Registration Dossier

Administrative data

Description of key information

Two in vitro studies are available to assess skin corrosion/irritation. The substance was considered to be non-corrosive to the skin in the EpiDerm™ Human Skin Model. Results from the EPISKIN™ reconstructed human epidermis model showed the substance to be non-irritant.

No effects on the skin were observed in an acute dermal study.

An in vivo skin irritation study does not need to be conducted because adequate data from accepted in vitro skin irritation studies are available.

For effects on the eyes a BCOP test was performed. The findings from this study, supported by histopathology and information on the liberation of hydrochloric acid and extreme low pH resulting from dissolution of the test item in water, supports a conclusion that the test item has the potential to be irritating to eyes. It also causes a red coloring of the eyes.

According to an assessment of the water solubility of the test item (Envigo Study Number FD03TF), no determination of the water solubility of the parent test item was possible or relevant due to rapid hydrolysis in contact with water ) supported by Toney et al (1985).

Analysis of saturated solutions resulted in a mean solution concentration equivalent to 103g/L of solution at 20.0+0.5 ◦C, monitoring the response attributed to soluble hydrolysis products formed on dissolution. Extreme low pH values for the solution were noted, (saturated aqueous solutions have a pH of 0.8), consistent with the liberation of hydrochloric acid during hydrolysis.

Therefore, the substance is considered as corrosive to the eye in a workplace setting where the eyes are rinsed when the substance gets in the eyes and hydrochloric acid is liberated. Further in vivo testing is not justified based on the fact that hydrochloric acid is liberated in contact with moisture.

For hydrochloric acid the following information is available:

Based on BCOP: 5% are eye damage Cat.1: BCOP and SkinEThic: < 1% no classification for eye irritation. The BCOP data however, leaves uncertainty for concentrations between 1-5%. It was decided to go for the most conservative approach, and to classify Cat 1 severe eye damage, H318 (Causes serious eye damage) from concentrations of 1% and higher.

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:
Experimental start date 10 August 2016 Experimental completion date 25 August 2016
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)
Deviations:
yes
Remarks:
Due to concern that the colored test item may interfere with MTT endpoint, a procedure using viable color correction tissues was performed to measure any possible interference. Negligible interference occurred, no affect on integrity/validity of study.
Qualifier:
according to
Guideline:
other: • Method B.40bis of Commission Regulation (EC) No 440/2008, of 30 May 2008, laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on REACH
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Identification: Dichlorobis(η-cyclopentadienyl)titanium (CAS# 1271-19-8)
Physical state/Appearance: Red crystalline powder
Batch Number: 0708501022
Purity (GLC): 99.3%
Expiry Date: 04 March 2019
Storage Conditions: Room temperature in the dark
Test system:
human skin model
Remarks:
EPIDERM Reconstructed Human Epidermis model Kit
Source species:
other: EPIDERM Reconstructed Human Epidermis model Kit
Cell type:
other: Epidermal
Cell source:
other: EPIDERM Reconstructed Human Epidermis model Kit
Source strain:
other: EPIDERM Reconstructed Human Epidermis model Kit
Details on animal used as source of test system:
EpiDerm™ Reconstructed Human Epidermis Model Kit
Supplier : MatTek
Date received : 23 August 2016
EpiDermTM Tissues (0.63cm2) lot number : 23351
Assay Medium lot number : 081816TMA
Upon receipt of the EpidermTM tissues, the sealed 24 well plate was stored in a refrigerator until use.
Justification for test system used:
This model incorporates several features, which make it advantageous in the study of potential dermal corrosivity. The target cells are epithelial, derived from human skin, and formed into a stratified, cornified epithelium. Test items are applied to the culture surface, at the air interface, so that undiluted and/or end use dilutions can be tested directly.
Vehicle:
unchanged (no vehicle)
Details on test system:
25 mg of the test item was added to 1 mL of a freshly prepared 1.0 mg/mL MTT solution. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 60 minutes. Untreated MTT solution was tested concurrently to act as a control.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
25 mg of the test item was added to 1 mL of a freshly prepared 1.0 mg/mL MTT solution.
Duration of treatment / exposure:
3 minutes and 60 minutes. The plate was returned to the incubator (37 °C, 5% CO2) for the 60 Minute exposure period
Duration of post-treatment incubation (if applicable):
Immediate observation following exposure
Number of replicates:
two x 24-well plates
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minute exposure
Value:
103.3
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

