Registration Dossier

Administrative data

Description of key information

Skin Irritation

Two in vitro and one in vivo skin irritation studies are available for the Substance and are summarised as follows:

 

OECD 431 (in vitro corrosion: RHE):               Non corrosive

OECD 439 (In vitro skin irritation: RHE):          Non irritant

OECD 404 (In vivo rabbit):                             Non irritant

Based on the weight of evidence the data indicates that the test material should not be classified as a skin irritant.

 

Eye Irritation

Two in vivo eye irritation studies and two in vitro eye irritation studies are available for the Substance. The studies are summarised as follows:

 

OECD 437 (BCOP):                                         Equivocal

OECD 492 (RhCE):                                          Non irritant

OECD 405 (in vivo Rabbit):                               Irritant

Summary study (in vivo Rabbit):                         Irritant

 

Based on the weight of evidence the data indicates that the test material should be classified as an eye irritant.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 October 2015 - 19 November 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Batch No: E00974-38D&F
Retest Date: 22 June 2018
Purity: 100%
Physical Description: Pale amber waxy solid
Storage Conditions: Ambient / dark
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: The human keratinocytes came from healthy consenting donors
Justification for test system used:
The EpiSkin® in vitro corrosion test uses EpiSkin® tissues supplied by SkinEthic. The Episkin® reconstructed human epidermis model has been accepted as a valid model for skin corrosion assessment by the OECD.
Vehicle:
unchanged (no vehicle)
Details on test system:
The human keratinocytes came from healthy consenting donors. HIV 1 & 2, HEP B and HEP C tests were carried out on the donors as well as verification of the bacteriological and fungal sterility of the cells and absence of mycoplasma. After a period of culture (13 days) a
well differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum were detectable. The surface area was 0.38 cm2.

The reproducibility of each batch was checked by histological analysis taking into account the general organisation, structure, and the reproducibility of the response of each EpiSkin batch was tested against a reference irritant: SDS.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
20 mg ± 2 mg
Duration of treatment / exposure:
3 min, 1 h +/- 5 min, or 4 h +/- 10 min
Number of replicates:
3 replicates per exposure time
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 mins
Value:
ca. 75.52
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 hour
Value:
ca. 118.44
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
4 hours
Value:
ca. 102.55
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The Substance did not reduce MTT.

The Substance did not become coloured upon mixing with ultrapure water.

See attached background documents

Interpretation of results:
GHS criteria not met
Conclusions:
Substance was demonstrated to be non-corrosive when tested in the EpiSkin in vitrocorrosion assay.
Executive summary:

In this study the corrosion potential of Substance was evaluated using the EpiSkin®in vitrocorrosion test.

 

Prior to the conduct of the corrosion assay, two preliminary tests were conducted to assess the intrinsic ability of the test item to reduce methylthiazoldiphenyl-tetrazolium bromide (MTT) to formazan, a measure of cell viability in the assay, and to assess the potential of the test item to cause colour interference. Substance did not reduce MTT to formazan or have the potential to cause colour interference, therefore, would not compromise the assay.

 

To assess dermal corrosion Substance was applied (20 mg ± 2 mg) to the exposed surface of EpiSkin®tissues for exposure periods of 4 h, 1 h and 3 min (3 tissues per exposure time). Due to the waxy nature of the test item it was heated toca 75°C then pipetted onto small filter papers, flattened onto the paper with a spatula and weighed. Filter papers were carefully placed on the exposed surface of the EpiSkin®tissue. The EpiSkin®surface area was 0.38 cm2, therefore the application rate wasca 52.6 mg/cm2. At the end of the exposure period, the filter papers were carefully removed and the surface of the Episkin®washed using Dulbecco’s phosphate-buffered saline (DPBS). The Episkin®tissues were transferred to MTT solution (0.3 mg/mL in assay medium) and incubatedin a humidified incubator set to maintain atemperature of 37°C and a CO2level of 5% for 3 h. Biopsies of the Episkin®membranes were removed, added to acidified isopropanol and left overnight, protected from light in order to extract formazan. The formazan production (cell viability) was assessed by measuring the optical density of the extracts at a wavelength of 570 nm.

