Registration Dossier

Administrative data

Description of key information

Skin Irritation/corrosion:


Warren (2011a), OECD 439, Skin Irrit. 2


Warren (2011b), OECD 431, Inconclusive  


Eye irritation:


Sanders (2011), ICCVAM REET acute eye irritation, Irritating 


Khalepo (1968), Acute eye irritation, Inconclusive 


 


Khalepo (1968) was determined to be inadequate for classification of the test substance due to the extremely limited information reported (Klimisch score 4). The ICCVAM REET acute eye irritation study conducted by Sanders (2011) concluded that the test substance has the potential to be an eye irritatant. However the study does not meet the information requirements withing REACH and therefore is not adequate for classification, the results can be used in a weight if evidence approach in the classification of the substance. Therefore, an additional study is required in order to determine the eye irritation/corrosion potential of the test substance.


The in vitro skin corrosion study (Warren, 2001b) conducted in accoradance with  OECD TG 431 indicated the test material was not corrosive to the skin. In order to determine classification an additional in vitro skin irritation study (Warren, 2011a) was conducted in accordance with OECD TG 439 where the test substane was determined to be a skin irritant Category 2 classification according to the CLP Regulation (EC) No 1272/2008. 


 


 

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:
08 December 2010 to 10 December 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was conducted in accordance with OECD Guideline 431 "In Vitro Skin Corrosion: Human Skin Model Test". This study was performed in compliance with UK GLP standards (SI 1999/3106 as amended by SI 2004/0994)).
Qualifier:
according to guideline
Guideline:
other: OECD Guideline for the Testing of Chemicals No. 431 "In Vitro Skin Corrosion: Human Skin Model Test" (adopted 13 April 2004)
Deviations:
yes
Remarks:
The acceptance criteria for the negative control deviated from that outlined in the OECD TG.
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: Source: not stated, Batch number: 101009
- Expiration date of the lot/batch: not supplied
- Purity test date: not supplied

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Store at room temperature in the dark
- Stability under storage conditions: Not stated
- Stability under test conditions: Stable
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: Not required as test substance is applied neat
- Reactivity of the test substance with the solvent/vehicle /test medium (if applicable): Not Applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: No pre treatment was conducted
- Preliminary purification step (if any): N/A
- Preparation of a nanomaterial dispersion (incl. dilution): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Not stated
Source strain:
not specified
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Model Kit 0.38cm3
- Tissue batch number(s): Not stated
- Production date: Not stated
- Shipping date: Not stated
- Delivery date: 07 December 2010
- Date of initiation of testing:08 December 2010

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room Temperature
- Temperature of post-treatment incubation (if applicable): Room Temperature

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Number of washing steps were not indicated. The samples were washed for 40 seconds
- Observable damage in the tissue due to washing: Not stated
- Modifications to validated SOP: Not stated

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/ml
- Incubation time: 3 hours
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 540 nm
- Filter: not stated
- Filter bandwidth: Not Stated
- Linear OD range of spectrophotometer: Not stated

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Not stated
- Barrier function: Not stated
- Morphology: Not stated
- Contamination: Not Stated
- Reproducibility: Not Stated

NUMBER OF REPLICATE TISSUES: Study reports use of Duplicate tissues

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues - Not stated
- Procedure used to prepare the killed tissues (if applicable): Not stated
- N. of replicates : Not stated
- Method of calculation used: Percentage tissue viability

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be non-corrosive to skin if after 240 minutes the relative mean tissue viability (% of negative control) is ≥ 35% .
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 ul of test material was applied
- Concentration (if solution): neat, 100%

VEHICLE
- Amount(s) applied (volume or weight with unit): N/A
- Concentration (if solution): N/A
- Lot/batch no. (if required): N/A
- Purity: N/A

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50ul
- Concentration (if solution): 0.9% sodium chloride solution

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50ul
- Concentration (if solution): Concentration of glacial acetic acid not stated
Duration of treatment / exposure:
3 minutes, 60 minutes, 240 minutes
Duration of post-treatment incubation (if applicable):
3 hours
Number of replicates:
Duplicated tissues stated
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minutes exposure
Value:
205.6
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minutes exposure
Value:
122.9
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
240 minutes exposure
Value:
58.9
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: Not stated
- Direct-MTT reduction: the test item did not reduce MTT.
- Colour interference with MTT: the MTT solution containing the test item did not turn blue/purple.