Direct MTT Reduction

The MTT solution containing the test item did not turn blue/purple. This was taken to indicate the test item did not reduce MTT.

Assessment of Color Interference with the MTT endpoint

The solution containing the test item was a red color. This color was considered to have the potential to cause color interference and therefore additional tissues were incorporated into the test to correct for this interference. However, the results obtained showed a negligible amount of color interference occurred and it was therefore considered unnecessary to use the results of the color correction tissues for reporting purposes or for quantitative correction of the results.

Test Item, Positive Control Item and Negative Control Item

Mean OD562values and viabilities for the negative control, positive control and test item are given in theappendix.

The relative mean viabilities for each treatment group were as follows:

Exposure Period

Percentage Viability

Negative Control

Positive Control

Test Item

3 minute

100*

5.3

103.3

60 minute

100*

5.2

92.0

*The mean viability of the negative control tissues is set at 100%

Quality Criteria

The mean OD562for the negative control treated tissues was 2.470 for the 3‑Minute exposure period and 2.463 for the 60‑Minute exposure period. The negative control acceptance criteria were therefore satisfied.

The relative mean tissue viability for the positive control treated tissues was 5.2% relative to the negative control following the 60‑Minute exposure period. The positive control acceptance criterion was therefore satisfied.

In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.

Interpretation of results:
GHS criteria not met
Conclusions:
The test item was considered to be non-corrosive to the skin.
Executive summary:

 Summary

Introduction

The purpose of this test is to evaluate the corrosivity potential of the test item using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes.

Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test item.

Methods

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. The test item was found to have the potential to cause color interference with the MTT end point and therefore additional tissues were incorporated into the testing for correction purposes. Negative and positive control groups were treated for each exposure period. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT‑loading. After MTT loading each tissue was placed in 2 mL Isopropanol for MTT extraction.

At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 mL samples were transferred to the appropriate wells of a pre-labeled 96‑well plate. The optical density (OD) was measured at 562 nm (OD562).

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

Results

The relative mean viabilities for each treatment group were as follows:

Exposure Period

Percentage Viability

Negative Control

Positive Control

Test Item

3 minute

100*

5.3

103.3

60 minute

100*

5.2

92.0

*The mean viability of the negative control tissues is set at 100%

Quality criteria: The quality criteria required for acceptance of results in the test were satisfied.

 

Conclusion

The test item was considered to be non-corrosive to the skin.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date 02 November 2016 Experimental completion date 07 November 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
yes
Remarks:
Due to concern that the colored test item may interfere with MTT endpoint, a procedure using viable color correction tissues was performed to measure any possible interference. Negligible interference occurred, no affect on integrity/validity of study.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Identification: Dichlorobis(η-cyclopentadienyl)titanium
(CAS# 1271-19-8)
Batch Number: 0708501022
Purity (GLC): 99.8%
Physical state / Appearance - Sponsor: Red crystalline powder
Physical state / Appearance - Envigo: Red powder
Expiry Date: 04 March 2019
Storage Conditions: Room temperature, in the dark
Test system:
human skin model
Source species:
other: reconstructed human epidermis model
Cell type:
other: Epidermal
Cell source:
other: reconstructed human epidermis model
Source strain:
other: reconstructed human epidermis model
Details on animal used as source of test system:
EPISKIN™ Reconstructed Human Epidermis Model Kit
Supplier : SkinEthic Laboratories, Lyon, France
Date received : 02 November 2016
EpiSkinTM Tissues (0.38cm2) lot number : 16-EKIN-044
Maintenance Medium lot number : 16-MAIN3-074
Assay Medium lot number : 16-ESSC-047
Justification for test system used:
Following a full validation study the EpiSkinTM reconstructed human epidermis model showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation when the endpoint is measured by MTT reduction and for being used as a replacement for the Draize Skin Irritation Test for the purpose of distinguishing between Irritating and Non-Irritating test items.
Vehicle:
unchanged (no vehicle)
Details on test system:
10 mg of the test item was added to 2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37 C, 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
10 mg of the test item was added to 2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium.
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours post exposure
Number of replicates:
12 well plates
Irritation / corrosion parameter:
% tissue viability
Value:
68.2
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The relative mean viability of the test item treated tissues was 68.2% after a 15 Minute exposure period and 42 Hour post exposure incubation period.
It was considered unnecessary to perform IL-1 analysis as the results of the MTT test were unequivocal.

