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Description of key information

The substance was not irritating in th eeye when tested in rabbits. In vitro testing for dermal irritation yielded negative results as well.

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:
24 Jan 2012 - 08 Feb 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Qualifier:
according to guideline
Guideline:
other: Commission Regulation (EU) No. 761/2009 of 23 July 2009 amending, for the purpose of its adaptation to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006.
GLP compliance:
yes (incl. QA statement)
Remarks:
Experimental Toxicology and Ecology, BASF SE, 67056 Ludwigshafen, Germany
Test system:
human skin model
Remarks:
Reconstructed human epidermis model EpiDermTM
Source species:
human
Details on animal used as source of test system:
Tissue model: Epi-200
Origin of tissue: MatTek Corporation, Ashland MA, USA
Details on test system:
The tissues were transferred to sterile 6-well plates with 0.9 mL assay medium and preconditioned in the incubator at 37°C. After 1 hour the preincubation medium was replaced with fresh medium and preconditioning continued for 18 ± 3 hours.
The test substance was applied minimally moistened with PBS.
Control samples:
other: Negative control (NC): PBS, sterile. Positive control (PC): 5% (w/v) sodium dodecyl sulfate (SDS, Sigma, Germany) in deionized water, sterile.
Amount/concentration applied:
25 μL of the solid ground test material (about 20 mg) was applied with a sharp spoon.
Duration of treatment / exposure:
The tissues were kept under the laminar flow hood at room temperature for 25 minutes overall and for 35 minutes in the incubator.
Rinsed tissues were blotted on sterile absorbent paper and transferred into new 6-well plates, pre-filled with 0.9 mL fresh medium. When all tissues were rinsed, the surface of each tissue was carefully dried with a sterile cotton swab. Subsequently, the tissues were incubated in the incubator at 37°C for 24 ± 2 hours. After 24 ± 2 hours the tissues were transferred into new 6-well plates pre-filled with 0.9 mL of fresh medium and placed into the incubator for additional 18 ± 2 hours post-incubation period.
Duration of post-treatment incubation (if applicable):
REMOVAL OF TEST SUBSTANCE
The tissues were washed with sterile PBS to remove residual test material 1 hour after start of application.

SCORING SYSTEM:
The present test is based on the experience that irritant chemicals produce cytotoxicity in human reconstructed epidermis. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity. The mitochondrial dehydrogenase reduces the yellow colored water-soluble 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to the insoluble blue colored formazan.
After the postincubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours. After incubation, the tissues were washed with PBS to stop the MTT-incubation. The formazan that was metabolically produced by the tissues was extracted by incubation of the tissues in isopropanol. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically.
Number of replicates:
Three tissues were treated with the test substance, the PC and NC, respectively.
Vehicle:
unchanged (no vehicle)
Irritation / corrosion parameter:
% tissue viability
Value:
100

EXPERIMENTAL RESULTS

tissue 1 tissue 2 tissue 3 mean SD
negative control mean OD570 1.932 2.087 1.96 1.993

viability

[% of NC]

96.9 104.7 98.3 100 4.15
test material mean OD570 1.958 2.046 1.959 1.988

viability

[% of NC]

98.2 102.7 98.3 100 2.54
positive control mean OD570 0.13 0.126 0.13 0.129

viability

[% of NC]

6.5 6.3 6.5 6 0.11
Interpretation of results:
GHS criteria not met
Conclusions:
Based on the observed results and applying the evaluation criteria cited it was concluded, that the test substance does not show a skin irritation potential in the EpiDermTM skin irritation test under the test conditions chosen.
Executive summary:

