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Description of key information

In an in vitro skin irritation study, performed according to OECD/EC test guidelines, the substance was found to be irritant to the skin. In an in vitro skin corrosion study, performed according to OECD/EC test guidelines, the substance was found not to be corrosive to the skin. In an in vitro skin irritation study with reconstructed human epidermis, performed according to OECD guidelines no indication for skin irritation was observed.
An in vitro eye irritation study, an in vitro corrosion study and an in vivo eye irritation test are available, performed in accordance with the OECD guideline. Based on irreversibel effects to the eye, the test substance is classified cat. 1 for eye irritancy.

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:
November 13th - December 24th, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
adopted 24 August 2009
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
adopted 22 July 2010
Deviations:
no
GLP compliance:
yes
Species:
human
Details on test animals and environmental conditions:
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 36.9 - 37.8
- Humidity (%): 69 - 95
Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
TEST MATERIAL
- Amounts applied: The liquid test substance was applied undiluted (25 μl) directly on top of the tissue. No correction was made for the purity/composition of the test compound.

NEGATIVE CONTOL:
- Amount applied: 25 µl Phosphate buffered saline.

POSITIVE CONTROL
- Amount applied: 25 µl
- Concentration: 5% (aq) Sodium dodecyl sulphate in PBS.
Duration of treatment / exposure:
15 minutes
Details on study design:
TEST SITE
- EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 12-EKIN-032). This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum. The EPISKIN human epidermis was obtained from SkinEthic Laboratories, Lyon, France.

REMOVAL OF TEST SUBSTANCE
- Washing: phosphate buffered saline
- Time after start of exposure: 15 minutes

POST INCUBATION PERIOD
- 43 hours

SCORING SYSTEM:
- Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
15-min exposure
Value:
23
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
31%

Accelerator (PT 25E or PT 25E/2) was checked for possible direct MTT reduction by adding the test substance to MTT medium. Because a colour change was observed it was concluded that Accelerator (PT 25E or PT 25E/2) did interact with MTT. In addition to the normal procedure, three killed tissues treated with test substance and three killed non treated tissues were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by Accelerator (PT 25E or PT 25E/2) was 0.6% of the negative control tissues. The net OD of the treated killed tissues was subtracted from the ODs of the test substance treated viable tissues.

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Remarks:
Category 2 classification according to EC Regulation 1272/2008
Conclusions:
An in vitro skin irritation test was conducted according to OECD 439 guideline and GLP principles. It is concluded that this test is valid and that the test substance is irritating in the in vitro skin irritation test.
Executive summary:

In an in vitro skin irritation test using a human skin model ( EPISKIN Standard Model) according to OECD 439 guideline and GLP principles, the influence of Accelerator (PT 25E or PT 25E/2) on the viability of human skin was tested. The test substance was applied directly to 0.38 cm2 cultured skin (25 μl). After 15 minutes, the substance was removed and cells were cultured for 43 hours. The viability of the cells was tested by reduction of MTT. Survival of unexposed skin was set at 100%, the positive control had a mean cell viability of 31% whereas the test substance showed cell viability of 23%. Since the mean relative tissue viability after exposure to the test substance was below 50%, it can be concluded that the test substance is irritating in the in vitro skin irritation test.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2012-11-09 - 2013-01-24
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)
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No 640/2012
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
other: EpiDerm™ 200 kit
Details on test animals and environmental conditions:
TEST SYSTEM:
- Source: MatTek Corp., Ashland MA, USA

Three dimensional human epidermis model
The EpiDerm™ model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm™ tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs®, 10 mm ∅) and commercially available as kits (EpiDerm™ 200), containing 24 tissues on shipping agarose.
Based on the results of ECVAM (European Center for Validation of Alternative Methods) funded validation studies, it was concluded by the ECVAM Scientific Advisory Committee that the EpiDerm™ human epidermis model is suitable to be used for distinguishing between corrosive and non-corrosive chemicals (ECVAM: ESAC statement on the application of the Epiderm™ human skin model for skin corrosivity testing of 14-15 Mar 2000) as well as between irritant and non-irritant chemicals (ECVAM: ESAC statement on the scientific validity of in-vitro tests for skin irritation testing of 5 Nov 2008).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 37
Vehicle:
unchanged (no vehicle)
Controls:
other: Concurrent treatment with 30 µL PBS (negative control, NC) or 30 µL of 5% SDS (positive control, PC)
Amount / concentration applied:
- Amount(s) applied (volume or weight with unit): 30 µL
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.
Observation period:
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.
Details on study design:
EXPERIMENTAL PROCEDURE
Pre-Test MTT reduction:
The test substance was added to 0.9 mL of the MTT solution. The mixture was incubated in the dark at about 37 °C for 55 to 65 minutes. A negative control (de-ionized water) was tested concurrently. If the MTT solution color or, in case of water-insoluble test substances the border to the water-phase, turned blue / purple, the test substance was presumed to directly reduce MTT. The direct reduction of MTT by a test substance interferes with the color density produced by metabolic capacity of the tissue and would falsify the test results. In case that direct MTT reduction occurred and visible residues of the test substance remained on the tissues after washing, subsequent testing of killed controls (one freeze-killed control tissue (KC)) was considered. Killed controls are treated with, each, the test article and the negative control, in the same way as described in section “Experimental procedure” (3.6), additionally.