The relative mean tissue viability for the positive control treated tissues was 9.8% relative to the negative control treated tissues and the standard deviation value of the viability was 3.5%. The positive control acceptance criteria were therefore satisfied.

The mean OD562for the negative control treated tissues was 0.774 and the standard deviation value of the viability was 0.056%. The negative control acceptance criteria were therefore satisfied.

The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 15.5%. The test item acceptance criterion was therefore satisfied.

Interpretation of results:
GHS criteria not met
Conclusions:
The test item was classified as non-irritant. The following classification criteria apply:
EU DSD & CLP Not classified for Irritation.
UN GHS Not classified for Irritation (category 3 can not be determined).
Executive summary:

Introduction

The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKINTMreconstructed human epidermis model after a treatment period of 15 minutes followed by a post‑exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric 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 tissues relative to the negative controls. 

Method

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. The test item was shown to produce a colored solution therefore additional color correction tissues were included in parallel to the main test. At the end of the post exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre labeled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT loaded tissues. 

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a pre‑labeled 96‑well plate. The optical density was measured at 562 nm.

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

Results

The relative mean viability of the test item treated tissues was 68.2% after the 15‑Minute exposure period and 42‑Hours post‑exposure incubation period.

Quality criteria: The quality criteria required for acceptance of results in the test were satisfied.

Conclusion

The test item was classified as non-irritant. The following classification criteria apply:

EU DSD and CLP Not classified for Irritation.

UN GHS Not classified for Irritation (category 3 can not be determined).

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:
Experimental start date: 15 November 2016. Experimental completion date 15 November 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)
Deviations:
yes
Remarks:
please see any other information on materials and methods section
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
yes
Remarks:
please see any other information on materials and methods section
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Identification: Dichlorobis(η-cyclopentadienyl)titanium (CAS#1271-19-8)
Batch: 0708501022
Purity: 99.3%
Physical state/Appearance: red crystalline powder
Expiry Date: 04 March 2019
Storage Conditions: room temperature in the dark
Species:
other: Eyes from adult cattle
Strain:
other: not applicable
Details on test animals or tissues and environmental conditions:
Source of Bovine Eyes
Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.
Vehicle:
other: sodium chloride solution 0.9% w/v.
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
0.75 mL of the test item preparation or control items were applied.
For the purpose of this study the test item was prepared as a 20% w/v solution in sodium chloride 0.9% w/v.
The negative control item, sodium chloride 0.9% w/v, was used as supplied.
The positive control item, Imidazole, was used as a 20% w/v solution in sodium chloride 0.9% w/v.
Duration of treatment / exposure:
240 minutes
Number of animals or in vitro replicates:
3
Details on study design:
Preparation of Corneas
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 65 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

Selection of Corneas and Opacity Reading
The medium from both chambers of each holder was replaced with fresh complete EMEM.
A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated.
Three corneas with opacity values close to the median value of all corneas were allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.

