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Diss Factsheets

Administrative data

Description of key information

Skin irritation:

In a determination of skin irritation potential study using the EPISKIN™ reconstructed human epidermis model,the relative mean tissue viability of the test item was 23.8%, less than 50%, and therefore, the test item demonstrated the ability to cause a positive response as a skin irritant.

In a supporting study, following application to the skin of rabbits under a closed patch, a single application of the test item produced no eschar formation, and minimal transient edema in one of six rabbits. Erythema could not be evaluated due to an intense green staining of the skin.

Eye irritation:

Based on the results of the BCOP test, no prediction of eye irritation can be made for the test item.

In asupporting study, a single application of the test item to the eyes of albino rabbits produced mild conjunctivitis in four animals and moderate conjunctivitis in the remaining two animals that did not meet GHS criteria for classification.  No effects on the cornea or iris were noted.

No information is available on respiratory irritation.

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:
26 October 2017 to 16 April 2018
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)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
CAS Registry Number: 72030-25-2
Purity: 100%; This substance has an Unknown or Variable composition, is a Complex reaction product, or a Biological material (UVCB)
Physical state/Appearance: Dark green liquid
Test system:
human skin model
Source species:
human
Cell type:
other: reconstructed human epidermis tissues
Cell source:
other: not specified
Vehicle:
unchanged (no vehicle)
Details on test system:
Triplicate tissues were treated with the test item for an exposure period of 15 minutes. Triplicate tissues treated with 10 µL of Dulbecco’s Phosphate Buffered Saline (DPBS) served as the negative controls and triplicate tissues treated with 10 µL of SDS 5% w/v served as the positive controls. The plates were kept in the biological safety cabinet at room temperature for 15 minutes. At the end of the exposure period, each tissue was removed from the well using forceps, rinsed using DPBS with Ca++ and Mg++, transferred to 3 wells containing maintenance medium, and incubated at 37 °C, 5% CO2 in air for 42 hours.

MTT Loading/Formazan Extraction (Day 3)
Following the 42 hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenize the released mediators in the maintenance medium. Maintenance medium from beneath each tissue was transferred to pre labeled micro tubes and stored in a freezer at 14 to 30 ºC for possible inflammatory mediator IL-1α determination.

MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plates. The tissues were transferred to the MTT filled wells, incubated for 3 hours at 37 °C, 5% CO2 in air and then placed onto absorbent paper to dry. A total biopsy of the epidermis was made, the epidermis was carefully separated from the collagen matrix, and both parts (epidermis and collagen matrix) placed into labeled micro tubes containing acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and then the tubes were refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals.

Absorbance/Optical Density Measurements (Day 6)
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous colored solution. For each tissue, duplicate samples were transferred to the appropriate wells of a pre labeled 96 well plate. Acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at 570 nm (without a reference filter) using the LabTech LT 4500 microplate reader.
Control samples:
other: Negative Control: Dulbecco’s Phosphate Buffered Saline (DPBS) with Ca++ and Mg++ and Positive Control: Sodium Dodecyl Sulphate (SDS)
Amount/concentration applied:
10 µL (26.3 µL/cm^2) of the test item was applied to the epidermis surface.
Duration of treatment / exposure:
Triplicate tissues were treated with the test item for an exposure period of 15 minutes.
Duration of post-treatment incubation (if applicable):
At the end of the exposure period each tissue was rinsed before incubating for 42 hours.
Number of replicates:
Three.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
15 minute exposure period and 42 Hours post exposure incubation period.
Value:
23.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation

Direct MTT Reduction

The MTT solution containing the test item turned purple which indicated that the test item directly reduced MTT. 

 

Assessment of Color Interference with the MTT Endpoint

The solution containing the test item was a dark green color, therefore additional color correction tissues were incorporated into the testing procedure. The results obtained showed that negligible color interference occurred.  It was considered unnecessary to use the results of the color correction tissues for quantitative correction of results.

 

Test Item, Positive Control Item and Negative Control Item

The relative mean viability of the test item treated tissues was 23.8% (≤50%) after a 15 Minute exposure period and 42 Hour post exposure incubation period.

 

Quality Criteria

The relative mean viability for the positive control was 19.7% relative to the negative control treated tissues and the standard deviation value of the viability was 6.7% (≤18%). The positive control acceptance criteria were therefore satisfied. The mean OD570 for the negative control treated tissues was 0.874, within the range of 0.6 - 1.5 and the standard deviation value of the viability was 2.1% (≤18%). The negative control acceptance criteria were therefore satisfied. The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues were 2.8% (≤18%). The test item acceptance criterion was therefore satisfied.

