Reaction mass of disodium hexatitanium tridecaoxide and disodium trititanium heptaoxide

Endpoint summary

Description of key information

Disodium titanate has been tested in one in vitro skin irritation and one in vitro skin corrosion study as well as in one in vitro eye irritation study. All tests show a negative response, thus disodium titanate does not require classification either as skin or as eye irritant.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-03-29 to 2010-03-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP guideline study reliable without restrictions

Justification for type of information:

Disodium titanate substance (EC 234-802-9) has the molecular formula Na2TiO3 and its composition is expressed as (Na2O)x(TiO2), where x is ranging from 0.1 to 6 according to the SIP. This substance, Reaction mass of Disodium Hexatitanate and Sodium Metatitanate, has a value of x = 0.21, calculated from XRF results, has been identified as a mixture of two specific types of disodium titanate and is therefore within the scope of the disodium titanate SIP.
It is assessed therefore that disodium titanate is an acceptable read-across substance for Reaction mass of Disodium Hexatitanate and Sodium Metatitanate

Qualifier:

according to guideline

Guideline:

OECD Guideline 435 (In Vitro Membrane Barrier Test Method for Skin Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICCVAM Minimum Performance Standards: In Vitro Membrane Barrier Test Systems for Skin Corrosion, June 23, 2003

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICCVAM Recommended Performance Standards for in vitro Test Methods for Skin Corrosion, May 2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2009-03-30

Details on test animals or test system and environmental conditions:

Not applicable - Since this is a in vitro study there is no information on test animals.

Amount / concentration applied:

TEST MATERIAL
Qualify Test: approx. 100 mg of the test item were applied into the “Qualify Test Vial”
Categorisation Test: approx. 100 mg of the test item were applied into the “Category A Vial” as well as into the “Category B Vial”
Classification Test: approx. 500 mg of the test item were applied per bio-barrier

Duration of treatment / exposure:

Qualify Test: 1 minute incubation
Categorisation Test: 1 minute incubation
Classification Test: >60 minutes

Observation period:

not applicable

Number of animals:

not applicable

Details on study design:

CHEMICAL DETECTION SYSTEM (CDS)
Corrosive substances are able to disrupt the integrity of the bio-barrier, leading to penetration of the test item through the bio-barrier into the CDS located beneath. The presence of the test item in the CDS results in a colour change that is detected visually. The time it takes a test item to penetrate the bio-barrier into the CDS is inversely proportional to its corrosivity: the more corrosive the test item is, the shorter time required affects a colour change. Non-corrosive test items do not disrupt the bio-barrier, or disrupt the bio-barrier too slowly to be identified as corrosive.

PREPARATION OF THE BIO-BARRIER
Preparation of the synthetic macromolecular bio-barrier matrix (Corrositex TM test kit, Lot no. CT101408, Transia GmbH, 61239 Ober-Mörlen) was completed one day prior to testing and stored at 4 - 8 °C until assay performance. The bio-barrier powder was solved in the bio-barrier diluent and heated for 20  2 minutes at 68 – 70 °C in a water bath under continuous stirring. The temperature did not exceed 70 °C. The mixture was allowed to cool in the turned-off water bath for another 10 minutes. The mixture was then filled into the membrane holders, 200 µL per membrane holder. Air bubbles were avoided. The filled membrane holders were sealed with parafilm and were stored at 4 – 8 °C until further use.

QUALIFY TEST
In order to assess whether the test system is suitable for the test item, approx. 100 mg of test item were applied into the “Qualify Test Vial”. The vial was shaken until the solution appeared homogenous, and incubated for at least 1 minute. Afterwards, the colour change was noted. Since a change in colour was visible in the “Qualify Test Vial”, the test item was considered to be suitable for the next step.

CATEGORISATION TEST
Approx. 100 mg of test item were applied into the “Category A Vial” as well as into the “Category B Vial”. The vials were shaken until the solution appeared homogenous. After at least one minute the colour change was monitored. Based on the colour change obtained, a test item is assigned to a category. If an intense colour change (similar to the category 1 colour chart) is observed in “Category A Vial” or in “Category B Vial” the test item is assigned to category 1. If a less intense colour change is observed (similar to the category 2 colour chart) is observed in “Category A Vial” or in “Category B Vial” the test item will be assigned to category 2. If no colour change is observed in either of the vials, a confirmation test is conducted. For the confirmation test two drops of the confirm reagent are added to the “Category B Vial”. The vial would be shaken for 5 seconds. The colour of the solution would match one of the colours shown in the accompanying colour chart, confirming that the test item is a category 2 substance.