 

Three replicates of each control (physiological saline, negative control, and acetic acid, positive control) were tested in parallel to test item treated tissues. Positive control was tested with the 4 h exposure group only, negative control was tested for all 3 exposure periods. The viability of each individual EpiSkinÒtissue was calculated as a percentage of the appropriate mean negative control viability (defined as 100%).

 

The mean A570of all negative control treated tissues was within 0.6-1.5 and the mean viability of positive control treated tissues was <20% therefore satisfying the assay acceptance criteria. Mean tissue viability (± SD) for test item treated tissues was 102.55 ± 16.53% (4h exposure), 118.44 ± 6.43% (1h exposure), and 75.52 ± 7.31% (3 min exposure).

 

In conclusion, Substance was demonstrated to be non-corrosive when tested in the EpiSkin in vitrocorrosion assay.
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 October 2015 - 23 November 2015
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)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Batch No: E00974-38D&F
Retest Date: 22 June 2018
Physical Description: Pale amber waxy solid
Storage Conditions: Ambient / dark
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: The human keratinocytes came from healthy consenting donors
Justification for test system used:
The EpiSkin¿ RhE model has been accepted as a valid model for the assessment of skin irritation by the Organisation for Economic Co-operation and Development (OECD).
Vehicle:
unchanged (no vehicle)
Details on test system:
The human keratinocytes came from healthy consenting donors. HIV 1 & 2, HEP B and HEP C tests were carried out on the donors as well as verification of the bacteriological and fungal sterility of the cells and absence of mycoplasma. After a period of culture (13 days) a
well differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum were detectable. The surface area was 0.38 cm2.

The reproducibility of each batch was checked by histological analysis taking into account the general organisation, structure, and the reproducibility of the response of each EpiSkin batch was tested against a reference irritant: SDS.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
10 mg +/- 2 mg
Duration of treatment / exposure:
15 Minutes
Duration of post-treatment incubation (if applicable):
recovery period of 42 h
Number of replicates:
Three replicates.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
15 Minute Exposure
Value:
ca. 118.35
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The Substance did not reduce MTT.

The Substance did not become coloured upon mixing with ultrapure water.

See attached background documents

Interpretation of results:
GHS criteria not met
Conclusions:
Substance was demonstrated to be non irritant when tested in the Episkin in vitro irritation assay.
Executive summary:

In this study, the irritation potential of Substance was evaluated using the EpiSkin in vitroirritation test.

 

Prior to the conduct of the irritation assay, a preliminary test was conducted to assess the intrinsic ability of the test item to reduce methylthiazoldiphenyl-tetrazolium bromide (MTT) to formazan, which is a measure of cell viability in the assay. In addition, prior to the conduct of the irritation assay, the ability of the test item to cause colour interference was assessed.  Substance did not reduce MTT to formazan, and did not cause any colour interference, and, therefore, would not compromise the assay.

The dermal irritation potential was assessed by applying 10 mg ± 2 mg of the solid Substance to the exposed surface of three EpiSkin tissues for 15 min. Prior to applying the test item, the EpiSkin surface was pre-moistened with ultra-pure water (5 µL). The surface area of the EpiSkin was 0.38 cm2, therefore the application rate wasca 26.3 mg/cm2. After the 15 min exposure period, the test item was removed from the surface of the EpiSkin and the tissues were washed using Dulbecco’s phosphate-buffered saline (DPBS). The EpiSkin tissues were then incubated for a recovery period of 42 hin a humidified incubator set to maintain atemperature and CO2level of 37°C and 5%, respectively. Following incubation, the EpiSkin tissues were transferred to assay medium containing MTT (0.3 mg/mL) and returned to the incubator for3 h. Biopsies of the EpiSkin membranes were removed, added to acidified isopropanol, and refrigerated forca 72 h in order to extract the formazan. The formazan production (cell viability) was assessed by measuring the optical density of the extracts at a wavelength of 550 nm. Three replicates of the positive control, aqueous sodium dodecyl sulphate (SDS) solution (5%, w/v), and the negative control, DPBS, were tested in parallel to demonstrate the efficacy of the assay. The viability of each individual EpiSkin tissue was calculated as a percentage of the mean negative control viability (defined as 100%).