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD540 of the tissue replicates treated with sodium chloride solution was 0.231 which is within the acceptance criteria of ≥ 0.115 and ≤ 0.4.
- Acceptance criteria met for positive control: the relative mean tissue viability was 5.2% relative to the negative control treated tissues following the 240 minute exposure period which is within the acceptance criteria of 0 to 20% relative to the negative control following 240 minute exposure period
- Acceptance criteria met for variability between replicate measurements: Not stated
- Range of historical values if different from the ones specified in the test guideline: historical data not provided.

Table 1. Mean OD540 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

 Item  Exposure Period  Mean OD540 of Duplicate Tissues  Relative Mean Vaibility (%)
 Negative Control Item  240 Minutes  0.231  100
 Positive Control Item  240 Minutes  0.012  5.2
Test Item  240 Minutes  0.136  58.9
 60 Minutes  0.284  122.9
 3 Minutes  0.475  205.6
Interpretation of results:
other: The test item was considered to be non corrosive to the skin according to OECD TG 431
Conclusions:
Under the conditions of the conducted test, the test substance did not possess corrosive properties towards reconstructed human epidermis tissue in the EpiSKIN model. No prediction on the skin irritation potential can be made and therefore additional testing should be conducted for classification and labelling purposes.
Executive summary:

the study was performed to OECD TG 431 under GLP conditions to assess the corrosive potential of the test material to Reconstructed Human Epidermis (RHE) following  single applciation. A volume of 50ul of the test item wsa applied topically to duplicate tissues ensuring uniform coverage of the tissues. duplicate tissues were also treated with 50ul of negative control 0.9% sodium chloride solution and positive control glacial acetic acid respectively. The RHE tissues samples were exposed to the test substance for 3, 60 and 240 minutes respectively with RHE tissues exposed to negative and positive controls for 240 minutes. Following the respective exposure times the tissues were rinsed for 40 seconds with PBD and incubated for 3 hours plus/minus 5 minues at room temperature. Under the conditions of this study, the test item was considered to not to be corrosive to the skin. 

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 January 2011 to 24 January 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Remarks:
This study was conducted using an alternative testing method (EPISKIN reconstructed human epidermis model) considered to provide valid data for the skin irritation/corrosion endpoint. The study was also performed in compliance with UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)).
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Principles of method if other than guideline:
The principle of the assay is based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colourimetric MTT reduction assay. Cell 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 formazan salt (within the mitochondria of viable cells) in the test item treated tissue relative to the negative controls. The concentration of the inflammatory mediator IL-1alpha in the culture medium retained following the 42-hour post-exposure incubation period is also determined for test items which are found to be borderline non-irritant based on the MTT reduction endpoint. This complementary endpoint will be used to either confirm a non-irritant result or will be used to override the non-irritant result.

The EPISKIN model is a three dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13-day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: Batch number: 101009
- Expiration date of the lot/batch: Not stated
- Purity test date: Not stated

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Store at room temperature in the dark
- Stability under storage conditions: Not stated
- Stability under test conditions: Stable
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: N/A
- Reactivity of the test substance with the solvent/vehicle /test medium (if applicable): N/A

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: N/A
- Preliminary purification step (if any): N/A
- Preparation of a nanomaterial dispersion (incl. dilution): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN(TM)
- Tissue batch number(s): Not stated
- Production date: Not stated
- Shipping date: Not stated
- Delivery date: 18 January 2011
- Date of initiation of testing: 20 January 2011

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature
- Temperature of post-treatment incubation (if applicable): 37˚C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps:Volume not stated, tissues were rinsed for 40 seconds
- Observable damage in the tissue due to washing: Not stated
- Modifications to validated SOP: Not stated