The potential of the test substance to cause dermal irritation was assessed by a single topical application of 25 μL bulk volume (about 20 mg) of the test substance to a reconstructed three dimensional human epidermis model (EpiDerm™). Three EpiDerm™ tissue samples were incubated with the test substance for 1 hour followed by an about 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 100%. Based on the observed results and applying the evaluation criteria cited it was concluded, that the test article 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
Reference
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1964
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
according to guideline
Guideline:
other: Dermal Toxicity - J. H. Draize, Ph. D. , Chief, Skin Toxicity Branch. Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics, Association of the Food and Drug Officials of the United States. pp. 46-59, 1959.
Principles of method if other than guideline:
One hundred milligrams of undiluted test material was instilled into the conjunctival sac of the right eye of each of six test animals. The right eyes were not washed; the left eyes served as a controls. At 24 hour consecutive intervals examinations were made of the cornea, the iris and the conjunctiva and grades were recorded according to a standard Draize scoring system.
GLP compliance:
no
Species:
rabbit
Strain:
not specified
Details on test animals or tissues and environmental conditions:
no data
Vehicle:
unchanged (no vehicle)
Controls:
other: untreated left eye served as control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 100 mg
Duration of treatment / exposure:
Instillation of the test article into the conjunctival sac of the right eye of each animal, no further washing.
Observation period (in vivo):
3 days
Number of animals or in vitro replicates:
6
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): no

SCORING SYSTEM: Draize Scoring system

TOOL USED TO ASSESS SCORE: fluorescein
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
iris score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
3
Irritation parameter:
chemosis score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritant / corrosive response data:
Instillation of the test material produced no discernible effects in the eyes of the rabbits tested.
Interpretation of results:
GHS criteria not met
Conclusions:
The test article is not an ocular irritant.
Executive summary:

In an in vivo study performed in the pre-GLP and pre-OECD guideline era, the test substance was instilled into the conjunctival sac of the right eye of six albino rabbits to assess its irritating potential. The eyes were not rinsed and the animals were examined for occular reactions at 24 hour consecutive intervals for three days. Cornea, the iris and the conjunctiva of each animal were examined for irritation and grades were recorded according to a standard Draize scoring system. No discernible effects in the eyes of the rabbits tested could be observed. Therefore, the test substance is considered to be not irritating to the rabbit eye under the conditions presented.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation

The potential of the test substance to cause dermal irritation was assessed by a single topical application of 25 μL bulk volume (about 20 mg) of the test substance to a reconstructed three dimensional human epidermis model (EpiDerm™). Three EpiDerm™ tissue samples were incubated with the test substance for 1 hour followed by an about 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 test substance 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 100%. Based on the observed results and applying the evaluation criteria cited it was concluded, that the test article does not show a skin irritation potential in the EpiDerm™ skin irritation test under the test conditions chosen.

In a dermal acute toxicity test, five male and five female HanRcc:WIST (SPF) rats were treated with the test substance at 2000 mg/kg by dermal application. One day before treatment, the backs of the animals were clipped with an electric clipper, exposing an area of approximately 10 % of the total body surface. The test item was diluted in the vehicle (PEG 300) at a concentration of 0.5 g/ml and administered at a volume of 4 ml/kg for 24 hours. Twenty-four hours after the application the dressing was removed and the skin was flushed with lukewarm tap water and dried with disposable paper towels. Thereafter, the reaction sites were assessed. The fur of all animals was shaved on test days 7 and 13 just after the assessment of the reaction to facilitate the skin reading for the next day. Local signs were assessed once daily during days 2-15. No local signs of toxicity were observed during the course of the study.

Together with the negative results observed in the EpiDerm in vitro test, the substance is considered to have no irritation potential and does therefore not require classification. No further tests are deemed necessary.

Eye Irritation

In an in vivo study performed in the pre-GLP and pre-OECD guideline era, the test substance was instilled into the conjunctival sac of the right eye of six albino rabbits to assess its irritating potential. The eyes were not rinsed and the animals were examined for ocular reactions at 24 hour consecutive intervals for three days. Cornea, the iris and the conjunctiva of each animal were examined for irritation and grades were recorded according to a standard Draize scoring system. No discernible effects in the eyes of the rabbits tested could be observed. Therefore, the test substance is considered to be not irritating to the rabbit eye under the conditions presented.

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, classification for irritation is not warranted under Regulation (EC) No.1272/2008.