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Administrative data

Description of key information

The test substance was not irritating to the skin in an in vitro EpiDerm test (BASF, 2012) and in an in vivo test (IBT, 1972). The test substance was not irritating to the eye in two in vitro studies (EpiOcular, BCOP, BASF 2012) and in an in vivo study (IBT, 1972).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
14 Feb 2012 - 06 Mar 2012
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)
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
(2009)
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
GLP compliance:
yes (incl. certificate)
Remarks:
BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany
Test system:
human skin model
Remarks:
reconstructed three dimensional human epidermis model (EpiDerm™)
Source species:
human
Cell type:
non-transformed keratinocytes
Vehicle:
other: minimally moistened with PBS
Details on test system:
EpiDerm TM 200 kit: MatTek ln Vitro Life Science Laboratories, Bratislava, Slovakia containing: 24 Epi-200 tissues (reconstructed epidermis): surface 0.6 cm² cultured in Millicells® 0 1 cm
Tissue for MTTreduction control: Epi-200 tissue that is killed by freezing at -20°C
Assay medium: Dulbecco's modified eagle's medium (DMEM); for the assay and for diluting MTT
Wash buffer: Dulbecco's phosphate buffered saline (P8S), w/o Ca2+ , Mg2+
Extracting agent: lsopropanol p.a.
Detection agent: 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), 1.0 mg I ml assay medium
Amount/concentration applied:
25 μL de-ionized water was applied first. Thereafter, a bulk volume of 25 μL of the solid test material was applied with a sharp spoon and homogeneously distributed with the water.
Duration of treatment / exposure:
1h
Number of replicates:
three tissue samples used each for test sample and controls
Details on test animals and environmental conditions:
N/A
Controls:
other: Control tissue used for positive and negative controls
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean value of three tissues
Run / experiment:
assay with the test article
Value:
102
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

Results

Test material Tissue 1 Tissue 2 Tissue 3 mean SD
negative control (NC)

mean OD570

1.824 2.069 1.717 1.87

viability [% of NC]

97.5

110.7 91.8 100 9.67
test article

mean OD570

1.735 2.068 1.911 1.905

viability [% of NC]

92.8 110.6 102.2 102 8.92
positive control (PC)

mean OD570

0.165 0.168 0.171 0.168

viability [% of NC]

8.8 9 9.1 9 0.17
Interpretation of results:
GHS criteria not met
Conclusions:
Based on the observed results it was concluded, that the test substance does not show a skin irritation potential in the EpiDerm(TM) irritation test under the test conditions chosen.
Executive summary:

The test article's potential to cause dermal irritation was assessed in an in vitro irritation test according to OECD guideline 439 and in compliance with GLP. To that end, a single topical application of 25 μL bulk volume (about 14 mg) of the test substance to a reconstructed three dimensional human epidermis model (EpiDerm™) was analyzed. Three EpiDerm™ tissue samples were incubated with the test substance for 1 hour followed by a 42-hours post-incubation period. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/ post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the testsubstance treated epidermal tissues is compared to that of negative control tissues. The quotient of both values indicates the relative tissue viability. The EpiDerm skin irritation test showed the following results: The test substance is not able to reduce MTT directly. The mean viability of the test-substance treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 102%. Based on the observed results it was concluded, that the test substance does not show a skin irritation potential in the EpiDerm™ skin irritation test under the test conditions chosen.

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:
weight of evidence
Study period:
28 Feb 2012 - 06 Mar 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: MatTek Corporation, Ashland, MA 01721, USA: EpiOcularTM human cell construct: Procedure details, Version 3.1a of February 10, 2010
Qualifier:
according to
Guideline:
other: Harbell J.W. et al. (2009): COLIPA Program on Optimization of Existing In Vitro Eye Irritation Assays for Entry into Formal Validation: Technology Transfer and Intra/Inter Laboratory Evaluation of EpiOcular Assay for Chemicals. Poster # 378, Society of To
GLP compliance:
yes (incl. certificate)
Remarks:
BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany
Species:
other: Reconstructed human cornea model EpiOcular(TM)
Strain:
other: Tissue model: OCL-200
Details on test animals or tissues and environmental conditions:
N/A
Vehicle:
other: tissue was pretreated with PBS, test item added unchanged
Controls:
other: Two tissues were each treated with positive and negative controls
Amount / concentration applied:
50 µl of test material
Duration of treatment / exposure:
90 minutes
Number of animals or in vitro replicates:
Two tisssues per sample
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes, with sterile PBS
- Time after start of exposure: 90 minutes
Irritation parameter:
other: tissue viability in %
Run / experiment:
test material
Value:
98
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