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/ml
- Incubation time: 3hours
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 540 nm
- Filter: Not stated
- Filter bandwidth: Not stated
- Linear OD range of spectrophotometer: Not stated

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Not stated
- Barrier function:Not stated
- Morphology: Not stated
- Contamination: Not stated
- Reproducibility: Not stated

NUMBER OF REPLICATE TISSUES: Three

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues - Not stated
- Procedure used to prepare the killed tissues (if applicable): N/A
- N. of replicates : Not stated
- Method of calculation used: Relative mean viability (5)

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritating to skin if the mean percent tissue viability after
exposure and post-treatment incubation is less than or equal (≤) to 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
10ul of test substance, negative control and positive control was applied
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
Three
Irritation / corrosion parameter:
% tissue viability
Remarks:
three tissue replicates
Run / experiment:
15 minutes exposure
Value:
5
Vehicle controls validity:
not specified
Negative controls validity:
valid
Remarks:
PBS was used as a negative control
Positive controls validity:
valid
Remarks:
5% SDS was used as a positive control
Remarks on result:
positive indication of irritation
Remarks:
Relative mean +/- SD viability (%) for three replicate tissues was 5.0 +/- 4.3, indicating the test substance is positive for skin irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: Not stated
- Direct-MTT reduction: The test item is presumed to have reduced the MTT based on the results.
- Colour interference with MTT: Not stated

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean absolute OD540 of three negative controls was 0.720 which is within the acceptability criteria of ≥ 0.6 and ≤1.5 as indicated in the OECD TG 439 29 June 2020.
- Acceptance criteria met for positive control: The mean relative viability (%) of positive controls was 6.4 (with the acceptance criteria being <40% OECD TG 439 29 June 2020)
- Acceptance criteria met for variability between replicate measurements: Standard deviation of viability (%) was 4.3.
- Range of historical values if different from the ones specified in the test guideline: Not stated

 


 


TEST ITEM, POSITIVE CONTROL ITEM AND NEGATIVE CONTROL ITEM


The individual and mean OD540 values, standard deviations and tissue viabilities for the test item, negative control item and positive control item are presented in Table 1. The mean viabilities and standard deviations of the test item and positive control, relative to the negative control are also presented in Table 1.


 


Table 1. Mean OD540 Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item.


































































 Item



 OD540 of Tissues



 Mean OD540 of Triplicate Tissues



 SD of OD540



 Relative Individual Tissue Viability (%)



 Relative Mean Vaibility (%)



SD of Relative Mean Viability (%) 



Negative control Item



0.375



0.720



0.017



102.1



100+



2.3



0.722



100.3



0.702



97.5



Positive control Item



0.055



0.046



0.011



7.6



6.4



1.5



0.050



6.9



0.034



4.7



Test Item



0.072



0.036



0.031



10.0



5.0



4.3



0.016



2.2



0.021



2.9



 


The relative mean viability of the test item treated tissues was 5.0% after a 15 -minute exposure period.


 


QUALITY CRITERIA


The relative mean tissue viability for the positive control treated tissues was less than or equal to 40% relative to the negative control treated tissues and the SD value of the percentage viability was less than or equal to 18 %. The positive control acceptance criterion was therefore satisfied.


The mean OD540 for the negative control treated tissues was greater than or equal to 0.6 and the SD value of the percentage viability was less than or equal to 18 %. The negative control acceptance criterion was therefore satisfied.


The SD calculated from individual percentage tissue viabilities of the three identically treated tissues was less than or equal to 18 %. The test item acceptance criterion was therefore satisfied.

Interpretation of results:
irritating
Remarks:
The test substance has an irritating potential according to the study, which is equivalent/similar to OECD TG 439.
Conclusions:
Under the conditions of the RHE test method the test substance showed irritant properties. The mean relative tissue viability was <50% (5%) after 15 minute exposure to the test substance. the available data on skin irritation of the test substance does meet the criteria for classification according to the CLP Regulation (EC) 1271/2008 and therefore is sufficient for classification.

