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Description of key information

Skin irritation: not irritating (in vitro reconstructed human epidermis model test, OECD 439/ EU B.46, GLP)

Eye irritation: serious eye damage (in vitro bovine corneal opacity and permeability test, OECD 437/EU B.47, GLP)

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:
2017-05-05 - 2017-06-02
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)
Version / remarks:
adopted July 28, 2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
adopted July 06, 2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
other: three-dimensional reconstructed human epidermis model EpiDermTM, comprised of normal, human-derived epidermal keratinocytes
Cell source:
other: human
Justification for test system used:
recommended by guideline
Vehicle:
unchanged (no vehicle)
Details on test system:
TEST SYSTEM
EpiDermTM (EPI-200, Lot no. 25815) MatTek In Vitro Life Science Laboratories, s.r.o, Mlynské Nivy 73, 821 05 Bratislava II, Slovak Republic.
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
Normal human keratinocytes were used to construct the epithelium. Multiple layers of viable epithelial cells (basal layer, stratum spinosum, stratum granulosum) were present under a functional stratum corneum. The Stratum corneum was multilayered containing the essential lipid profile to produce a functional barrier with robustness to resist rapid penetration of the cytotoxic marker substance sodium dodecyl sulphate (SDS). The barrier function is assessed either by determination of the concentration at which a marker substance reduces the viability of the tissues by 50% (IC50) after a fixed exposure time, or by determination of the exposure time required to reduce cell viability by 50% (ET50) upon application of the marker substance at a specified, fixed concentration. The containment properties of the model prevented the passage of material around the stratum corneum to the viable tissue, which would lead to poor modelling of skin exposure. The skin model was free of contamination by bacteria, viruses, mycoplasma, or fungi.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: The whole exposure period for the used EpiDermTM skin model was 60 minutes. The incubation conditions were 37°C, 5% CO2 and 95% relative humidity for the first 35 minutes followed by 25 minutes at room temperature under a sterile hood.
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: At the end of the exposure period, the test item was carefully washed from the skin surface with Dulbecco's phosphate buffered saline (D-PBS)

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Tecan Sunrise Magellan Version 7.2
- Wavelength: 540 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The preferred assay for determining the magnitude of viability is the MTT assay. The optical density (OD) of the extraction solvent alone should be sufficiently small, i.e. OD < 0.1. The tissue treated with the negative control (NC) should exhibit stability in culture (provide similar viability measurements) for the duration of the test exposure period.
- Quality controls (QC) of the model: The EpiDerm™ System was manufactured according to defined quality assurance procedures. All biological components of the epidermis and the culture medium were tested by manufacturer for viral, bacterial, fungal and mycoplasma contamination. The supplier of the test system determines the ET50 value following exposure to Triton X-100 (1%) for each EpiDerm lot. The ET50 must fall within a range established based on a historical database of results. A respective certificate of analysis regarding cell source, analysis for potential biological contaminants and analysis for tissue functionality and quality is added to the study report.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Not applicable. Prior to testing it was evaluated that there is no possible interference with the MTT measurement (OD 540 nm), due to colour changes or direct interacting with the MTT assay reagent.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 3

PREDICTION MODEL / DECISION CRITERIA
mean tissue viability ≤ 50% Irritant (I); further testing is required to distinguish between Category 1 or Category 2
mean tissue viability > 50% non-irritant (NI)
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 25 mg of test item were applied to the skin model and uniformly covered the skin surface with an area of 0.63 cm².

VEHICLE
- The test item is a fine powder. For better contact of the test item to the skin, the skin surface was moistened with 25 µL Dulbecco’s phosphate buffered saline.

NEGATIVE CONTROL
- Amount(s) applied: 30 µL Dulbecco's phosphate buffered saline (D-PBS)
- Concentration (if solution): n.a.