 

Mean Viabilities of the EPISKIN™ Tissues

Treatment

OD570
(Mean ± SD)

Relative Tissue Viability
(Mean ± SD)

Test Item

0.208 ± 0.024

23.8% ±2.8%

DPBS (Negative Control)

0.874± 0.019

100%±2.1%

0.5% SDS (Positive Control)

0.172 ± 0.058

19.7% ±6.7%

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Remarks:
Supporting evidence of non-corrosive results in supporting study and eye irritation results.
Conclusions:
The relative mean tissue viability of the test item was 23.8%, less than 50%, and therefore, the test item demonstrated the ability to cause a positive response as skin irritant. However this study does not allow the conclusion on whether the test item is Category 1 or Category 2 of Regulation (EC) No. 1272/2008 Classification, Labelling and Packaging of Substances and Mixtures (EU CLP).
Executive summary:

The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKINTMreconstructed human epidermis model after a treatment period of 15 minutes followed by a post exposure incubation period of 42 hours. Tissue viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3 [4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue/purple formazan salt in the test item treated tissues relative to the negative controls. 

 

Triplicate tissues were treated with the test item for 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. The test item was found to directly reduce MTT; therefore, additional non-viable tissues were incorporated into the testing for correction purposes. The test item was also found to have the potential to cause color interference with the MTT endpoint therefore additional tissues were incorporated into the testing to correct for this. A third set of controls was included, comprising water killed tissues, to prevent a double correction from a colored test item that also reduces MTT. At the end of the post exposure incubation period the maintenance medium from beneath each tissue was transferred to pre labeled micro tubes and stored in a freezer for possible inflammatory mediator IL-1α determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a pre labeled 96 well plate. The optical density was measured at 570 nm. Data was presented in the form of percentage viability (MTT reduction in the test item or positive control treated tissues relative to negative control tissues). The relative mean viability of the test item treated tissues was 23.8% after the 15 minute exposure period and 42 hours post exposure incubation period. The quality criteria required for acceptance of results in the test were satisfied.

 

Under the conditions of the study, the relative mean tissue viability of the test item was 23.8%, less than 50%, and therefore, the test item demonstrated the ability to cause a positive response as skin irritant. However this study does not allow the conclusion on whether the test item is Category 1 or Category 2 of Regulation (EC) No. 1272/2008 Classification, Labelling and Packaging of Substances and Mixtures (EU CLP), and United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS).

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
28 September 1965 to 16 December 1965
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: Regulations (Federal Register, August 12, 1961 et seq) under the Federal Hazardous Substances Labeling Act
Deviations:
not specified
GLP compliance:
no
Species:
rabbit
Strain:
other: albino
Type of coverage:
occlusive
Preparation of test site:
clipped
Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
0.5mL
Duration of treatment / exposure:
24 hours
Observation period:
24 and 72 hours
Number of animals:
Six
Details on study design:
The test item was applied under a 1-inch x 1-inch surgical gauze patch to an intact-skin area. The application sites were prepared by clipping the hair from the saddle area of the rabbits. Each patch was held in place with two strips of 1-inch adhesive tape. After application of the patches the trunk of each rabbit was wrapped and the animals were immobilized in restraining stocks for 24 hours. At the end of the 24-hour exposure period, the patches were removed and any residual test item was removed by sponging with a moistened towel.

The reactions were scored immediately after removal of the patches (24-hour reading) and again two days later (72- hour reading) according to the scale given in the Regulations (United States Federal Register, August 12, 1961) under the Federal Hazardous Substances Labeling Act.
Irritation parameter:
primary dermal irritation index (PDII)
Basis:
mean
Time point:
24 h
Score:
0
Reversibility:
not specified
Remarks on result:
no indication of irritation
Remarks:
PII cannot really be calculated because the erythema cannot be scored due to green staining of the skin.
Irritation parameter:
primary dermal irritation index (PDII)
Basis:
mean
Time point:
72 h
Score:
0
Reversibility:
not specified
Remarks on result:
no indication of irritation
Remarks:
PII cannot really be calculated because the erythema cannot be scored due to green staining of the skin.
Irritation parameter:
erythema score
Basis:
mean
Time point:
other: 24h and 72h
Score:
0
Reversibility:
not specified
Remarks on result:
no indication of irritation

Intense green staining of the skin made readings of erythema impossible. No eschar formation was noted; the only gross signs of irritation noted consisted of minimal edema in one rabbit at the 24-hour reading.