Treatment of Corneas
The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item preparation or control items were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 240 minutes. At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed three times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A posttreatment opacity reading was taken and each cornea was visually observed.

Application of Sodium Fluorescein
Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.

Permeability Determinations
After incubation the medium in the posterior chamber of each holder was decanted and retained.
360 µL of medium representing each cornea was applied to a designated well on a 96-well plate and the optical density at 492 nm (OD492) was measured using the Anthos 2001 microplate reader.

Histopathology
The corneas were retained after testing for histopathology. Each cornea was placed into a pre-labeled tissue cassette fitted with a histology sponge to protect the endothelial surface.
The cassette was immersed in 10% neutral buffered formalin.
Following fixation in neutral buffered formalin, the cassettes containing the corneas were dispatched to the Test Site (histology processing), sectioned and stained. Prepared slides were sent to the Test Site study pathologist, for histopathological examination.
Samples (two samples of each cornea) were processed to paraffin wax block, sectioned at approximately 4-5µm and stained with Haematoxylin and Eosin (H&E). Semi-quantitative microscopic examination of the H&E-stained corneas were evaluated according to the BCOP histopathological grading system to this document and the criteria described below.
Paperwork, blocks and slides were labelled with Envigo (Shardlow) study number and corneal reference/number.
Grade Description of histopathological findings
0 Absence of any significant epithelial changes.
1 Superficial cellular alterations characterized by sloughing of cells with epithelial loss and focal erosion.
2 Vacuolation of cells and nuclei in upper layers.
3 Vacuolation and disruption of basal cells leading to detachment of upperlayers.
4 Cell coagulation/necrosis.
5 Complete necrosis of all cell layers and/or disintegration.
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean
Value:
61.6
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: >55
Remarks:
Category 1. UN GHS or EU CLP Causes serious eye damage
Other effects / acceptance of results:
Corneal Epithelium Condition
The corneas treated with the test item were slightly cloudy and stained orange post treatment. The corneas treated with the negative control item were clear post treatment. The corneas treated with the positive control item were cloudy post treatment.

Criteria for an Acceptable Test
The positive control In Vitro Irritancy Score was above the range of 50.8 to 100.4. The positive control acceptance criterion was therefore not satisfied. This is reported as a deviation.
The negative control gave opacity of ≤5.4 and permeability ≤0.070. The negative control acceptance criteria were therefore satisfied.

DISCUSSION
All test item treated eyes were stained orange which may have influenced the opacity values observed and increased the overall IVIS score. At least two of the test item treated corneas were consistent with the negative control scores for permeability whilst the other was marginally higher but considerably less than the positive control scores for permeability.
Histopathological evaluation of the changes observed in corneas of eyes administered the test item showed test item related findings in all the eyes presented. Changes consistent with a grading of 4 were apparent in all positive controls, and no changes (grade 0) were seen in the negative controls.

Individual and Mean Corneal Opacity and Permeability Measurements

Treatment

Cornea Number

Opacity

Permeability (OD)

In Vitro Irritancy Score

Pre-Treatment

Post-Treatment

Post-Treatment-Pre‑Treatment

Corrected Value

 

Corrected Value

Negative Control

4

3

4

1

 

0.055

 

 

6

4

6

2

 

0.047

 

 

7

4

5

1

 

0.063

 

 

 

 

 

1.3*

 

0.055¨

 

2.2

Positive
Control

1

5

87

82

80.7

1.993

1.938

 

2

3

87

84

82.7

1.265

1.210

 

3

3

93

90

88.7

2.026

1.971

 

 

 

 

 

84.0·

 

1.706·

109.6

Test Item

5

5

75

70

68.7

0.118

0.063

 

8

2

62

60

58.7

0.040

0.000

 

9

5

63

58

56.7

0.055

0.000

 

 

 

 

 

61.3·

 

0.021·

61.6


OD= Optical density           * = Mean of the post-treatment -pre‑treatment values             ¨= Mean permeability                     ·= Mean corrected value                               

Histopathology assessment of corneas - tabulation of test scores for the test item.