 

Exposure to Substance resulted in a mean EpiSkin viability of 118.35% ± 9.57% of the negative control value. Exposure to the positive control, aqueous SDS solution (5%, w/v), resulted in a mean EpiSkin viability of 9.71% ± 4.88% of the negative control value. Cell viability values below a threshold of 50% of the negative control value indicate that the test item (or positive control treatment) is an irritant.

 

In conclusion, Substance was demonstrated to be non-irritant when tested in the EpiSkin in vitroirritation assay.

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 February 2000 - 25 February 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
GLP compliance:
yes
Specific details on test material used for the study:
Expiration Date: September 2004
Description:. Amber solid
Storage Condition: Room temperature

Adjustments in the dose calculations were not made to correct for the purity of the
test substance. The test substance, as received, was considered the “pure” substance.
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
Temperature: 64 to 72 degrees Fahrenheit
Humidity: 30 to 70 percent relative humidity
Lighting: Approximately 12 hours light (0700 to 1900) and 12 hours
dark (1900 to 0700) by automatic timer.

Monitored twice per day Monday through Friday and once daily on Saturday,
Sunday and holidays.
Type of coverage:
occlusive
Preparation of test site:
clipped
Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
0.5 mL
Duration of treatment / exposure:
4 hour Exposure
Observation period:
60 minutes, 24, 48 and 72 hours
Number of animals:
3 animals
Details on study design:
This study was conducted to evaluate the dermal irritation potential of the test substance in the rabbit. The test substance was administered to the clipped backs of three New Zealand White rabbits as a single 0.5 ml dose introduced under a gauze patch. The patch was secured with non-irritating tape and was loosely held in contact with the skin by means of a semi-occlusive dressing. After an exposure period of approximately 4 hours, the dressing and gauze patch were removed and residual test substance was wiped from the skin using
peanut oil and paper towels. The test site was graded for erythema, edema and other signs of dermal irritation approximately 60 minutes, 24, 48, and 72 hours following patch removal. All dermal scoring was made according to the modified Draize method (Draize, 1959).

All animals survived to study termination and all animals gained weight during the test period.
Irritation parameter:
erythema score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
fully reversible
Irritation parameter:
edema score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
fully reversible
Irritant / corrosive response data:
Topical application of the test substance did not elicit any signs of irritation. Erythema and edema were not observed in any animal at any interval. Since all animals were free of irritation at the 72-hour observation the study was terminated.
Other effects:
Supplemental dermal observations were not detected during the study.

Animal Number

Dermal Observation

Body Weight (kg)

Day 0

24 hours

48 Hours

72 Hours

ER

ED

SD

ER

ED

SD

ER

ED

SD

ER

ED

SD

Day 0

Term

JEK708M

0

0

 

0

0

 

0

0

 

0

0

 

2.47

2.62

JEK697M

0

0

 

0

0

 

0

0

 

0

0

 

2.32

2.57

JEK698M

0

0

 

0

0

 

0

0

 

0

0

 

2.33

2.60

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Primary Irritation Index: 0.0

NOTE

ER

=

Erythema

 

ED

=

Edema

 

SD

=

Supplemental Dermal Observation

Interpretation of results:
GHS criteria not met
Conclusions:
Based on these findings and a primary irritation index of 0.0, the test substance was considered a non-irritant to rabbit skin.
Executive summary:

This study was conducted to evaluate the dermal irritation potential of the test substance in the rabbit. The test substance was administered to the clipped backs of three New Zealand White rabbits as a single 0.5 ml dose introduced under a gauze patch. The patch was secured with non-irritating tape and was loosely held in contact with the skin by means of a semi-occlusive dressing. After an exposure period of approximately 4 hours, the dressing and gauze patch were removed and residual test substance was wiped from the skin using peanut oil and paper towels. The test site was graded for erythema, edema and other signs of dermal irritation approximately 60 minutes, 24, 48, and 72 hours following patch removal.