Executive summary:

The study was performed according to OECD TG 439 under GLP to assess the irritancy potential of the test material to reconstructed human epidermis skin (EPISKIN(TM)) following a single application. A volume of 10ul of test items was applied to the epidermis surface of the model in triplicate. Additional triplicate tissues were treated with 10ul of PBS serving as a negative control and 5% SDS as a positive control respectively. Tissues were exposed to either the test substance, negative or positive control for 15 minutes where they were removed and rinsed for 40 seconds with PBS solution where they were then incubated (in maintenance medium) at 37 ºC, 5% CO2 in air for 42 hours. The relative mean viability(%) of the test substance was determined to be 5.0%, under the conditions of this study the test item was considered to be irritating to the skin.


The OECD TG 439 does not have the capacity to resolve between UN GHS Categories 1 and 2 therefore information on the skin corrosive potential of the test chemical is required to determine the classification. Results from the OECD TG 431 (Warren, 2011) determined the test chemical to be non-corrosive with the mean tissue viability being greater or equal to 50%. Following the assessment of results obtained from OECD TG 439, the test substance was determined to be an irritant to the skin in accordance with UN GHS Category 2.  


 

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:
20 MAY 2011
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
This study has been undertaken in line with the ICCVAM - Recommended Test Method Protocol isolated Rabbit Eye Test method with some deviations identified. The study does not include key information such as the sex and strain of the rabbit used, there is no use of a positive or negative control, additionally the cut off value for maximum corneal opacity (cloudiness x area) is greater than that outlined in the ICCVAM protocol. ICCVAM has indicated that this IRE test method protocol is for non-regulatory use it does allow for a first stage assessment of the ocular irritancy potential of the test substance and to substantiate the overall irritant potential of the test chemical. The study is performed under GLP conditions in accordance with directive 2004/9/EC.
Qualifier:
equivalent or similar to guideline
Guideline:
other: ICCVAM - Test method protocol Isolated Rabbit Eye Test method
Version / remarks:
ICCVAM-Recommended Test Method Protocol Isolated Rabbit Eye Test Method
Originally published as Appendix B5 of “ICCVAM Test Method Evaluation Report: Current Validation Status of In Vitro Test Methods Proposed for Identifying Eye Injury Hazard Potential of Chemicals and Products”
NIH Publication No. 10-7553 – Published 2010
Available at: http://iccvam.niehs.nih.gov/methods/ocutox/MildMod-TMER.htm
Deviations:
yes
Remarks:
no use of a positive/negative control, source of enucleated eyes differ, cut off value for maximum corneal opacity (cloudiness x area) is greater than that outlined.
Principles of method if other than guideline:
- Principle of test: The purpose of the protocol is to provide details of the essential procedures required to (1) insure induction of corneal irritancy in the enucleated eye of the rabbit by a potentially irritating test substance, (2) evaluate the degree of irritancy, and (3) enable assignment of an appropriate regulatory classification on the potential ocular irritancy of a test substance. ocular corrosion or severe irritation.
- Short description of test conditions: the donor rabbits were sacrificed by intravenous administration of an overdose of sodium pentobarbitone. Immediately afterwards, two/three doses of saline solution was applied to the cornea to prevent desiccation during excision. The eye was removed, positioned in a perspex clamp and placed within the chamber of the superfusion apparatus, with the saline drip at the rear of the chamber to allow for irrigation of the surface of the cornea. The eyes were allowed to equilibrate for approximately 30 minutes where they were re-examined to ensure they had not been damaged during excision. Corneal thickness was measured with an ultrasonic pachymeter, with any eyes where the corneal swelling was greater than 10% relative to the pre-enucleation measurement were rejected. Three eyes were treated with the test item, with two additional eyes remaining untreated for control purposes. The test item was used undiluted as supplied. A volume of 0.1 ml of the test item was applied evenly over the surface of the cornea , after 10 seconds the test item was washed off the cornea using a minimum of 20ml saline solution. Immediately after washing of the corneal surface, the treated eye was returned to the superfusion chamber and the saline drop repositioned to irrigate the eye. The untreated eyes were similarly washed and used for control purposes.
- Parameters analysed / observed: Toxic effects in the isolated rabbit eye are measured by (1) subjective assessment of changes in corneal opacity, (2) uptake of fluorescein dye within the cornea (permeability), (3) increased corneal thickness (swelling), and (4) corneal epithelial changes (pitting, sloughing, mottling, etc.) which are evaluated macroscopically or by slit- lamp. The opacity, swelling, and permeability assessments following exposure to a test substance are assessed individually and are used to determine if the test substance has the potential to induce
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Batch number: 101009
- Purity, including information on contaminants, isomers: Not stated