POSITIVE CONTROL
- Amount(s) applied: 30 µL sodium dodecyl sulphate (SDS) solution
- Concentration (if solution): 5%
Duration of treatment / exposure:
60 min
Duration of post-treatment incubation (if applicable):
Posttreatment incubation period of the rinsed tissues in fresh assay medium of 42 hours
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean value
Value:
64.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
DEMONSTRATION OF TECHNICAL PROFICIENCY:
Viability: The preferred assay for determining the magnitude of viability is the MTT assay. The optical density (OD) of the extraction solvent alone should be sufficiently small, i.e. OD < 0.1. The tissue treated with the negative control (NC) should exhibit stability in culture (provide similar viability measurements) for the duration of the test exposure period.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The absolute optical density (OD) of the negative control (NC) tissues (treated with sterile PBS buffer) in the MTT test is an indicator of tissue viability obtained in the testing laboratory after shipping and storing procedures and under specific conditions of use. The assay meets the acceptance criterion if the mean OD of the NC tissues is ≥ 1.0 and ≤ 2.5. The mean optical density (OD) of 3 negative control tissues was 1.794 and was well within the acceptable range of ≥ 1.0 to ≤ 2.5.
- Acceptance criteria met for positive control: A 5% SDS (in H2O) solution was used as a positive control (PC) and tested concurrently with the test chemicals. Concurrent means here that the PC has to be tested in each assay, but only one PC is required per testing day. The assay meets the acceptance criterion if the mean viability of PC tissues expressed as % of the negative control tissues is ≤ 20%. The viability of cells treated with the positive reference item, 5% SDS, was 4.8% of the negative control and fulfilled the acceptance criterion of ≤ 20%.
The standard deviation determined for all triplicates
- Acceptance criteria met for variability between replicate measurements: Since in each test skin irritancy potential is predicted from the mean viability determined on 3 single tissues, the variability of tissue replicates should be acceptably low. The assay meets the acceptance criterion if the standard deviation (SD) calculated from individual % tissue viabilities of the 3 identically treated replicates is ≤ 18%. The standard deviation determined for all triplicates was below the limit of acceptance of 18%.
Interpretation of results:
GHS criteria not met
Conclusions:
n-Butyltriphenylphosphonium bromide was not irritating in the in vitro skin irritation reconstructed human epidermis model test.
According to the criteria of REGULATION (EC) No 1272/2008, n-Butyltriphenylphosphonium bromide does not have to be classified and has no obligatory labeling requirement for skin irritation.
According to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations, n-Butyltriphenylphosphonium bromide does not have to be classified for skin irritation.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-05-05 - 2017-05-18
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)
Version / remarks:
dated December 08, 2010
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: slaughterhouse
- Characteristics of donor animals (e.g. age, sex, weight): Bovine eyes from cattle in the age range of 6 to 12 months; no data on sex and weight
- indication of any existing defects or lesions in ocular tissue samples: Upon arrival at the laboratory, the eyes were examined for defects such as but not limited to increased opacity, scratches, and neovascularisation. Only corneas from eyes free of defects were used. The quality of each cornea was also evaluated at later steps in the assay. Corneas that had opacity greater than seven opacity units or equivalent for the opacitometer and cornea holders used after an initial one hour equilibration period had to be discarded.
- Indication of any antibiotics used: To minimize deterioration and bacterial contamination, on collection the eyes were completely submerged in Hanks’ Balanced Salt Solution, (HBSS) containing penicillin at 100 IU/mL and streptomycin at 100 μg/mL; no further data
Vehicle:
physiological saline
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): 20% suspension in 0.9% sodium chloride solution (w/v)

VEHICLE
- 0.9% sodium chloride solution
Duration of treatment / exposure:
240 minutes
Duration of post- treatment incubation (in vitro):
n.a.
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
Bovine eyes from cattle in the age range of 6 to 12 months were obtained from a slaughterhouse. The corneas were dissected with a 2 to 3 mm rim of sclera.

QUALITY CHECK OF THE ISOLATED CORNEAS
Upon arrival at the laboratory, the eyes were examined for defects such as but not limited to increased opacity, scratches, and neovascularisation. Only corneas from eyes free of defects were used. The quality of each cornea was also evaluated at later steps in the assay. Corneas that had opacity greater than seven opacity units or equivalent for the opacitometer and cornea holders used after an initial one hour equilibration period had to be discarded.

NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
Solvent control was used as negative control

SOLVENT CONTROL USED: yes; 0.9% sodium chloride solution

POSITIVE CONTROL USED: yes ; 20% Imidazole (CAS no. 288-32-4) in 0.9% sodium chloride solution

APPLICATION DOSE AND EXPOSURE TIME: 750 µL, 240 minutes

TREATMENT METHOD: The corneas were mounted in corneal holders with anterior (epithelium) and posterior (endothelium) chambers. Beginning with the posterior chambers, the chambers were filled to excess with pre-warmed Eagle’s Minimum Essential Medium (EMEM), while preventing bubble formation. The corneal holder was equilibrated at 32 ± 1°C for at least one hour.

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the exposure period of 240 minutes the test item, the negative and positive controls, were removed from each chamber. Subsequently, the epithelium was washed with EMEM containing phenol red at least three times. Washing was repeated until no test item or discolouration (yellow or purple) of phenol red was visible. The corneas were rinsed a final time with EMEM only to remove any remaining phenol red from the chamber. The chamber was then filled with EMEM without phenol red.

- POST-EXPOSURE INCUBATION: no

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was determined by the amount of light transmission through the cornea measured quantitatively with the aid of an opacitometer resulting in opacity values measured on a continuous scale.
- Corneal permeability: To determine the corneal permeability 1 mL sodium fluorescein solution (5 mg/mL in 0.9% sodium chloride solution) was added to the anterior chamber (epithelial surface) while the posterior chamber (endothelial surface) was refilled with fresh EMEM. The holder was incubated in a horizontal position at 32 ± 1°C for 90 ± 5 minutes. The amount of sodium fluorescein that crossed from the anterior to the posterior chamber was measured quantitatively using a microplate reader (Tecan Sunrise Magellan Version 6.410). Measurements at 490 nm were recorded as optical density (OD490). The fluorescein permeability values were determined using OD490 values based upon a visible light spectrophotometer (Tecan Sunrise) using a standard 1 cm path length.

- Others: After the equilibration period, fresh pre-warmed EMEM was added to both chambers and baseline opacity readings were taken for each cornea. Corneas exhibiting macroscopic tissue damage (e.g. scratches, pigmentation, neovascularisation) or an opacity >7 opacity units were discarded. The mean opacity of all equilibrated corneas was calculated by use of an opacitometer. A minimum of three corneas with opacity values close to the median value for all corneas were selected as negative control corneas. The remaining corneas were then distributed into treatment and positive control groups.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS):
After correcting the opacity and mean permeability (OD490) values for background opacity and the negative control permeability OD490 values, the mean opacity, and permeability OD490 values for each treatment group were combined in an empirically-derived formula to calculate an in vitro irritancy score (IVIS) for each treatment group as follows: IVIS = mean opacity value + (15 x mean permeability OD490 value)
> 3 and <= 55: No prediction can be made
> 55: corrosive or severe irritant

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used: yes
Irritation parameter:
in vitro irritation score
Remarks:
opacity
Run / experiment:
mean of three corneas
Value:
10.491
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other:
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for solvent (negative) control: The corneas treated with the negative control item 0.9% sodium chloride solution revealed a mean opacity value of 0.531 ± 0.161 and a mean permeability value of 0.017 ± 0.019. The calculated IVIS value of 0.791 ± 0.328 was well below the cut-off value of 3 (UN GHS no category).
- Acceptance criteria met for positive control: The corneas treated with the positive control item 20% Imidazole in 0.9% NaCl solution revealed a mean opacity value of 66.840 ± 5.811 and a mean permeability value of 2.381 ± 0.389 compared to the solvent control. The calculated IVIS value of 102.560 ± 11.271 was within two standard deviations of the current historical mean and well above the cut-off value of 55. Hence, the acceptance criteria for the test were fulfilled.

Following treatment with n-Butyltriphenylphosphonium bromide a mean opacity of 7.131 ± 2.342 and a mean permeability value of 0.224 ± 0.091 compared to the negative control were determined. The calculated IVIS of 10.491 ± 2.268 is above the cut-off value of 3 (UN GHS no category) and below the cut-off value of 55, identifying test substances as inducing serious eye damage (UN GHS Category 1).

Consequently no prediction concerning irritant or severely irritant potential of the test item can be made.