Test item/Reference Eye/Holder Number Grade Observations (if any in addition to grade)
Negative Contol
(0.9% soduium chloride)
1 0 Absence of any significant epithelial changes
2 0 Absence of any significant epithelial changes
3 0 Absence of any significant epithelial changes
Positive Control
(20% (w/v) imidazole)
4 4 Cell coagulation/necrosis
5 4 Cell coagulation/necrosis
6 4 Cell coagulation/necrosis
dichlorobis(n-cyclopentadienyl)
titanium (CAS#1271-19-8)
7 2 Vacuolation of cells and nuclei in upper layers
8 2 Vacuolation of cells and nuclei in upper layers
9 2 Vacuolation of cells and nuclei in upper layers
Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
Each cornea treated with a 20% w/v suspension of the test item in sodium chloride 0.9% w/v, appeared orange-colored at the end of the 240-minute exposure period. It is possible that this coloration was due to uptake of the red-colored test item by the cornea and that this may have contributed to the opacity values obtained for the test item-treated corneas, resulting in an overestimation of corneal opacity. However, from the available data it is not possible to conclude on the degree to which the coloration might have influenced the values for corneal opacity. For two of the test item-treated corneas, the values obtained for corneal permeability were consistent with those for the negative control corneas. The permeability value for the other test item-treated cornea was only slightly higher than the values for the negative control corneas, but considerably less than the values obtained for the corneas treated with the positive control item Imidazole, 20% w/v solution in sodium chloride 0.9% w/v.
The IVIS obtained for the test item was 61.6 and an IVIS of >55 requires that the test item is classified as Category 1 according to UN GHS or EU CLP. However, as there was doubt about the reliability of the assessment of corneal opacity, due to the orange-colored staining of the test item-treated corneas, and the low permeability values for the test item-treated corneas, a decision was taken to conduct histopathological assessment of the three corneas treated with test item, as well as those used as negative and positive control corneas.
Histopathological changes were observed in each cornea that had been treated with the test item although these were confined to vacuolation of cells and nuclei in the upper layers (Grade 2). No histopathological changes were noted in the negative control corneas and changes consistent with a grading of 4 were observed in all positive control corneas.
According to an assessment of the water solubility of the test item (Envigo Study Number: FD03TF), no determination of the water solubility of the parent test item was possible or relevant due to rapid hydrolysis in contact with water (supported by Toney et al (1985)). Analysis of saturated solutions resulted in a mean solution concentration equivalent to 103g/L of solution at 20.0 + 0.5 ◦C, monitoring the response attributed to soluble hydrolysis products formed on dissolution. Extreme low pH values for the solution were noted, consistent with the liberation of hydrochloric acid during hydrolysis.
The findings of the BCOP test, supported by histopathology and information on the liberation of hydrochloric acid and extreme low pH resulting from dissolution of the test item in water, supports a conclusion that the test item has the potential to be irritating to eyes. It may be corrosive to the eye in a workplace setting where the eyes are rinsed when the substance gets in the eyes and hydrochloric acid is liberated (information from sponsor).
Executive summary:

Introduction

The purpose of this test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability. The test method can correctly identify test items (both chemicals and mixtures) inducing serious eye damage as well as those not requiring classification for eye irritation or serious eye damage, as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Items (GHS) and EU Classification, Labelling and Packaging (CLP) of chemicals (Regulation (EC) No 1272/2008), and it was therefore endorsed as scientifically valid for both purposes. Test items inducing serious eye damage are classified as UN GHS and EU CLP Category 1. Items not classified for eye irritation or serious eye damage are defined as those that do not meet the requirements for classification as UN GHS/EU CLP Category 1 or 2 (2A or 2B), i.e. they are referred to as UN GHS/EU CLP No Category.