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at room temperature in the dark
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage:N/A
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: Not stated
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: Not stated
- Reactivity of the test material with the incubation material used (e.g. plastic ware): Not stated

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): NA
- Preliminary purification step (if any): N/A
- Final concentration of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid (e.g. stock crystals ground to fine powder using a mortar and pestle): N/A
Species:
rabbit
Strain:
other: The strain of rabbit eye used in this in vitro study was not reported
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Not stated
- Number of animals: Three
- Characteristics of donor animals (e.g. age, sex, weight):Not stated
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): N/A
- Time interval prior to initiating testing: Not stated, the eyes were allowed to equilibrate for approximately 30 minutes following enucleation
- Indication of any existing defects or lesions in ocular tissue samples: No defects were noted
- Indication of any antibiotics used: Not stated
- Selection and preparation of corneas: Prior to enucleation, the eyes of provisionally selected rabbits were examined for evidence of occular irritation or defects following the application of Fluorescein Sodium drops BP (1% w/v). Examination was aided with the Kowa SL-5 slit lamp biomicroscope (Keeler Ltd, Windsor, Berks; UK). Corneal thickness values were also recored using the DGH-55 Ultrasonic pachymeter (DGH Technology Inc, Solana Beach, CA). Only animals whose eyes showed no evidence of ocular irritation or defect were used for testing purposes
- Quality check of the isolated corneas: Corneal thickness was measured using the ultrasonic pachymeter, with any eyes with swelling greater than 10% relative to the pre-enucleation measurement rejected.
Vehicle:
unchanged (no vehicle)
Controls:
other: Control eyes were used in the study
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 0.1 ml
- Concentration (if solution): test material is applied neat

VEHICLE
- Amount(s) applied (volume or weight with unit): NA
- Concentration (if solution): NA
- Lot/batch no. (if required): NA
- Purity: NA
Duration of treatment / exposure:
10 seconds
Duration of post- treatment incubation (in vitro):
60, 120, 180 and 240 minutes
Number of animals or in vitro replicates:
Three replicate enucleated eyes were used
Details on study design:
METHODS
Pre-Test Procedures
Superfusion Chamber
The water heating circulator (Julabo MP5, Jencons (Scientific) Ltd., Leighton Buzzard,
Beds, UK), was adjusted so that the temperature of the water flowing through the water
jacket of the superfusion apparatus, gave a stable temperature, of 32 ±1.5°C, within the
chambers of the apparatus. A peristaltic pump (205S/BA, Watson Marlow Ltd, Falmouth,
Cornwall; UK) was used to supply saline solution at a flow rate of 0.15 to 0.4 ml/minute
(at approximately 30°C) into the rear of each chamb er of the apparatus in order to
irrigate the surface of the cornea.

Selection of Eyes
Prior to enucleation, the eyes of the provisionally selected rabbits were examined for
evidence of ocular irritation or defect, following application of Fluorescein Sodium drops
BP (1% w/v). Examination was aided with the Kowa SL-5 slit-lamp biomicroscope
(Keeler Ltd, Windsor, Berks; UK). Corneal thickness values were also recorded using
the DGH-55 Ultrasonic pachymeter (DGH Technology Inc, Solana Beach, CA). Only
animals whose eyes showed no evidence of ocular irritation or defect were used for
testing purposes (Appendix 1).