All dermal scoring was made according to the modified Draize method (Draize, 1959). All animals survived to study termination and all animals gained weight during the test period. V Topical application of the test substance did not elicit any signs of irritation during the study. Since all animals were free of irritation at the 72-hour observation, the study was terminated after that interval.

Based on these findings and a primary irritation index of 0.0, the test substance was considered a non-irritant to rabbit skin.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not 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:
07 March 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
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)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Batch /Lot /Notebook Ref: E00974-38D&F
Label Lot: E00350-288
Retest Date: 22 June 2018
Physical Description: Pale amber waxy solid
Purity: 100%
Storage Conditions: Ambient / dark
Species:
other: Bovine
Details on test animals or tissues and environmental conditions:
Cow eyes were collected from freshly slaughtered cattle, and placed into containers containing Hank’s balanced salt solution (HBSS). Transport to the lab was on the same day as slaughter. Eyes were kept cool during transport. HBSS and transport materials were provided by the lab.

Corneas were prepared and used for testing on the day of collection. Upon receipt, eyes were rinsed with HBSS at ambient temperature and examined for defects (scratches, opacity or neovascularisation) prior to use. Any eyes showing defects were rejected from further use. Corneas from undamaged eyes were dissected, leaving a sclera margin of 2 to 3 mm. Collected corneas were placed epithelial side down in HBSS at ambient temperature until use
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
75 mg per disc
Duration of treatment / exposure:
incubator set to maintain a temperature of 32C (actual incubator temperature 31.5+/-C) for 10 +/- 1 min
Duration of post- treatment incubation (in vitro):
2 h +/- 5 min in an incubator set to maintain a temperature of 32C (actual incubator temperature was 31.0C)
Number of animals or in vitro replicates:
3 replicate for negative and postive control
4 replicate for substance
Details on study design:
Bovine corneas were prepared and then mounted into corneal holders. Once mounted, both chambers of the cornea holders were filled with pre-warmed (ca 32ºC) Minimum Essential Medium (MEM) without phenol red. Holders were then allowed to equilibrate for =1 h in an incubator set to maintain a temperature of 32¿C. At the end of the equilibration period, baseline opacity readings were then taken using an Opacitometer. Damaged corneas (opacity >7 opacity units) were rejected from further use.

The negative control, physiological saline, was applied to the surface of three corneas, undiluted. Ethanol was used as a positive control and was applied to the surface of three corneas each, undiluted. The Substance was applied undiluted as a solid disc to 4 corneas. The dosed units were then returned to an incubator set to maintain a temperature at a temperature of 32¿C for 10 ¿ 1 min.

Following the exposure incubation, the chambers were opened and underwent a rinsing procedure. The corneas were then incubated for 2 h ¿ 5 min in an incubator maintained at a temperature of 32¿C and allowed to recover. Following the recovery period, the opacity of each cornea was measured. Gross damage or other changes were observed by visual assessment and recorded in the study files.

Following the measurement of opacity, a permeability test was conducted. The cornea were rotated to the horizontal position, and incubated for 90 ¿ 5 min in an incubator at a temperature of 32¿C

At the end of the permeability test, MEM from the posterior chambers was collected. These samples were stored in a refrigerator set to maintain a temperature of 4¿C overnight. Following overnight storage, triplicate aliquots from each sample were transferred into a 96 well plate, and analysed with a Multiskan Spectrum plate reader at 490 nm. Three blank wells containing MEM without phenol red were analysed on the same plate, along with two aliquots of a quality control solution (sodium fluorescein; 10 µg/mL).
Irritation parameter:
in vitro irritation score
Remarks:
IVIS
Value:
ca. 6.07
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable

See attached background document

Interpretation of results:
other: No prediction can be made
Conclusions:
In conclusion, the BCOP assay was unable to categorise the Substance, giving a result of “No prediction can be made”.
Executive summary:

Evaluation of ocular irritation is part of the Human Health Hazard Assessment required for registration of a chemical. The objective of this study was to evaluate the ocular irritation potential of substance using the Bovine Corneal Opacity and Permeability (BCOP) assay.

