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Description of key information

Skin irritation in vitro: not irritant

OECD 439: negative

Skin irritation in vivo: insufficient for classification according to CLP (UN/GHS)

Eye irritation in vitro: irritant Cat 2

OECD 437: inconclusive, not cat 1

OECD 492: cat 1 or 2

Eye irritation in vivo: insufficient for classification according to CLP (UN/GHS)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 28, 2017 - August 22, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
Commission Regulation (EU) No. 640/2012 amending, for the purpose of its adaption to technical progress, Regulations (EC) No. 440/2008 laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and the council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
July 28, 2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Skinethic skin irritation test -42bis Standard operating procedure (SOP) 2009
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
No pre-treatment; the test item was applied neat to the tissues.
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from a single donor
Justification for test system used:
standard model
Vehicle:
unchanged (no vehicle)
Remarks:
No vehicle used in this study; The test item was applied neat to the tissues.
Details on test system:
CELL CULTURE
- Supplier: EpiSkin/SkinEthic Laboratories, Lyon, France)
- Source: human keratinocytes cultured on a polycarbonate filter in conditions which permit their terminal differentiation
- Format: 24 well plate
- Batch: 17-RHE-083
- Expires: August 7, 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment: room temperature
- Temperature of post-treatment incubation: 37°C

REMOVAL OF THE TEST MATERIAL AND CONTROL
After the end of the treatment interval, the residual test item was removed immediately by gently rinsing with a minimum volume of 25 mL DPBS using a pipette. Excess DPBS was removed by gently shaking the inserts and blotting the bottom with blotting paper.

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer:ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany at 570 nm


MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany
- Wavelength: 570 nm
- Linear OD range of spectrophotometer: 0 - 3.0

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA

Acceptance Criteria Result
Cell viability (570 nm optical density, MTT test) OD >0.7 OD 1.2 (CV = 5.5 %)

Barrier function integrity test
(Exposure time inducing 50%
viability using Triton X-100 1%) 4.0h ≤ ET50 ≤ 10.0h 4.4h

Histological observation
(HES stained vertical paraffin sections) Number of cell layers ≥ 4 5.5 cell layers
Absence of significant
histological abnormalities. Absence of significant histological abnormalities.

Well differentiated epidermis Satisfactory
consisting of basal, spinous,
granular layers and a stratum c
orneum.


NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1 experiment

PREDICTION MODEL / DECISION CRITERIA:
A test item is considered as non-irritant to skin (UN GHS No Category) if the tissue viability after exposure and post-treatment incubation is > 50%.
A test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and post-treatment incubation is ≤ 50%. Since the in vitro skin irritation test according to OECD 439 cannot resolve between UN GHS Categories 1 and 2, further information on skin corrosion is required to decide on its final classification.
Control samples:
yes, concurrent negative control
Amount/concentration applied:
TEST MATERIAL: 16 mg of solid test material
NEGATIVE CONTROL: 16 µL (Dulbecco`s Phosphate-Buffered Saline)
POSITIVE CONTROL: 16 µL (5% aqueous solution of sodium dodecyl sulfate in deionised water)
Duration of treatment / exposure:
42 min (± 1 minute)
Duration of post-treatment incubation (if applicable):
42 hours (± 1 hour)
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Experiment 1 / Run 1
Value:
88.8
Vehicle controls validity:
not applicable
Remarks:
The test item was applied neat to the tissues
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: none
- Colour interference with MTT: none

ACCEPTANCE OF RESULTS:

Acceptability of the Quality Control Data of the Skin Model with Reference to Historical Batch Data:

Acceptance Criterion Result
Negative control OD ≥ 0.8 and ≤ 3.0 2.065 to 2.242

Acceptability of the Positive and Negative Control stated by Episkin/SkinEthic Laboratories:

Acceptance Criterion Result
Mean OD negative control ≥ 1.2 2.140
Mean viability positive control < 40% 1.3%
SD of group-mean value ≤ 18% 7.7% (positive control)
4.3% (negative control)

Acceptability of the Positive and Negative Control based on Historical Data of the Testing Laboratory:

Acceptance Criterion Result
Mean OD negative control ≥ 1.455 2.140
Mean viability positive control ≤ 2.97% 1.3%

Test Item Data Acceptance Criteria:

Acceptance Criterion Result
SD of group-mean value ≤ 18% 3.4%

The study met all acceptance criteria.