Main Test
On the day of arrival in the laboratory, 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. Three tissues were treated with the test substance, the PC and NC, respectively. Thirty microliter (30 μL) of the undiluted test substance was applied using a pipette. Control tissues were concurrently applied with 30 μL of sterile PBS (negative control, NC) or with 30 μL of 5% SDS (positive control, PC). A nylon mesh was placed carefully onto the tissue surface afterwards. The tissues were kept under the laminar flow hood at room temperature for 25 minutes overall and for 35 minutes in the incubator. The tissues were washed with sterile PBS to remove residual test material 1 hour after start of application. 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. 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. Blank values were established of 6 microtiter wells filled with isopropanol for each microtiter plate.

DATA EVALUATION
Principle
The OD570 values determined for the various tissues are measures of their viability. The quotient of the OD570 of tissues treated with the test material and the mean OD570 values of the NC (percent of control) is used for evaluating whether or not a test material is an irritant.

Calculation of individual and mean optical densities
The individual tissue OD570 is calculated by subtracting the mean blank value of the respective microtiter plate from the respective individual tissue OD570 value. The mean OD570 for a test group of three tissues treated in the same way is calculated.

Tissue viability
The quantification of tissue viability is presented as the quotient of the mean OD570 divided by the respective OD570 NC value in percent.

ACCEPTANCE CRITERIA
Assay acceptance criterion for the negative control (NC)
The absolute OD570 of the negative control tissues in the MTT test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is acceptable if the mean OD570 of the NC is ≥ 1.0. The mean OD570 of the NC should not exceed 2.5.

Assay acceptance criterion for the positive control (PC)
5% SDS is used as PC and reflects the sensitivity of the tissues used in the test conditions. A viability of ≤ 20% is acceptable.

Assay acceptance criterion for tissue variability
For every treatment, 3 tissues are treated in parallel. The intertissue variability is considered to be acceptable if the SD of %-viability is ≤ 20.

EVALUATION OF RESULTS
Irritant potential of the test materials is predicted from the mean relative tissue viabilities compared to the negative control tissues concurrently treated with sterile PBS. A chemical is considered as "irritant", if the mean relative tissue viability with a test material is less than or equal to 50%.
Irritation / corrosion parameter:
% tissue viability
Value:
99
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100% (mean OD570: 2.407)
Positive controls validity:
valid
Remarks:
4% (mean OD570: 0.097)
Other effects / acceptance of results:
Based on the observed results and applying the evaluation criteria, it was concluded, that the test substance does not show a skin irritation potential in the EpiDerm (TM) skin irritation test under the test conditions chosen.
Interpretation of results:
GHS criteria not met
Remarks:
Not classified according to EC Regulation 1272/2008.
Conclusions:
Based on the observed results and applying the evaluation criteria, it was concluded, that the test substance does not show a skin irritation potential in the EpiDerm (TM) skin irritation test under the test conditions chosen.
Executive summary:

The substance was tested in a reliable in vitro skin irritation test with reconstructed human epidermis, performed according to OECD guidelines and according to GLP principles. Based on the observed results and applying the evaluation criteria, it was concluded, that the test substance does not show a skin irritation potential in the EpiDerm (TM) skin irritation test under the test conditions chosen.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (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:
October 9th-30th, 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
GLP compliance:
yes (incl. certificate)
Remarks:
issued 31JAN1994
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: David Percival Ltd., Moston, Sandbach, Cheshire, UK
- Age at study initiation: 12 - 16 weeks old
- Weight at study initiation: 3.1 kg
- Housing: individually in a suspended metal cage
- Diet: ad libitum (STANRAB SQC Rabbit diet, Special Diets Services, Witham, Essex, UK)
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-21
- Humidity (%): 54 - 74
- Air changes (per hr): appr. 15
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
unchanged (no vehicle)
Controls:
not required
Amount / concentration applied:
0.1 ml
Duration of treatment / exposure:
Single instillation
Observation period (in vivo):
21 days
Number of animals or in vitro replicates:
1
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): no

SCORING SYSTEM: According to the OECD guideline.