 

Potential for causing eye damage is an important safety evaluation for chemicals and products. Classification is based on the Globally Harmonized System of Classification and Labelling of Chemicals (GHS), which differentiates severity of eye damage as follows: Serious Eye Damage is the production of tissue damage in the eye which is not fully reversible within 21 days of application, whereas Eye Irritation is the production of changes which are fully reversible within 21 days of application. Compounds causing irreversible eye damage are classified as GHS Category 1 and compounds causing reversible eye damage are classified as GHS Category 2. Compounds which cause no eye damage are Not Classified (NC).

 

Corneas were dissected from freshly obtained cow eyes, and mounted into corneal holders. Following a pre-dose equilibration period, and measurement of baseline opacity (using an opacitometer), the epithelial sides of the corneas were treated with the Substance (ca 75 mg), melted and weighed onto a filter paper, and tested by direct application to the cornea surface with saline (675 µL). The Substance was tested following the BCOP method for liquid and surfactant test items, with physiological saline and ethanol as negative and positive controls, respectively. The volume applied for the control treatments wasca 750µL.

 

Following exposure for 10 min ± 1 min, the test item filter paper discs were removed. The corneas were then rinsed. The corneas then underwent an additional recovery period of 2 h ± 5 min. The opacity of each cornea was then determined, followed by measurement of permeability by assessment of the passage of sodium fluorescein through the cornea. Irritancy was assigned on the basis of in vitro irritancy scores (IVIS). The IVIS of the substance was determined to be 6.07, resulting in an outcome of “No prediction can be made”.

 

In conclusion, the BCOP assay was unable to categorise the substance, giving a result of “No prediction can be made”.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 February 2016 - 25 February 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Batch No: E00974-38D&F
Purity: 100%
Retest Date: 22 June 2018
Physical Description: Pale amber waxy solid
Storage Conditions: Ambient / dark
Species:
human
Strain:
other: derived from normal human keratinocytes
Details on test animals or tissues and environmental conditions:
The MatTek EpiOcular™ corneal model is derived from normal human keratinocytes, cultured to form a stratified, 3D structure similar to that found in the cornea. Each batch of EpiOcular™ tissues is safety tested for HIV, Hepatitis-B, Hepatitis-C and bacteria, yeast and
fungi by the manufacturer. Cells are cultured on 9 mm diameter permeable cell culture inserts.
Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
The weight of the applied test item was 50 mg ± 2 mg (mean ± SD = 51.59 mg ± 0.62 mg).
Duration of treatment / exposure:
6 h ± 15 min
Duration of post- treatment incubation (in vitro):
25 ± 2 min
Number of animals or in vitro replicates:
Triplicate
Details on study design:
The procedure was conducted as described in the MatTek EpiOcular™ EIT Protocol[3]. Test item was applied to the upper surface of the EpiOcular™ tissue, equivalent to the anterior surface of the human cornea. Tissues were exposed to test item for a defined period and then
rinsed in Dulbecco’s phosphate buffered saline, without calcium or magnesium (PBS-). Following post soak and recovery periods, cell viability was then assayed as a measure of ocular irritation potential.

The endpoint of the EpiOcular™ EIT is the estimation of cell viability by MTT assay. In viable cells, mitochondrial dehydrogenase activity reduces MTT (methylthiazolyldiphenyltetrazolium bromide), which is yellow, to a formazan metabolite, which is blue-purple.
Following a 3 h incubation with MTT solution, the formazan was extracted into isopropanol and the optical absorbance 570 nm (A570) was read using a plate reader.
Irritation parameter:
in vitro irritation score
Value:
ca. 83.89
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The Substance did not reduce MTT, therefore, no additional non-viable tissue controls were required.

The colourant status of the Substance in water was determined by visual observation,no additional control tissues were required

See attached background documents

Interpretation of results:
GHS criteria not met
Conclusions:
The EpiOcular™ EIT categorised substance as “No Category” (non-irritant).
Executive summary:

The irritation potential of the Substnace was evaluated using the EpiOcularEye Irritation Test (EIT) provided by MatTek.

 

The procedure was conducted as described in the MatTek EpiOcularEIT Protocol. Test item was applied to the upper surface of the EpiOculartissue, equivalent to the anterior surface of the human cornea. Tissues were exposed to test item for a defined period and then rinsed in Dulbecco’s phosphate buffered saline, without calcium or magnesium (PBS-). Following post soak and recovery periods, cell viability was then assayed as a measure of ocular irritation potential.