Enucleation of Eyes
The donor rabbits were sacrificed by intravenous administration of an overdose of
sodium pentobarbitone. Immediately afterwards, two to three drops of saline solution
(approximately 32°C) were applied to the cornea to prevent desiccation during excision.
The eye was then carefully removed, positioned in a perspex clamp and placed within
the chamber of the superfusion apparatus, with the saline drip at the rear of the chamber
adjusted so that saline solution was allowed to irrigate the surface of the cornea. The
eyes were then allowed to equilibrate for approximately thirty minutes. Following the
equilibration period, the eyes were re-examined to ensure they had not been damaged
during excision. Corneal thickness was also measured using the ultrasonic pachymeter.
Any eyes in which the corneal swelling was greater than 10% relative to the preenucleation
measurement, or in which the cornea was stained with fluorescein, were
rejected. The post equilibration corneal thickness values for each eye were recorded
(Appendix 1).

PROCEDURE
Test Item Administration
Three eyes were treated with test item, two additional eyes remained untreated for
control purposes. The treatment eye was removed from the superfusion apparatus
whilst still being held in the perspex clamp. The clamp/eye was then placed horizontally
into a petri dish.
The test item was used undiluted as supplied. A volume of 0.1 ml of the test item was
applied as evenly as possible to the surface of the cornea. After ten seconds the test
item was washed off the cornea using a minimum of 20 ml of saline solution
(approximately 32°C).
Immediately following washing of the corneal surface, the treated eye was returned to
the superfusion chamber and the saline drip repositioned to irrigate the eye.
The untreated eyes were similarly washed and used for control purposes.

Observations
Assessment of corneal cloudiness was made pre-enucleation, post equilibration and
approximately 60, 120, 180 and 240 minutes following treatment, according to the
numerical evaluation given in Appendix 2 adopted from Advances in Modern Toxicology:
Dermatoxicology, 4th Ed, (F Marzulli and H Maibach, eds) Hemisphere Publishing
Corporation, Washington DC, 1991, pp 749-815.
Examination of the eye was facilitated by use of a slit-lamp biomicroscope. The
thickness of the cornea was measured using an ultrasonic pachymeter. For each
enucleated eye a measurement was made at the optical centre, and at a further four
locations at the apex of the cornea. A mean value for corneal thickness was then
calculated. Measurements for corneal thickness were carried out pre-enucleation, post
equilibration and approximately 60, 120, 180 and 240 minutes following treatment.
The condition of the corneal epithelium was assessed approximately 60, 120, 180 and
240 minutes following treatment. Assessment was facilitated by the use of the slit-lamp
biomicroscope.
The uptake of fluorescein by the corneal epithelium was assessed pre-enucleation, post
equilibration and approximately 240 minutes following treatment, according to the
numerical evaluation given in Appendix 2. This was carried out using the cobalt blue
filter of the slit-lamp biomicroscope, following application of Fluorescein Sodium drops.
Irritation parameter:
cornea opacity score
Run / experiment:
Maximum score obtained in 3 eyes
Value:
8
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
not examined
Remarks on result:
positive indication of irritation
Irritation parameter:
other: fluoroscein uptake
Run / experiment:
Maximum score obtained in 3 eyes
Value:
8
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
not examined
Remarks on result:
positive indication of irritation
Irritation parameter:
percent corneal swelling
Run / experiment:
Mean swelling calculated for 3 eyes - 4 hours following treatment
Value:
115.1
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
not examined
Remarks on result:
positive indication of irritation

Corneal Opacity

Individual scores for corneal opacity are given in Table 1 of the raw data provided in the attached background material.

Some loss of transparency was noted in one test eye and moderate loss of transparency

was noted in two test eyes at the 60-Minute observation with moderate loss of

transparency noted in all test eyes at the 120, 180 and 240 minute observations. No

corneal effects were noted in the control eyes during the study period.

Corneal Thickness

Individual and mean corneal thickness measurements and corneal swelling calculations

are given in Table 2 and Table 3 of the raw data provided in the attached background

material.

Corneal swelling of the test eyes during the study period was considerably greater than

that observed in the control eyes over the same period.

Corneal Condition

The condition of the corneal epithelium following treatment is given in Table 4 of

the raw data provided in the attached background material.

Sloughing of the corneal epithelium was noted in all test eyes. The condition of the

corneal epithelium of the control eyes appeared normal during the study period.