Method

The test item was applied at a concentration of 20% w/v in sodium chloride 0.9% w/v for 240 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

Data Interpretation

The test item is classified according to the prediction model as follows:

IVIS

Classification

≤ 3

No category. Not requiring classification to UN GHS or EU CLP

> 3; ≤55

No prediction of eye irritation can be made

> 55

Category 1. UN GHS or EU CLP Causes serious eye damage

Results

The In Vitro irritancy scores are summarized as follows:

Treatment

In Vitro Irritancy Score

Test Item

61.6

Negative Control

2.2

Positive Control

109.6

Discussion

All test item treated eyes were stained orange which may have influenced the opacity values observed and increased the overall IVIS score. At least two of the test item treated corneas were consistent with the negative control scores for permeability whilst the other was marginally higher but considerably less than the positive control scores for permeability.

Histopathological evaluation of the changes observed in corneas of eyes administered the test item showed test item related findings in all the eyes presented. Changes consistent with a grading of 4 were apparent in all positive controls, and no changes (grade 0) were seen in the negative controls.

Conclusion

Each cornea treated with a 20% w/v suspension of the test item in sodium chloride 0.9% w/v, appeared orange-colored at the end of the 240-minute exposure period. It is possible that this coloration was due to uptake of the red-colored test item by the cornea and that this may have contributed to the opacity values obtained for the test item-treated corneas, resulting in an overestimation of corneal opacity. However, from the available data it is not possible to conclude on the degree to which the coloration might have influenced the values for corneal opacity. For two of the test item-treated corneas, the values obtained for corneal permeability were consistent with those for the negative control corneas. The permeability value for the other test item-treated cornea was only slightly higher than the values for the negative control corneas, but considerably less than the values obtained for the corneas treated with the positive control item Imidazole, 20% w/v solution in sodium chloride 0.9% w/v.

The IVIS obtained for the test item was 61.6 and an IVIS of >55 requires that the test item is classified as Category 1 according to UN GHS or EU CLP. However, as there was doubt about the reliability of the assessment of corneal opacity, due to the orange-colored staining of the test item-treated corneas, and the low permeability values for the test item-treated corneas, a decision was taken to conduct histopathological assessment of the three corneas treated with test item, as well as those used as negative and positive control corneas.

Histopathological changes were observed in each cornea that had been treated with the test item although these were confined to vacuolation of cells and nuclei in the upper layers (Grade 2). No histopathological changes were noted in the negative control corneas and changes consistent with a grading of 4 were observed in all positive control corneas.

According to an assessment of the water solubility of the test item (Envigo Study Number: FD03TF), no determination of the water solubility of the parent test item was possible or relevant due to rapid hydrolysis in contact with water. Analysis of saturated solutions resulted in a mean solution concentration equivalent to 103g/L of solution at 20.0 + 0.5 ◦C, monitoring the response attributed to soluble hydrolysis products formed on dissolution. Extreme low pH values for the solution were noted, consistent with the liberation of hydrochloric acid during hydrolysis as which is supported by Toney et al (1985).

The findings of the BCOP test, supported by histopathology and information on the liberation of hydrochloric acid and extreme low pH resulting from dissolution of the test item in water, supports a conclusion that the test item has the potential to be irritating to eyes. It may be corrosive to the eye in a workplace setting where the eyes are rinsed when the substance gets in the eyes and hydrochloric acid is liberated (information from sponsor).

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

References:

Toney et al., (1985) Hydrolysis chemistry of the metallocene dichlorides M(ƞ5-C5H5)2Cl2, M = titanium, vanadium, or zirconium. Aqueous kinetics, equilibria, and mechanistic implications for a new class of antitumor agents.  J. Am. Chem. Soc., 1985, 107 (4), pp 947–953.

 

Justification for classification or non-classification

Based on the available in vitro studies the substance is not classified for effects on the skin. The substance is classified as corrosive to the eyes, Eye Damage 1, based on the fact that hydrochloric acid is liberated in contact with moisture.