RESULTS

tissue 1 tissue 2 mean inter-tissue variability [%]
negative control

mean OD570

1.488 1.665 1.577
viability [% of NC] 94.4 105.6 100 11.2
test material

mean OD570

1.419 1.685 1.522
viability [% of NC] 90 106.9 98 16.9
positive control

mean OD570

0.296 0.327 0.311
viability [% of NC] 18.7 20.7 20 2
Interpretation of results:
GHS criteria not met
Conclusions:
Based on the observed results it was concluded, that the test substance does not show an eye irritation potential in the EpiOcular™ eye irritation test under the test conditions chosen.
Executive summary:

The potential of the test article to cause ocular irritation was assessed by a single topical application of 50 μL bulk volume (about 19 mg) of the test substance to a reconstructed three dimensional human cornea model (EpiOcular™). Two EpiOcular™ tissue samples were incubated with the test substance for 90 minutes followed by a 18-hours post-incubation period. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the testsubstance treated epidermal tissues is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability. The EpiOcular™ eye irritation test showed the following results:

The test substance is not able to reduce MTT directly. The mean viability of the test-substance treated tissues was 98%. Based on the observed results it was concluded, that the test substance does not show an eye irritation potential in the EpiOcular™ eye irritation test under the test conditions chosen.

Endpoint:
eye irritation: in vitro / ex vivo
Remarks:
in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
06 Feb 2012 - 06 Mar 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
GLP compliance:
yes (incl. certificate)
Remarks:
BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany
Species:
cattle
Strain:
other: Isolated bovine cornea
Details on test animals or tissues and environmental conditions:
Bovine eyes are obtained as a by-product of freshly slaughtered cattle (age of the animals: minimum 12 months, maximum 60 months).
Supplier: Schlachthof Bensheim, Am Schlachthof 7-9, 64625 Bensheim
Vehicle:
water
Remarks:
deionized
Controls:
other: control corneas treated with control substances.
Amount / concentration applied:
TEST MATERIAL
- Concentration (if solution): 20% (w/v) suspension in de-ionized water

VEHICLE
- Amount(s) applied (volume or weight with unit): 750 µl
Duration of treatment / exposure:
4 h
Number of animals or in vitro replicates:
Each treatment group (test substance, NC and PC) consisted of 3 corneas.
Irritation parameter:
in vitro irritation score
Run / experiment:
test substance
Value:
4.6
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

RESULTS

Test substance mean opacity score mean permeability score In Vitro irritancy Score
test article 4.5 0.006 4.6
negative control 5.5 -0.001 5.5
positive control 80.2 2.804 122.2
Conclusions:
Based on the observed results it was concluded, that the test material does not cause serious eye damage in the Bovine Corneal Opacity and Permeability Test (BCOP Test) under the test conditions chosen.
Executive summary:

The potential of the test article to cause serious damage to the eyes was assessed by a single topical application of 750 μL of a 20% test substance preparation to the epithelial surface of isolated bovine corneas. Three corneas were treated with the test-substance preparation for an exposure period of 4 hours. Corneal opacity was measured quantitatively as the amount of light transmission through the cornea. Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea. Both measurements were used to calculate an In Vitro Irritancy Score of the test substance relative to the control corneas. There was no difference between test material and negative controls, therefore it was concluded, that the test article does not cause serious eye damage in the Bovine Corneal Opacity and Permeability Test (BCOP Test) under the test conditions chosen.