TOOL USED TO ASSESS SCORE: ophthalmoscope
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
24/48/72 h
Score:
1.3
Max. score:
4
Reversibility:
other: Ectropion and circumcorneal vascularisation persisted at the end of observation. Effects were considered irreversible
Remarks on result:
other: irreversible effects
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
1
Max. score:
2
Reversibility:
other: Ectropion and circumcorneal vascularisation persisted at the end of observation. Effects were considered irreversible
Remarks on result:
other: irreversible effects
Irritation parameter:
conjunctivae score
Remarks:
(redness)
Basis:
animal #1
Time point:
24/48/72 h
Score:
2
Max. score:
3
Reversibility:
other: Ectropion and circumcorneal vascularisation persisted at the end of observation. Effects were considered irreversible
Remarks on result:
other: irreversible effects
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
2
Max. score:
4
Reversibility:
other: Ectropion and circumcorneal vascularisation persisted at the end of observation. Effects were considered irreversible
Remarks on result:
other: irreversible effects
Irritation parameter:
other: discharge
Basis:
animal #1
Time point:
24/48/72 h
Score:
1.67
Max. score:
3
Irritant / corrosive response data:
Areas of diffuse corneal opacity were noted in the treated eye one hour after treatment and at the 24 and 48-hour observations with areas of translucent corneal opacity noted at the 72-hour and 7-day observations. Diffuse corneal opacity was also noted at the 14-day observation.

Iridial inflammation was noted in the treated eye one hour after treatment and at the 24, 48, 72-hour and 7-day observations with minimal conjunctival irritation noted at the 14-day observation.
Other effects:
Ectropion was noted in the treated eye at the 7, 14 and 21-day observations with circumcorneal vascularisation invading o to 2 mm onto the cornea also noted at these times. These reactions were considered to be irreversible.
Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
An in vivo eye irritation test was performed according to OECD guideline and GLP principles. Based on irreversibel effects to the eye, BISOMER PTE is classified cat. 1 for eye irritancy.
Executive summary:

An in vivo eye irritation test was performed according to the OECD guideline and GLP principles. One rabbit was treated and observed for 21 days. The average scores for observations at 24, 48 and 72 hours were 1.3 (cornea score), 1 (iris score) and 2 (chemosis score). At the end of the observation period of 21 days, ectropion was noted in the treated eye with circumcorneal vascularisation invading up to 2 mm onto the cornea. Since these reactions were considered to be irreversible, BISOMER PTE is classified cat. 1 for eye irritancy (corrosive).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In an in vitro skin irritation test using a human skin model ( EPISKIN Standard Model) according to OECD 439 guideline and GLP principles, the influence of Accelerator (PT 25E or PT 25E/2) on the viability of human skin was tested. The test substance was applied directly to 0.38 cm2cultured skin (25 μl). After 15 minutes, the substance was removed and cells were cultured for 43 hours. The viability of the cells was tested by reduction of MTT. Survival of unexposed skin was set at 100%, the positive control had a mean cell viability of 31% whereas the test substance showed cell viability of 23%. Since the mean relative tissue viability after exposure to the test substance was below 50%, it can be concluded that the test substance is irritating in the in vitro skin irritation test.

In an in vitro skin corrosion test using a human skin model (EpiDerm Skin Model) according to OECD guideline 431 and GLP principles, the influence of Accelerator (PT 25E or PT 25E/2) on the viability of human skin was tested. The test substance was applied directly to 0.6 cm2cultured skin (25mg, in presence of 25 μl Milli-Q water). After 3 minutes or 1 hour, the substance was removed and cells were cultured for 3 hours in the presence of MTT. The viability of the cells was tested by reduction of MTT. Survival of unexposed skin was set at 100%, the positive control had a mean cell viability of 14% and 8% after resp. 3 minutes or 1 hour exposure whereas the test substance showed cell viability of 85% and 56% resp. Since the mean relative tissue viability after exposure to the test substance was above 50% or 15% after resp. 3 minutes or 1 hour exposure, it can be concluded that the test substance is not corrosive in the in vitro skin corrosion test.

The substance was tested in a reliable in vitro skin irritation test with reconstructed human epidermis, performed according to OECD guidelines and according to GLP principles. Based on the observed results and applying the evaluation criteria, it was concluded, that the test substance does not show a skin irritation potential in the EpiDerm (TM) skin irritation test under the test conditions chosen.

An in vivo eye irritation test was performed according to the OECD guideline and GLP principles. One rabbit was treated and observed for 21 days. The average scores for observations at 24, 48 and 72 hours were 1.3 (cornea score), 1 (iris score) and 2 (chemosis score). At the end of the observation period of 21 days, ectropion was noted in a treated eye with circumcorneal vascularisation invading up to 2 mm onto the cornea. Since this reaction was considered to be irreversible, BISOMER PTE is classified cat. 1 for eye irritancy (corrosive).


Justification for selection of skin irritation / corrosion endpoint:
Two studies were selected as key studies: an in vitro skin irritation study (WIL 501366) and an in vitro skin corrosion study (WIL 501367). Both studies are needed for the endpoint conclusion. Both studies were conducted according to OECD guideline and GLP principles (both reliability 1).

Effects on skin irritation/corrosion: irritating

Effects on eye irritation: corrosive

Justification for classification or non-classification

According to the available data, the test substance is irritating to the skin, but not corrosive and is classified skin irritant cat. 2 according to CLP Regulation (EC) No. 1272/2008.

According to the available data, it is concluded that the test substance is corrosive to the eye, and is classified cat.1 according to CLP Regulation (EC) No. 1272/2008.