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
The test of n-Butyltriphenylphosphonium bromide in a Bovine Corneal Opacity and Permeability Test resulted in an in vitro irritancy score (IVIS) of 10.491 ± 2.268 indicating serious effects to the eyes. However, the method is not validated to discriminate between Serious Eye Damage Cat. 1 and Serious Eye Irritation Cat. 2. In accordance with the ECHA Guidance document Chapter R7.a, Version 6.0 –July 2017, R.7.2.8.1, Figure R. 7.2-5 5, n-Butyltriphenylphosphonium bromide is classified in Category 1 (irreversible effects on the eye).
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In an in vitro skin irritation reconstructed human epidermis model test performed in accordance with OECD Guideline 439 (adopted July 28, 2015) and EU Method B.46 (adopted July 06, 2012), n-Butyltriphenylphosphonium bromide was applied to the three-dimensional human epidermis model EpiDermTM for an exposure period of 60 minutes in triplicates. 25 mg of test item were applied to the skin model and uniformly covered the skin surface with an area of 0.63 cm². The test item is a fine powder. For better contact of the test item to the skin, the skin surface was moistened with 25 µL Dulbecco’s phosphate buffered saline (D-PBS). At the end of the exposure period, the test item was carefully washed from the skin surface with D-PBS. Subsequently the tissue constructs were incubated for 42 h at 37°C. Cytotoxicity (irritancy) was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue (MTT).The positive (5% sodium dodecyl sulphate (SDS)) and negative (D-PBS) control gave responses that were within the acceptance criteria and as such demonstrated the validity of the study. The relative mean tissue viability obtained after 60 minutes treatment with n-Butyltriphenylphosphonium bromide compared to the negative control tissues was 64.4%. Since the mean relative tissue viability for the test substance was above 50%, n-Butyltriphenylphosphonium bromide is identified to be not irritating.

In an in vitro Bovine Corneal Opacity and Permeability Test according to OECD guideline 437 (adopted July 26, 2013) and EU method B.47 (dated December 08, 2010), n-Butyltriphenylphosphonium bromide was applied for 240 minutes to three isolated corneas from bovine eyes. The solid test item was dissolved in a 0.9% sodium chloride solution with a final concentration of 20% n-Butyltriphenylphosphonium bromide as recommended in the test guideline 437 for non-surfactant solids. Possible damage by the test item was assessed by quantitative measurements of changes in corneal opacity and permeability.

The positive (20% Imidazole in 0.9% NaCl) and negative (solvent, 0.9% NaCl) control gave responses that were within the acceptance criteria and as such demonstrated the validity of the study. Following treatment with n-Butyltriphenylphosphonium bromide a mean opacity of 7.131 ± 2.342 and a mean permeability value of 0.224 ± 0.091 compared to the negative control were determined. The in vitro irritancy score (IVIS) was 10.491 ± 2.268 indicating serious effects to the eyes. However, the method is only validated for classification in EU CLP/UN GHS no category (in vitro irritancy score (IVIS<3) and EU CLP/GHS Category 1 (IVIS > 55). The method is not validated to discriminate between Serious Eye Damage Cat. 1 and Serious Eye Irritation Cat. 2.

In accordance with the ECHA Guidance document Chapter R7.a, Version 6.0 –July 2017, R.7.2.8.1, Figure R. 7.2-5 5, n-Butyltriphenylphosphonium bromide is classified in Category 1 (irreversible effects on the eye).

Justification for classification or non-classification

n-Butyltriphenylphosphonium bromide was not irritating in the in vitro skin irritation reconstructed human epidermis model test.

According to the criteria of REGULATION (EC) No 1272/2008, n-Butyltriphenylphosphonium bromide does not have to be classified and has no obligatory labeling requirement for skin irritation.

According to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations, n-Butyltriphenylphosphonium bromide does not have to be classified for skin irritation.

The test of n-Butyltriphenylphosphonium bromide in a Bovine Corneal Opacity and Permeability Test resulted in an in vitro irritancy score (IVIS) of of 10.491 ± 2.268 indicating on serious effects to the eyes. However, the method is not validated to discriminate between Serious Eye Damage Cat. 1 and Serious Eye Irritation Cat. 2. In accordance with the ECHA Guidance document Chapter R7.a, Version 6.0 –July 2017, R.7.2.8.1, Figure R. 7.2-5 5, n-Butyltriphenylphosphonium bromide is classified in Category 1 (irreversible effects on the eye).