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

Additional information

Skin irritation

The test article's potential to cause dermal irritation was assessed in an in vitro irritation test according to OECD guideline 439 and in compliance with GLP. To that end, a single topical application of 25 μL bulk volume (about 14 mg) of the test substance to a reconstructed three dimensional human epidermis model (EpiDerm™) was analyzed. Three EpiDerm™ tissue samples were incubated with the test substance for 1 hour followed by a 42-hours post-incubation period. Tissue destruction was determined by measuring the reduction of mitochondrial dehydrogenase activity by reduced formazan production after incubation with a tetrazolium salt (MTT). The test substance is not able to reduce MTT directly. The mean viability of the test-substance treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 102%. Based on the observed results it was concluded, that the test substance does not show a skin irritation potential in the EpiDerm™ skin irritation test under the test conditions chosen.

In addition, the test article was assessed in vivo with six New Zealand White rabbits (IBT, 1972). In this study, only minimal reactions below the threshold of regulatory significance were observed. Considering the history of IBT (reporting of fake data), the reliability of this study is questionable and an accurate Klimisch rating is impossible. Consequently, the study is rated with Klimisch 4. However, since falsified data was predominantly reported for studies with repeated exposure, the data from this IBT study was taken into account in a weight of evidence approach. The in vivo data supports the findings observed in the in vitro study, therefore it is concluded that the test article is not irritating to skin.

Eye irritation

The potential of the test article to cause serious damage to the eyes was assessed by a single topical application of 750 μL of a 20% test substance preparation to the epithelial surface of isolated bovine corneas. The study was performed according to OECD test guideline 437 and compliant to GLP principles. Three corneas were treated with the test-substance preparation for an exposure period of 4 hours. Corneal opacity was measured quantitatively as the amount of light transmission through the cornea. Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea. Both measurements were used to calculate an In Vitro Irritancy Score of the test substance relative to the control corneas. There was no difference between test material and negative controls, therefore it was concluded, that the test article does not cause serious eye damage in the Bovine Corneal Opacity and Permeability Test (BCOP Test) under the test conditions chosen. The test method according to the regulatory accepted protocol at the time of reporting does not allow for the evaluation of eye irritation. The result does not exclude an irritation potential of the test substance. Therefore, an additional eye irritation study was performed as described below.

To assess the test article's potential to cause ocular irritation, an EpiOcular irritation test was performed. The study was conducted in compliance to GLP guidelines and followed the methods described by MatTek Corp., 2010 and Harbell et al., 2009. In this assay, a single topical application of 50 μL bulk volume (about 19 mg) of the test substance to a reconstructed three dimensional human cornea model (EpiOcular™) was analyzed. Two EpiOcular™ tissue samples were incubated with the test substance for 90 minutes followed by a 18-hours post-incubation period. Tissue destruction was determined by measuring the reduction of mitochondrial dehydrogenase activity by reduced formazan production after incubation with a tetrazolium salt (MTT). The formazan production of the test substance treated epidermal tissues is compared to that of negative control tissues. The test substance is not able to reduce MTT directly. The mean viability of the test-substance treated tissues was 98%. Based on the observed results it was concluded, that the test substance does not show an eye irritation potential in the EpiOcular™ eye irritation test under the test conditions chosen.

In addition, an in vivo eye irritation study performed with 6 New Zealand White rabbits is available (IBT, 1972). In this study, only minimal reactions below the level of regulatory significance were observed and the test material was considered to be not irritating.Considering the history of IBT (reporting of fake data), the reliability of this study is questionable and an accurate Klimisch rating is impossible. Consequently, the study is rated with Klimisch 4. However, since falsified data was predominantly reported for studies with repeated exposure, the data from this IBT study was taken into account in a weight of evidence approach. The in vivo data supports the findings observed in the in vitro studies, therefore it is concluded that the test article is not irritating to the eye.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for the purpose of classification under Regulation (EC) No.1272/2008. Based on the data, the test substance is not classified as skin or eye irritant.