CLASSIFICATION TEST
7 vials containing the CDS were pre-warmed to room temperature. 4 vials were used for quadruplicate measurement of the test item, the vial labelled (+) was used for the positive control (single measurement, Sulfuric acid 95-98%), and the vial labelled (-) was used for the negative control (single measurement, Citric acid (10% solution in deionised water)). The vial labelled C was used as colour reference for the CDS.
Application of the test item formulations and the controls was performed staggered to ensure accurate reaction times to be recorded.
The prepared bio-barriers were placed atop the CDS vials (not longer than 2 min prior to application) and approx. 500 mg of the test item or 500 µL of the controls, respectively, were applied per bio-barrier. Colour change or precipitation in the CDS solution were recorded in the raw data file.

Irritation / corrosion parameter:

penetration time (in minutes)

Remarks:

Change in colour in the Chemical Detection System (CDS) solution

Run / experiment:

See details on "Details on Study Design" section

Value:

> 60

Vehicle controls validity:

valid

Remarks:

Qualify test: approx. 100 mg of test item applied into the "Qualify Test Vial". Vial shaken until solution appeared homogenous and incubated for at least 1 minute. Afterwards, the colour change was noted.

Negative controls validity:

valid

Remarks:

Classification test: vial labelled as (-). Single measurement, citric acid (10% solution in deionised water). Colour change in CDS solution was not observed after 60 minutes.

Positive controls validity:

valid

Remarks:

Classification test: vial labelled as (+). Single measurement, sulfuric acid -%. Colour change in CDS solution observed after 1.15 minutes.

Remarks on result:

other: Non corrosive to skin

Remarks:

No change in colour of CDS solution after > 60 minutes.

Other effects / acceptance of results:

The test item was classified as non corrosive.

Qualify Test

The test item induced a change in colour in the qualify test after 1 minute incubation. Since a change in colour was visible in the “Qualify Test Vial”, the test item was considered to be suitable for the categorisation test.

 

Categorisation Test

The test item induced a change in colour in the Category A vial but not in the Category B vial after 1 minute incubation. Therefore, a confirmation experiment did not have to be performed. Therefore, the test item was classified as category II.

Classification Test

 

Test Group	Time to colour change (minutes)	DOT Packing Group	R-Sentence	GHS
Negative Control	Colour change was not observed after 60	-	-	-
Positive Control	1.15	I	R35	1A
Test item	> 60	Non corrosive	-	-

 

Interpretation of results:

other: non corrosive

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item Disodium titanate is non corrosive to skin.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Remarks:

validated "in vitro" test method

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-03-30 to 2010-04-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP guideline study reliable without restrictions

Justification for type of information:

Disodium titanate substance (EC 234-802-9) has the molecular formula Na2TiO3 and its composition is expressed as (Na2O)x(TiO2), where x is ranging from 0.1 to 6 according to the SIP. This substance, Reaction mass of Disodium Hexatitanate and Sodium Metatitanate, has a value of x = 0.21, calculated from XRF results, has been identified as a mixture of two specific types of disodium titanate and is therefore within the scope of the disodium titanate SIP.
It is assessed therefore that disodium titanate is an acceptable read-across substance for Reaction mass of Disodium Hexatitanate and Sodium Metatitanate

Qualifier:

according to guideline

Guideline:

other: Commision regulation (EC) No. 440/2008 B.46

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ECVAM international validation study on in vitro tests for acute skin irritation (Altern Lab Anim. 2007 Dec; 35 (6): 559-601)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for the testing of chemicals, Draft proposal for a new guideline, in vitro skin irritation: Reconstructed Human Epidermis (RhE) Test method, 11 December 2009, Vers. 4

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2009-03-30

Details on test animals or test system and environmental conditions:

Not applicable - Since this is a in vitro study there is no information on test animals.

Vehicle:

other: deionised water

Amount / concentration applied:

TEST MATERIAL
15 mg of the test item were applied to each of triplicate tissues and wetted with 25 µL deionised water and spread to match the tissue size.
No further information on the amount/concentration applied was stated.

Duration of treatment / exposure:

15 +/- 1 min

Observation period:

not applicable

Number of animals:

not applicable

Details on study design:

CELL CULTURE:
EpiSkin TM kits (Lot No.: 10-EKIN-011) are purchased from Skinethic Laboratories (06000 Nice, France). The EpiSkin TM tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiSkin TM tissues (surface 0.38 cm^2) are cultured on specially prepared cell culture inserts.