 

The endpoint of the EpiOcularEIT is the estimation of cell viability by MTT assay. In viable cells, mitochondrial dehydrogenase activity reduces MTT (methylthiazolyldiphenyl-tetrazolium bromide), which is yellow, to a formazan metabolite, which is blue-purple. Following a 3 h incubation with MTT solution, the formazan was extracted into isopropanol and the optical absorbance 570 nm (A570) was read using a plate reader.

 

In accordance with the OECD Guideline, irritation potential for the test item was assigned based on the mean viability expressed as a percentage of the negative control viability. The test item is considered to be “No Category” if the final viability is >60%. If the final viability is =60%, the test item is Category 1/2.

 

In conclusion, the EpiOcularEIT was run successfully with all acceptance criteria being met. The mean percentage viability of the EpiOculartissues treated with the Substance was 83.89% compared to the negative control. The EpiOcularEIT categorised the Substance as “No Category” (non-irritant).

 

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
7 March 2000 - 28 March 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
GLP compliance:
yes
Specific details on test material used for the study:
Date Received: September 13, 1999
Expiration Date: September 2004
Description: Amber solid
Storage Condition: Room temperature
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
Species: Rabbit
Strain/stock: New Zealand White
Supplier: Covance Research Products Inc., Denver, PA
Age at initiation of dosing: Approximately 16 weeks
Weight at initiation of dosing: 2.67 to 3.04 kilograms
Temperature: 64 to 72 degrees Fahrenheit
Humidity: 30 to 70 percent relative humidity
Lighting: Approximately 12 hours light (0700 to 1900) and 12 hours dark (1900 to 0700) by automatic timer.

Monitored twice per day Monday through Friday and once daily on Saturday and Sunday.
Vehicle:
unchanged (no vehicle)
Controls:
not required
Amount / concentration applied:
0.1 mL
Observation period (in vivo):
Observations for signs of ocular irritation were made using the naked eye at approximately 1, 24, 48, and 72 hours post-instillation and on Days 7, 10, 14, and 21.
Number of animals or in vitro replicates:
3 replicates
Details on study design:
No more than 24 hours prior to test substance application, both eyes of each animal were examined using the naked eye for the presence of dye retention of the cornea using sodium fluorescein dye under UV light. Immediately prior to test substance instillation, the eyes were re-examined without sodium fluorescein. Animals exhibiting corneal or conjunctival injury or irritation were not placed on study.

The test substance was administered using an eyecup as a single 0.1 ml dose into the lower conjunctival sac of the right eye. The upper and lower lids were gently held together for approximately 1 second to prevent loss of the substance. The contralateral eye served as the control. The test substance was administered to one animal and this animal was observed for at least 5 minutes. Since no extreme reaction was displayed, the remaining animals were dosed in the same manner as the first. The treated eyes of the three animals remained unwashed.

The animals were examined for viability twice daily Monday through Friday, and once daily weekends and holidays (if applicable).

Body weights were recorded on the day of dosing.

Observations for signs of ocular irritation were made using the naked eye at approximately 1, 24, 48, and 72 hours post-instillation and on Days 7, 10, 14, and 21. At each interval, the treated and control eyes were examined and scored for ocular reactions according to the Draize Standard Eye Irritation Grading Scale (Draize, 1959), which is presented in Key A. Observations also were made for unusual effects, such as pannus, blistering of the conjunctivae, ulceration, or other effects indicative of a corrosive action.