 Group Time / [min]  Mean OD  Mean Relative viability / [%]
 Negative Control 42  2.140 100 
 Positive Control 42

0.028

1.3

 Test Material

42

1.846

88.8

Interpretation of results:
GHS criteria not met
Conclusions:
This study was performed according to GLP and the methods applied are fully compliant with OECD TG 439. Under the conditions of the present study, the test item is not considered to possess an irritant potential to skin (UN GHS: No Category).
Executive summary:

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 439. Under the conditions of the present study, the test item is not considered to possess an irritant potential to skin (UN GHS: No Category).

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1981-08-11 to 1981-08-14
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
Only a brief summary of the study is available
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
not applicable
GLP compliance:
not specified
Specific details on test material used for the study:
Appearance: yellow crystals

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Before application, the test material was moistened with 0.4 mL 0.9% NaCl solution
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
-Source: Hoechst AG, Frankfurt, Germany
-Weight at study initiation: 1.9- 2.7 kg
-Housing: single
-Diet: ad libitum
-Water: ad libitum
Type of coverage:
occlusive
Preparation of test site:
other: clipped and scarified
Vehicle:
physiological saline
Controls:
not required
Amount / concentration applied:
0.5 g per animal
Duration of treatment / exposure:
24 hours
Observation period:
up to 8 days
Number of animals:
6
Details on study design:
TEST SITE
-Area of exposure: 6*3 cm on the flank
-Type of wrap if used: adhesive plaster (Hansamed) and polyethylene foil

REMOVAL OF TEST SUBSTANCE
-Method: not specified
-Time after start of exposure: 24 hours

OBSERVATION TIME POINTS
-After removal of the plaster and after 48 and 72 hours
d then daily up to experimental day 8

SCORING SYSTEM
-According to OECD GL 404
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
other: not further specified
Score:
1.6
Reversibility:
not specified
Remarks on result:
probability of mild irritation
Irritant / corrosive response data:
In two rabbits effects on the skin (dry, rough, cracked, eschar formation) was noted

Study design

In this study performed equivalent to OECD GL 404, 500 mg of the test item was mixed with physiological saline soution to ensure good contact to the skin. Afterwards, the test material was applied to clipped and scarified skin for a 24 hours period under occlusive conditions. Scoring was performed after pathch removal (24 h), and after 48, and 72 hours.

Results

Under the conditions of the present study, the treatment caused a mild irritation (mean score: 1.6).

Conclusion

A mild irritating potential could be detected.

Interpretation of results:
study cannot be used for classification
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1981
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
Only a brief summary of the study is available
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
not applicable
GLP compliance:
not specified
Specific details on test material used for the study:
Description: yellow crystals
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
-Source: Hoechst AG, Frankfurt, Germany
-Weight at study initiation: 1.9 - 2.7 kg
-Housing: groupwise
-Diet: ad libitum
-Water: ad libitum
Vehicle:
physiological saline
Controls:
no
Amount / concentration applied:
100 mg test item moistened with 0.04 mL physiological saline solution
Duration of treatment / exposure:
24 hours
Observation period (in vivo):
up to 72 hrs
Number of animals or in vitro replicates:
6
Details on study design:
A volume of 100 mg of the test material was instilled into the conjunctival sac of the left eye, while the right eye remained untreated and was used for control purposes.

REMOVAL OF TEST SUBSTANCE
-The eyes were rinsed 24 hrs after application.

SCORING SYSTEM: according to OECD GL 405

The 48/72 hrs score was obtained after installation of one drop of a 0.01% fluorescein sodium.
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
other: 7 hours
Score:
6
Reversibility:
not specified
Remarks on result:
no indication of irritation
Interpretation of results:
GHS criteria not met
Conclusions:
The test material gave no indication for an eye irritation potential in rabbits.
Executive summary:

This study was performed equivalent to OECD GL 405 and gave no indication for an eye irritation potential in rabbits.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 28, 2017 - August 14, 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:
2013
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
Commission Regulation (EU) 2017/735 of 14 February 2017 amending, for the purpose of its adaption to technical progress, the Annex to Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
PREPARATION OF THE TEST MATERIAL
The test item was prepared as a 20% (w/v) solution in a 0.9% sodium chloride solution. The stability in the vehicle was not investigated. The test item preparation was administered within <1 hour after preparation.
Vehicle:
physiological saline
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL: 750 µL (i.e. 150mg/750µL)

NEGATIVE / VEHICLE CONTROL: 750 µL

Designation: 0.9% sodium chloride solution
Supplier: B. Braun Melsungen AG, Germany
Batch: 16435011
Storage: 2 to 8°C
Released until: September 2019


POSITIVE CONTROL: 750 µL

Designation: Art. 814223
Synonym: Imidazole
Supplier: Merck KGaA, Germany
Batch: S6746923
Purity (GC) 99.8% (a/a)
Storage: At room temperature
Released until: August 31, 2018

Imidazole was dissolved with 0.9% sodium chloride solution to a concentration of 20% (w/v).