Fluorescein Uptake

Individual scores for fluorescein uptake are given in Table 5 of the raw data provided

in the attached background material.

Fluorescein uptake was noted in the test eyes 240 minutes following test item

application. No fluorescein uptake was noted in the control eyes 240 minutes following

treatment.

Interpretation of results:
other: The test item was considered to have the potential to cause severe ocular irritancy in vivo
Conclusions:
Following assessment of the data for all endpoints, the test item was considered to have the potential to cause severe ocular irritancy in vivo.
Executive summary:

The study was performed under GLP conditions in accordance with the ICCVAM - Test method protocol isolated rabbit eye (IRE) test method, with deviations noted.


The deviations noted inlcude key information such as the sex and strain of the rabbit used, there is no use of a positive or negative control and additionally the cut of value for maximum corneal opacity (cloudiness x area) is greater than that outlined in the ICCVAM protocol.


ICCVAM have indicated that the use of IRE test method protocol is not for use in regulatory testing straregies but is used as a first stage assessment of the ocular irritancy potential of a test substance and to substantiate the overall irritant potential of a test chemical. The EURL ECVAM have indicated that the rabbit eye enuclation test is not a validated test method to determine the endpoint of eye irritation but the results can be used as a weight of evidence in the classification of the substance. 


Application of the test substance to the enucleated eye resulted in all parameters; Corneal Opacity, Fluorscein and Corneal Swelling (%) exceeding the REET cut off values. It was therefore concluded that the test substance has the potential to cause severe ocular irritancy in vivo.

Endpoint:
eye irritation: in vivo
Type of information:
other: Published paper
Adequacy of study:
disregarded due to major methodological deficiencies
Study period:
Not stated
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Qualifier:
no guideline available
Principles of method if other than guideline:
Not stated
GLP compliance:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Not stated
- Purity, including information on contaminants, isomers, etc.: Not stated

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Not stated
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Not stated
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: Not stated
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: Not stated
- Reactivity of the test material with the incubation material used (e.g. plastic ware): Not stated

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): Not stated
- Preliminary purification step (if any): Not stated
- Final concentration of a dissolved solid, stock liquid or gel: Not stated
- Final preparation of a solid (e.g. stock crystals ground to fine powder using a mortar and pestle): Not stated
Species:
rabbit
Strain:
not specified
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Not stated
- Age at study initiation: Not stated
- Weight at study initiation: Not stated
- Housing: Not stated
- Diet (e.g. ad libitum): Not stated
- Water (e.g. ad libitum): Not stated
- Acclimation period: Not stated

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Not stated
- Humidity (%): Not stated
- Air changes (per hr): Not stated
- Photoperiod (hrs dark / hrs light): Not stated

Vehicle:
not specified
Controls:
not specified
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): Not stated
- Concentration (if solution): Not stated

VEHICLE
- Amount(s) applied (volume or weight with unit): Not stated
- Concentration (if solution): Not stated
- Lot/batch no. (if required): Not stated
- Purity:Not stated
Duration of treatment / exposure:
Single application of test material
Observation period (in vivo):
1 month
Duration of post- treatment incubation (in vitro):
NA
Number of animals or in vitro replicates:
Not stated
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): Not stated
- Time after start of exposure: Not stated

SCORING SYSTEM: Not stated

TOOL USED TO ASSESS SCORE: Not stated
Irritation parameter:
other: keratoconjunctivitis
Basis:
other: No animal no. stated
Time point:
other: Not stated
Reversibility:
fully reversible within: 1 month
Remarks on result:
not determinable because of methodological limitations
Irritant / corrosive response data:
Not stated
Interpretation of results:
study cannot be used for classification
Conclusions:
Under the limited conditions of this study outlined in the published paper the test material has an was determined to be irritating to the eye.
Executive summary:

The published paper pre-dates the existence of nationally or internationally recognised test methods. No specific details on the test method were provided. The limited information presented indicates that the application of test material m-Aminobenzotrifluoride (m-ABTF, a synonym for alpha,alpha,alpha-trifluoro-m-toluidine) in a single exposure was applied to the eye conjunctival sac of a rabbit. Keratoconjunctivitis was noted with recovery observed one month after exposure. Due to the limited information available on the study conducted this data is not considered reliable for use in the classification of the substance according to CLP Regulation (EC) No 1272/2008.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irreversible damage)