TREATMENT:
The negative control (deionised water (Lot no. 230310); 15 µL were applied to each of triplicate tissues) and positive control (5% Sodium lauryl sulphate (Lot no. 1353471 51508322; Sigma, 82024 D-Taufkirchen) solution in deionised water; 15 µL were applied to each of triplicate tissues), and the test item were added into the insert atop the concerning EpiSkin TM triplicate tissues. The plates were placed into the incubator for 15+/- 1 min at 37 +/- 1.5 °C, 5 +/- 0.5 % CO2.
After the end of the treatment interval the inserts were removed immediately from the plate. Using a wash bottle the tissues were gently rinsed with PBS to remove any residual test material. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper. The inserts were placed in the plates with 2 mL maintenance medium. The tissues were incubated for 42 +/- 1 hour at 37 +/- 1.5 °C, 5 +/- 0.5 % CO2.

CELL VIABILITY TEST:
Cell viability is measured by dehydrogenase conversion of MTT [(3-4, 5-dimethyl thiazole 2-yl) 2,5-diphenyl-tetrazoliumbromide], present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison of untreated negative controls is used to predict skin irritation potential.
After the treatment procedure was completed for all tissues of each time point cell culture inserts were transferred from the holding plates to plates containing 2 mL assay medium containing 0.3 mg/mL MTT per well. After a nearly 3 hour incubation period (37 +/- 1.5 °C, 5 +/- 0.5 % CO2) MTT solution was aspirated from the wells and wells were rinsed three times with PBS. Tissues samples were cut out of the inserts with a biopsy punch and transferred into plastic vials. The tissue samples were immersed into extractant solution by gently pipetting 0.5 mL extractant solution (isopropanol) into each vial. The tissue samples were completely covered by isopropanol. The vials were sealed to inhibit isopropanol evaporation. The formazan salt was extracted for 2 hours 45 minutes while shaking (~120 rpm) at room temperature.
Per each tissue sample 2 * 200 µL aliquots of the formazan blue solution were transferred into a 96-well flat bottom microtiter plate. OD was read in a microplate reader (Versamax® Molecular Devices, D-85737 Ismaning) at 570 nm without reference filter. Mean values were calculated from the 2 wells per tissue sample.

EVALUATION OF RESULTS:
The mean OD of the three negative control tissues was calculated. This value corresponds to 100% tissue viability in the current test. For each individual tissue treated with the test item or the positive control the individual relative tissue viability is calculated according to the following formula:
Relative viability (%) = [OD test item / OD negative control] * 100
For the test item and the positive control the mean relative viability +/- standard deviation of the three individual tissues are calculated and used for classification according to the following prediction model:
For the current test, an irritation potential of a test item according to EU classification R38 (acc. to Directive 67/548/EEC) and Category 2 (acc. to Regulation 1272/2008/EC) is predicted if the mean relative tissue viability of three individual tissues is ≤ 50% of the negative control.

ACCEPTABILITY OF THE ASSAY:
The absolute OD 570 nm of the negative control tissues in the MTT test is an indicator of tissue viability obtained after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is meeting the acceptance criterion if the mean OD of the three tissues is ≥ 0.6 till ≤ 1.5.
The standard deviations in between tissues of the same treatment group should be ≤ 18%.
An assay is meeting the acceptance criterion if mean relative tissue viability of the positive control is ≤ 40%.

TEST FOR DIRECT MTT REDUCTION:
For correct interpretation of results it was necessary to assess the ability of the test item to directly reduce MTT. To test for this ability approximately 15 mg of the test item were added to 1 mL of MTT solution and the mixture was incubated in the dark at room temperature for 60 minutes. Untreated MTT medium was used as control. If the MTT solution colour turned blue/purple, the test item was presumed to have reduced the MTT.
A colour change could not be observed.
No further information on the study design was stated.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

See details on "Details on Study Design" section

Value:

ca. 93.8

Vehicle controls validity:

valid

Remarks:

Vehicle controls: 15mg of the test item were applied to each of triplicate tissue and wetted with 25 μL deionised water and spread to match the tissue sized. No further information on the amount/concentration applied was stated.

Negative controls validity:

valid

Remarks:

Negative controls: Deionised water (Lot no. 230310); 15μL applied to each of triplicate tissues

Positive controls validity:

valid

Remarks:

Positive controls: 5% sodium lauryl sulphate (Lot no. 1353471 51508322, Sigma, 82024 D-Taufkirchen) solution in deionised water; 15μL applied to each of triplicate tissues.

