any signs of eye irritation.

Scores for ocular lesions:

*Individual mean score: Only the scores from the readings after 24, 48 and 72 hours are included in the

calculation of the individual mean scores.

 Rabbit No/Weight per kg  Parameter  Individual mean score*
 1926/2.4  cornea opacity  0.00
   iris  0.00
   conjunctiva redness  0.00
   conjunctiva chemosis  0.00
 1928/2.4  cornea opacity  0.00
   iris  0.00
   conjunctiva redness  0.33
   conjunctiva chemosis  0.00
 1929/2.4  cornea opacity  0.00
   iris  0.00
   conjunctiva redness  0.33
   conjunctiva chemosis  0.00
 1930/2.4  cornea opacity  0.00
   iris  0.00
   conjunctiva redness  0.33
   conjunctiva chemosis  0.33
Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008
Conclusions:
The test item is not to be classified as eye irritating according to CLP regulation.
Executive summary:

In the current study the eye irritant effects of the test item were investigated according to the method recommended in the OECD Guideline No. 405, and EEC Guideline B.5. The study was performed in accordance with GLP.

Four female albino rabbits were exposed to 0.1 mL of the test article in one eye, while the other eye remained untreated and served as control. The eyes were examined and the changes were graded according to a numerical scale 1, 24, 48 and 72 hours after dosing.

Only very slight signs of irritation were observed on the treated eyes. Compared to the classification criteria indicated in the CLP regulation, the test item shall not be classified as eye irritating.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation

For this endpoint there is one study available in which the irritation potential of the test item was assessed according to OECD 439 and GLP.

In this study 3 tissues of the human skin model EpiDermTM were used and treated with the test item, the negative control (DPBS) or the positive control (5% SLS) for 60 minutes. Hereafter the skin tissues were washed and further incubated for about 42 hours, whereafter the tissues were treated with MTT for 3 hours following about 2.7 hours extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm.

The negative control had absorbance values well within the required range of the acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues. The positive control induced a sufficient decrease in the relative absorbance compared to the negative control, thus assuring the validity of the test system. The relative standard deviations were below the threshold, thus ensuring the validity of the study.

Treatment with the test item resulted in a mean relative absorbance value of 99.8% 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 irritating potential.

Eye irritation

There is one study available that assesses the possible irritation or corrosion potential of the test substance to the eye. The study was according to GLP and OECD 405.

Four female albino rabbits were exposed to 0.1 mL of the test article in one eye, while the other eye remained untreated and served as control. The eyes were examined and the changes were graded according to a nurnerical scale one hour, 24, 48 and 72 hours after dosing. No or only very slight signs of irritation were observed on the treated eyes. Compared to the classification criteria indicated in the CLP regulation, the test item shall not be classified as eye irritating.

Justification for classification or non-classification

Skin irritation

The result of an OECD 439 study was a mean relative absorbance value of 99.8% 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 irritating potential.

Eye irritation

According to the EU Regulation No. 1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures (CLP) a substance has irreversible effects on the eye (Category 1) if, when applied to the eye of an animal, a substance produces

- at least in 1 animal effects on the cornea, iris or conjunctiva that are not expected to reverse or have not fully reversed within an observation period of normally 21 days; and/or

- at least in 2 or 3 tested animals, a positive response is seen of corneal opacity >= 3 and/or iritis > 1.5

calculated as the mean scores of gradings at 24, 48 and 72 hours after installation of the test material.

A substance is considered irritating to the eyes (Category 2) if, when applied to the eye of an animal, a substance produces

- at least in 2 or 3 tested animals a positive response of corneal opacity >= 1 and/or iritis >= 1 and/or conjunctival redness >= 2 and/or conjunctival oedema (chemosis) >= 2

calculated as the mean scores of gradings at 24, 48 and 72 hours after installation of the test material, and which are fully reversible in an observation period of 21 days.

No or only very slight signs of irritation were observed with a mean score below the given threshold values, and the observed effects were fully reversible. Therefore, the substance is not to be classified as eye irritant according to the criteria set out in CLP regulation.