Interpretation of results:
GHS criteria not met
Conclusions:
Following application to the skin of rabbits under a closed patch, a single application of the test item produced no eschar formation, and minimal transient edema in one of six rabbits. Erythema could not be evaluated due to an intense green staining of the skin.
Executive summary:

A study was conducted according to the Regulations (Federal Register, August 12, 1961 et seq) under the Federal Hazardous Substances Labeling Act to determine the potential of the test item to cause irritation to the skin.

 

0.5 mL of the test item was applied under a 1-inch x 1-inch surgical gauze patch to an intact-skin area on six albino rabbits. The application sites were prepared by clipping the hair from the saddle area of the rabbits.

 

Each patch was held in place with two strips of 1-inch adhesive tape. After application of the patches the trunk of each rabbit was wrapped and the animals were immobilized in restraining stocks for 24 hours. At the end of the 24-hour exposure period, the patches were removed and any residual test item was removed by sponging with a moistened towel. The reactions were scored immediately after removal of the patches (24-hour reading) and again two days later (72- hour reading) according to the scale given in the Regulations (United States Federal Register, August 12, 1961) under the Federal Hazardous Substances Labeling Act.

 

Intense green staining of the skin made readings of erythema impossible. No eschar formation was noted; the only gross signs of irritation noted consisted of minimal edema in one rabbit at the 24-hour reading. Under these conditions it was not possible to calculate the Primary Irritation Index.

 

In conclusion, application of the test item to the skin of rabbits under a closed patch, a single application of the test item produced no eschar formation, and minimal transient edema in one of six rabbits. Erythema could not be evaluated due to an intense green staining of the skin.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (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:
26-September-2017 to ****
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)
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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
CAS Registry Number: 72030-25-2
Purity: This substance has an Unknown or Variable composition, is a Complex reaction product, or a Biological material (UVCB)
Physical state/Appearance: Dark green liquid
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised after slaughter and placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL), and transported to the test facility over ice packs on the same day of slaughter. The corneas were refrigerated on arrival and used within 24 hours of receipt.
Vehicle:
unchanged (no vehicle)
Controls:
other: Negative control: Sodium chloride 0.9% w/v; Positive control: Ethanol
Amount / concentration applied:
0.75 mL of the test item was applied to the corneas.
Duration of treatment / exposure:
Incubated (at 32 ± 1 ºC) for 10 minutes.
Duration of post- treatment incubation (in vitro):
90 minutes.
Number of animals or in vitro replicates:
Three.
Details on study design:
Preparation of Corneas
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling, and the iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders. The chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red, plugged and incubated at 32 ± 1 ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects, only corneas free of damage were used.

Selection of Corneas and Opacity Reading
The medium from both chambers of each holder was replaced with fresh complete EMEM and a pre treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated.

Treatment of Corneas
The EMEM was removed from the anterior chamber of the BCOP holder and the test item or control items were applied to the appropriate corneas and each holder was incubated at 32 ± 1 ºC for 10 minutes.
At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The chamber was then refilled with fresh complete EMEM without phenol red. A post treatment opacity reading was taken and each cornea was visually observed.
The holders were incubated at 32 ± 1 ºC for 120 minutes. After incubation, the medium from both chambers was replaced with fresh complete EMEM and a final opacity reading was taken. Each cornea was visually observed.

Application of Sodium Fluorescein
Following the final opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium was removed and replaced with sodium fluorescein solution (4 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.

Permeability Determinations
After incubation the medium in the posterior chamber of each holder was decanted and retained.
360 µL of media representing each cornea was dispensed into the appropriate wells of a pre labeled 96 well plate. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader.
Irritation parameter:
in vitro irritation score
Value:
28
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Corneal Epithelium Condition
The corneas treated with the test item were partly cloudy post treatment and cloudy post incubation. The corneas treated with the negative control item were clear post treatment and post incubation. The corneas treated with the positive control item were cloudy post treatment and post incubation.

Criteria for an Acceptable Test
The positive control In Vitro Irritancy Score was within the historical range of 29.6 to 65.1; the positive control acceptance criterion was satisfied. The negative control gave opacity of ≤2.3 and permeability ≤0.041; the negative control acceptance criteria were satisfied.

Test Item
The in vitro irritancy score of the test item was 28.0, based on this result, no prediction of eye irritation can be made for the test item.