Additional information

Assessment of the test substance for skin irritation/corrosion and eye irritation was published within a paper which reported toxicity of fluorine derivatives of toluene (multiple related substances, of which alpha,alpha,alpha-trifluoro-m-toluidine was one) following single exposures in a number of animal models (Khalepo, 1968). The irritation/corrosion testing within Khalepo (1968) did not provide adequate detail on the studies, therefore these studies were considered to be unreliable and assigned a Klimisch Score of 4. As such, it was not possible to determine a classification using these studies alone, and additional testing according to the currently accepted testing strategy under REACH was required.


In accordance with the accepted testing strategy under REACH, an in vitro skin corrosion/irritation endpoint study was performed. The in vitro assessment of skin corrosion was undertaken according to OECD TG 431 (in vitro skin corrosion in the EPISKINTM Reconstructed human Epidermis Model (RhE)) (Warren, 2011b). At 240 minutes of exposure, relative mean tissue viability was 58.9%. As the relative mean tissue viability (%) was greater than or equal to 50% it did not meet the UN GHS criteria for classification as a skin corrosive substance. 


In line with the testing strategy, it was necessary to complete an in vitro skin irritation study (Warren, 2011a). The study was performed to OECD TG 439 (In vitro skin irritation: Reconstructed Human Epidermis Test method) to assess the skin irritancy potential of the test material following the single application of the test substance following a 15 minute exposure. Assessment of the test item’s skin irritatancy potential was made following an incubation period of 42 hours. The test material produced a relative mean viability (%) of 5.0 which is below the mean tissue viability for an irritant of <50% therefore the test material was considered to be a skin irritant. Since the RhE test methods outlined in the OECD TG 439 cannot resolve between UN GHS Skin irritant Categories 1 and 2, consideration of the results from the OECD TG 439 skin corrosion test are required in order to determine classification. The result of the OECD TG 431 determined the test chemical to be non-corrosive, with a mean tissue viability after exposure of  ≥50%, therefore the test chemical is considered to be irritant to the skin Category 2 in accordance with UN GHS criteria as per the CLP Regulation (EC) No 1272/2008. 


Assessment of the test substance for potential eye irritation effects conducted in vivo by Khalepo (1968) indicated that under the conditions of the test outlined in the published paper the test substance showed irritant effects in the eye. However, no prediction on the potential for serious eye damage could be made, therefore additional testing required in order to conclude on the classification of the substance.


In line with this requirement, a Rabbit Eye Enucleated Eye Test (REET) was conducted (Sanders 2011). The study was performed under GLP conditions in accordance with the ICCVAM - Test method protocol isolated rabbit eye test method, with deviations noted. Application of the test substance to the enucleated eye resulted in all parameters; Corneal Opacity, Fluorscein and Corneal Swelling (%) exceeding the REET cut off values. It was therefore concluded that the test substance was irritating to the eye. The REET is not a validated test method to determine the endpoint of eye irritation according to EURL ECVAM (ECVAM, 2020). However, they concluded that results of the REET test can be used as a weight of evidence in the classification of the test material. 


Using the results from the REET (Sander, 2011) and in vitro skin irritation (Warren, 2011a) the test chemical was determiend to be a skin irritant Category 2 in accordance with UN GHS criteria as per CLP Regulation (EC) No 1272/2008.


 


Reference: EURL ECVAM dataset on alternative methods to animal experimentation, Available via: https://ec.europa.eu/jrc/en/scientific-tool/database-alternative-methods-animal-experimentation (accessed December 2020). 

Justification for classification or non-classification




Results from the key studies OECD TG 439(Warren, 2011a) and 431 (Warren, 2011b) and results from the ICCVAM REET acute eye irritation study (Sanders, 2011) used as a weight of evidence were sufficient for Category 2 Eye Irritant classification according to CLP Regulation (EC) No 1272/2008.