Sodium fluorescein dye under UV light was used to aid in corneal examination starting with the 24-hour observation and at each subsequent observation until there was no dye retention in the cornea for 2 consecutive observation intervals.
After the Day 21 observations, all rabbits were sacrificed by intravenous injection of sodium pentobarbital solution and discarded without further examination.
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
24/48/72 h
Score:
ca. 0
Reversibility:
fully reversible
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
ca. 0.33
Max. score:
1
Reversibility:
fully reversible
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
ca. 0.67
Max. score:
1
Reversibility:
fully reversible
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
24/48/72 h
Score:
ca. 2.33
Max. score:
3
Reversibility:
fully reversible
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
24/48/72 h
Score:
ca. 1.67
Max. score:
2
Reversibility:
fully reversible
Irritation parameter:
iris score
Basis:
animal #2
Time point:
24/48/72 h
Score:
ca. 1
Max. score:
1
Reversibility:
fully reversible
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
24/48/72 h
Score:
ca. 2.67
Max. score:
3
Reversibility:
fully reversible
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24/48/72 h
Score:
ca. 1.67
Max. score:
2
Reversibility:
fully reversible
Irritation parameter:
cornea opacity score
Basis:
animal #3
Time point:
24/48/72 h
Score:
ca. 1.33
Max. score:
2
Reversibility:
not fully reversible within: 21 Days
Irritation parameter:
iris score
Basis:
animal #3
Time point:
24/48/72 h
Score:
ca. 1
Max. score:
1
Reversibility:
fully reversible
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
24/48/72 h
Score:
ca. 3
Max. score:
3
Reversibility:
not fully reversible within: 21 Days
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
24/48/72 h
Score:
ca. 1.33
Max. score:
2
Reversibility:
not fully reversible within: 21 Days

See attached background documents

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
In conclusion, ocular exposure to 0.1 ml of the test substance caused severe irritation and corrosion, in the conjunctiva, and cornea that did not clear within 21 days and substantial irritation in the iris. The maximum total Draize score was 49 (out of 110).
Executive summary:

The ocular irritation potential of the test substance was evaluated following a single 0.1 ml instillation to the right eye of three New Zealand White rabbits. The treated eyes of all animals remained unwashed. Observations for signs of ocular irritation were made at 1, 24, 48, and 72 hours post-instillation and on Days 7, 10, 14, and 21. All ocular reactions were graded according to the Draize Standard Eye Irritation Grading Scale (Draize, 1959).

 

Ocular instillation of the test substance elicited conjunctival ocular irritation in all three animals with irritation being observed at the 1, 24, 48, and 72-hour observations and on Day 7 in all animals, in two animals on Days 10 and 14, and in one animal on Day 21. At the 1-hour observations, all three animals displayed conjunctival redness, chemosis, and discharge with a maximum Draize conjunctiva score of 14. Conjunctival irritation remained essentially the same at the 24 and 48-hour observations except that necrosis was noted for one animal at the 48-hour observation, for two animals at the 72-hour observation, and in one animal on Days 7, 10, and 14. Conjunctival irritation (redness and chemosis) persisted on Day 21 in one animal.

 

Irritation of the iris was noted for all animals at the 24-hour observation, for two animals at the 48 and 72-hour observations and for one animal on Day 7. All irritation was Grade 1.

Irritation of the cornea consisting of opacity and/or ulceration was noted for all animals at the 24, 48, and 72-hour observation and on Days 10, 14, 21. On Day 7 irritation of the cornea was noted for two animals. Stippling was noted for one animal on Days 14 and 21 and pannus was noted for one animal on Day 21.

 

All animals survived to study termination and were free of clinical signs during the study.

In conclusion, ocular exposure to 0.1 ml of the test substance caused severe irritation and corrosion, in the conjunctiva, and cornea that did not clear within 21 days and substantial irritation in the iris. The maximum total Draize score was 49 (out of 110).