In Vitro Irritancy Score

Treatment

In Vitro Irritancy Score

Test Item

28.0

0.9% (w/v) Sodium Chloride (Negative Control)

0.1

Neat Ethanol (Positive Control)

42.5

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the results of the BCOP test, no prediction of eye irritation can be made for the test item.
Executive summary:

The purpose of this test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability. 

The test method can correctly identify test items (both chemicals and mixtures) inducing serious eye damage as well as those not requiring classification for eye irritation or serious eye damage, as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Items (GHS) and EU Classification, Labelling and Packaging (CLP) of chemicals (Regulation (EC) No 1272/2008), and it was therefore endorsed as scientifically valid for both purposes. Test items inducing serious eye damage are classified as UN GHS and EU CLP Category 1. Items not classified for eye irritation or serious eye damage are defined as those that do not meet the requirements for classification as UN GHS/EU CLP Category 1 or 2 (2A or 2B), i.e. they are referred to as UN GHS/EU CLP No Category.

 

The undiluted test item was applied for 10 minutes followed by an incubation period of 120 minutes. Negative (0.9% (w/v) Sodium Chloride) and positive (Neat Ethanol) control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS). 

 

Based on the results of the BCOP test, no prediction of eye irritation can be made for the test item.

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
28 September 1965 to 16 December 1965
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: Regulations (Federal Register, August 12, 1961 et seq) under the Federal Hazardous Substances Labeling Act
Deviations:
not specified
GLP compliance:
no
Species:
rabbit
Strain:
not specified
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Strain: Albino
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
0.1 mL
Observation period (in vivo):
Examinations for gross signs of eye irritation were made at 24, 48 , and 72 hours following application.
Details on study design:
The test item was applied to the right eye of each of six albino rabbits, whilst the left eye was untreated and served as a control. Examinations for gross signs of eye irritation were made at 24, 48, and 72 hours following application, in which corneal, iris, and conjunctival effects are scored.
Irritation parameter:
iris score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Reversibility:
not specified
Remarks on result:
no indication of irritation
Irritation parameter:
other: cornea
Basis:
mean
Time point:
24/48/72 h
Score:
0
Reversibility:
not specified
Remarks on result:
no indication of irritation
Irritation parameter:
other: Erythema (redness)
Basis:
mean
Time point:
24/48/72 h
Score:
1.1
Reversibility:
not specified
Remarks on result:
positive indication of irritation
Irritation parameter:
other: Edema (swelling)
Basis:
mean
Time point:
24/48/72 h
Score:
0.83
Reversibility:
not specified
Remarks on result:
positive indication of irritation
Irritant / corrosive response data:
No effects on the cornea or iris were observed. Four animals showed Grade 1 erythema and swellings; two remaining animals showed Grade 2 erythema for up to 48 hours.
Interpretation of results:
GHS criteria not met
Conclusions:
A single application of the test item to the eyes of albino rabbits produced mild conjunctivitis in four animals and moderate conjunctivitis in the remaining two animals that did not meet GHS criteria for classification. No effects on the cornea or iris were noted.
Executive summary:

A study was conducted according to the Regulations (Federal Register, August 12, 1961 et seq) under the Federal Hazardous Substances Labeling Act to determine the potential of the test item to cause irritation in the eye.

The test item was applied to the right eye of each of six albino rabbits; the left eye was untreated and served as a control.  Examinations for gross signs of eye irritation were made at 24, 48, and 72 hours following application.  Corneal, iris, and conjunctival effects are scored separately. 

No effects on the cornea or iris were observed.  Four animals showed Grade 1 erythema and swelling and the two remaining animals showed Grade 2 erythema for up to 48 hours.

A single application of the test item to the eyes of albino rabbits produced mild conjunctivitis in four animals and moderate conjunctivitis in the remaining two animals.  No effects on the cornea or iris were noted. In conclusion, the test item does not meet the GHS criteria for classification as an eye irritant.

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

Skin irritation:

In a determination of skin irritation potential study using the EPISKIN™ reconstructed human epidermis model, the relative mean tissue viability of the test item was 23.8%, less than 50%, and therefore, the test item demonstrated the ability to cause a positive response as a skin irritant. However this study does not allow the conclusion on whether the test item is Category 1 or Category 2 of Regulation (EC) No. 1272/2008 Classification, Labelling and Packaging of Substances and Mixtures (EU CLP).  Based upon a lack of corrosive effects noted in the supporting animal study and the absence of corrosive results in the eye study, Category 2 was determined to be most appropriate for this test material.