Duration of treatment / exposure:
240 minutes
Number of animals or in vitro replicates:
in vitro: triplicate design
Irritation parameter:
in vitro irritation score
Run / experiment:
Run 1 / Experiment 1
Value:
3.4
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable
Remarks:
According to OECD 437 no prediction can be made regarding the eye hazard potential of the test item.
Other effects / acceptance of results:
OTHER EFFECTS:
- All corneas treated with the test item preparation showed a slight yellowish stain after removing the test item preparation by washing with wash medium.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: After treatment with the negative control (0.9% sodium chloride solution) the calculated IVIS was 0.2 and, thus, within three standard deviations of the current historical mean of the negative control (IVIS: -1.4 – 3.2).
- Acceptance criteria met for positive control: After treatment with the positive control (20% Imidazole) the calculated IVIS was 106.4 and, thus, also within two standard deviations of the current historical mean of the positive control (IVIS 80.5 – 133.0).

Therefore, the study fulfilled the acceptance criteria.

Opacity
Permeability
IVIS
per cornea
per group
(mean value)
SD
Negative control
0.9% NaCl Solution
0.0 0.002
0.030
0.2
0.4
0.0
0.003
0.045
0.6
0.004
0.660
Positive control
20% Imidazole solution
71.0
2.506
108.590
106.4
2.3
71.2
2.352
106.480
72.4
2.112
104.080
Test item
Test item
4.7
0.017
4.955
3.4
1.5
2.8
0.015
3.025
1.8
0.018
2.070


Interpretation of results:
other: Under the conditions of the present study, the eye hazard potential of the test item cannot be predicted.
Conclusions:
Under the conditions of the present study, the eye hazard potential of the test item cannot be predicted.
Executive summary:

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 437. Under the conditions of the present study, the eye hazard potential of the test item cannot be predicted.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 17, 2017 - November 8, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD 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:
October 9, 2017
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: DB-ALM Protocol No. 164: Ocular Irritation Assay for Chemicals using EpiOcular™ EIT,
Version / remarks:
September 14, 2015
Deviations:
no
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; MatTek Corporation
Version / remarks:
June 29, 2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: No pre-treatment, the test item was applied neat to the tissues.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL: 50 mg per tissue

NEGATIVE / VEHICLE CONTROL: 50 µL per tissue

Sterile deionized water was used as negative control.


POSITIVE CONTROL: 50 µL per tissue

Designation: Methyl acetate
Supplier: MatTek In Vitro Life Science Laboratories
Lot-No.: 032817ISA
Catalog #: TC-MA
Purity (GC): 99.7%
Appearance: Colorless liquid
Expiration date: March 28, 2018
Storage: 15 to 30°C

Duration of treatment / exposure:
6 hours
Number of animals or in vitro replicates:
in vitro: duplicate design
Irritation parameter:
other: Viability %
Run / experiment:
Run 1 / Experiment 1
Value:
4.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Remarks:
According to OECD 492 the test item is labeled irritant (UN GHS Category 1 or Category 2).
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No observations

ACCEPTANCE OF RESULTS:
1. The negative control OD is >0.8 and <2.5 (1.184 and 1.186).
2. The mean relative viability of the positive control is below 50% of the negative control viability (17.3%).
3. The difference of viability between the two relating tissues of a single chemical is <20% (values between 0.1% to 1.5%) in the same run (for positive and negative control tissues and tissues of single chemicals).

The study met all acceptance criteria

   Mean OD  Mean Viability
 Negative Control 1.185 100.0% 
 Positive Control 0.204 17.3%
 Test Item 0.052 4.4%
Interpretation of results:
other: The test item is labeled irritant (UN GHS Category 1 or Category 2).
Conclusions:
Under the conditions of the present study, the test item did show an eye hazard potential. The test item is labeled irritant (UN GHS Category 1 or Category 2).
Executive summary:

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 492. Under the conditions of the present study, the test item did show an eye hazard potential. The test item is labeled irritant (UN GHS Category 1 or Category 2).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Based on the provided information there is no need for classification for skin irritation according to the EU Regulation (EC) No 1272/2008 on Classification, Labelling and Packaging of Substances and Mixtures, as amended for the 10th time in Regulation (EU) No 2017/776.

Based on the OECD 437 result eye damage cat 1 can be excluded. Taking all test results into account there is need for classification for eye irritation category 2 according to the EU Regulation (EC) No 1272/2008 on Classification, Labelling and Packaging of Substances and Mixtures, as amended for the 10th time in Regulation (EU) No 2017/776. Based on Test