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
10 September 1976
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
abstract
Justification for type of information:
The results indicate a study that has been conducted to a guideline normally used for that time. No methodology was detailed and no explaination regarding the scoring of the observations. However may be suitable as part of a weight of evidence
Qualifier:
no guideline available
Principles of method if other than guideline:
6 animals were treated and observed for a 14 Day period
GLP compliance:
not specified
Specific details on test material used for the study:
Physical Description: Brown Solid
Form Administered: Instillation (100 mg, undiluted)
Special Instruction: Eye unwashed
Species:
rabbit
Strain:
other: Albino Rabbits
Details on test animals or tissues and environmental conditions:
6 animals test and observed over a 14 Day period.
Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
100 mg
Duration of treatment / exposure:
Treated and unwashed over the 14 Day observation period
Observation period (in vivo):
Not Specified
Number of animals or in vitro replicates:
6 animals were used
Details on study design:
6 animals test with 100 mg of undiiluted material. Eye were unwashed and observed over a 14 Day period.
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
24/48/72 h
Score:
20
Max. score:
20
Reversibility:
not fully reversible within: 14 days
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
1.7
Max. score:
5
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
24/48/72 h
Score:
8
Max. score:
10
Reversibility:
fully reversible within: 14 days
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
24/48/72 h
Score:
1.7
Max. score:
5
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
iris score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
0
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
24/48/72 h
Score:
4.7
Max. score:
6
Reversibility:
fully reversible within: 7 days
Irritation parameter:
cornea opacity score
Basis:
animal #3
Time point:
24/48/72 h
Score:
6.7
Max. score:
10
Reversibility:
fully reversible within: 7 Days
Irritation parameter:
iris score
Basis:
animal #3
Time point:
24/48/72 h
Score:
1.7
Max. score:
5
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
24/48/72 h
Score:
7.3
Max. score:
10
Reversibility:
fully reversible within: 7 Days
Irritation parameter:
cornea opacity score
Basis:
animal #4
Time point:
24/48/72 h
Score:
11.7
Max. score:
15
Reversibility:
not fully reversible within: 14 Days
Irritation parameter:
iris score
Basis:
animal #4
Time point:
24/48/72 h
Score:
5
Max. score:
5
Reversibility:
fully reversible within: 14 Days
Irritation parameter:
conjunctivae score
Basis:
animal #4
Time point:
24/48/72 h
Score:
9.3
Max. score:
10
Reversibility:
fully reversible within: 14 Days
Irritation parameter:
cornea opacity score
Basis:
animal #5
Time point:
24/48/72 h
Score:
40
Max. score:
40
Reversibility:
not fully reversible within: 14 Days
Irritation parameter:
iris score
Basis:
animal #5
Time point:
24/48/72 h
Score:
5
Max. score:
5
Reversibility:
not fully reversible within: 14 Days
Irritation parameter:
conjunctivae score
Basis:
animal #5
Time point:
24/48/72 h
Score:
12
Max. score:
14
Reversibility:
not fully reversible within: 14 Days
Irritation parameter:
cornea opacity score
Basis:
animal #6
Time point:
24/48/72 h
Score:
0
Max. score:
0
Irritation parameter:
iris score
Basis:
animal #6
Time point:
24/48/72 h
Score:
0.7
Max. score:
2
Reversibility:
fully reversible within: 48 Hours
Irritation parameter:
conjunctivae score
Basis:
animal #6
Time point:
24/48/72 h
Score:
5.3
Max. score:
8
Reversibility:
fully reversible within: 14 Days
Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
This study concluded that the Substance is severely irritating.
Executive summary:

6 animals test with 100 mg of undiluted material. Eye were unwashed and observed over a 14 Day period.

This study concluded that the Substance is severely irritating.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

In vitro studies were conducted before the known existences of in vivo studies on the Substance were known. The results of the OECD 431 (in vitro corrosion: RHE) and OECD 439 (In vitro skin irritation: RHE) study considered the registration Substance to not classified as corrosive or irritant to the skin. This was supported by the in vivo study which also demonstrated no irritation effects in any animal tested. Based on the data and according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixture, the Substance is not classified for skin irritation.

 

Multiple in vitro and in vivo eye irritation studies were available. In vitro studies were conducted before the known existences of in vivo studies on the Substance were known. Result of the OECD 437 (BCOP) and 492 (RhCE) indicated that the Substance did not require classification. However, the in vivo OECD 405 (Acute Eye Irritation) study conducted with the substance was observed with data that met the GHS criteria for classification. Additional in vivo data was also available which also supported eye irritation effects by the Substance. Based on the decision logic for classification of substance, as detailed in the Guidance on the Application of CLP Criteria, version 4.1 (June 2015), existing data should be considered first. Based on the data the Substance was classified as an eye irritant according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixture. The Substance is classified for serious eye damage category 1 with the hazard statement, H318: Causes serious eye damage.