Remarks on result:

no indication of irritation

Remarks:

An irritation potential of a test item according to EU classification (Regulation 1272/2008/EC0 is predicted if the mean relative tissue viability of three individual tissues is <= 50% of the negative control.

Other effects / acceptance of results:

After treatment with the test item Disodium titanate the relative absorbance values were decreased to 93.8% (of the negative control). This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

Results after treatment with Disodium titanate:

Dose group	Treatment Interval	Absorbance 570 nm Tissue 1*	Absorbance 570 nm Tissue 2*	Absorbance 570 nm Tissue 3*	Mean Absorbance of 3 Tissues	Standard Deviation [%]	Rel. Absorbance[%] of Negative Control]**
Negative Control	15 min	1.109	0.993	1.034	1.045	5.6	100.0
Positive Control	15 min	0.326	0.212	0.322	0.287	6.2	27.4
Test Item	15 min	0.951	1.020	0.970	0.980	3.4	93.8

* Mean of the three replicate wells after blank correction

** Relative absorbance [rounded values]: 100*(absorbance test item) / (absorbance negative control)

Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.

Historical data:

Positive Control		Negative Control
Number of Studies	73	Number of Studies	73
Period	July 2007 – March 2010	Period	July 2007 – March 2010
Mean Viability	16.5%	Mean Viability	1.081
Standard Deviation	11.0%	Standard Deviation	0.262
Range of Viabilities	3% - 36%	Range of ODs	0.7 – 1.6*

 

* The upper OD value is outside of the range of 0.6 – 1.5 recommended by the OECD Guideline. Nevertheless since the OD value is only slightly above the required range, the historical Data can still be considered as valid.

Interpretation of results:

not irritating

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

After treatment with the test item Disodium titanate the relative absorbance values were decreased to 93.8%. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an iritant potential.
The test item should not be classified and labelled as skin irritant according to regulation (EC) No.: 1272/2008.

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:

2010-03-30 to 2010-03-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP guideline study reliable without restrictions

Justification for type of information:

Disodium titanate substance (EC 234-802-9) has the molecular formula Na2TiO3 and its composition is expressed as (Na2O)x(TiO2), where x is ranging from 0.1 to 6 according to the SIP. This substance, Reaction mass of Disodium Hexatitanate and Sodium Metatitanate, has a value of x = 0.21, calculated from XRF results, has been identified as a mixture of two specific types of disodium titanate and is therefore within the scope of the disodium titanate SIP.
It is assessed therefore that disodium titanate is an acceptable read-across substance for Reaction mass of Disodium Hexatitanate and Sodium Metatitanate

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for Testing of Chemicals 437: Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants, September, 2009

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: INVITTOX (UK) protocol no. 98 “The Bovine Corneal Opacity and Permeability Assay”, February 1994

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2009-03-30

Details on test animals or tissues and environmental conditions:

Not applicable - Since this is a in vitro study there is no information on test animals.

Vehicle:

physiological saline

Remarks:

0.9% (w/v) NaCl in deionised water

Amount / concentration applied:

Prior to the application of 0.75 mL the test item was suspended in saline (20% (w/v))

Duration of treatment / exposure:

Opacity measurement: incubation time: 240 min (+/- 5min)
Permeability of the corneae: 90 min

Observation period (in vivo):

not applicable

Number of animals or in vitro replicates:

not applicable

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): After the test item was rinsed off from the application side by changing cMEM three times.
- Time after start of exposure: 240 min (+/- 5 min)

SCORING SYSTEM:
The following formula was used to determine the in vitro score of the negative control:

In vitro Score = opacity value + (15 x OD490 value)

The following formula was used to determine the in vitro score of the positive control and the test item:

In vitro Score = (opacity value – opacity value mean negative control) + (15 x corrected OD490 value)

The in vitro score was calculated for each individual treatment and positive control cornea. The mean in vitro score value of each treated group was calculated from the individual in vitro score values.

TOOL USED TO ASSESS SCORE: fluorescein

Irritation parameter:

in vitro irritation score

Run / experiment:

See details on "Details on Study Design" section

Value:

ca. -0.26

Vehicle controls validity:

valid

Remarks:

Vehicle: physiological saline 0.9%(w/v) NaCl in deionised water. Prior to application of 0.75mL, the test item was suspended in saline (20%(w/v))

Negative controls validity:

valid

Remarks:

Negative control: 0.9% NaCl solution. Neither an increase of opacity not permeability of the cornea could be observed. The mean in vitro score was calculated as 1.84

Positive controls validity:

valid

Remarks:

Positive control: 10%(w/v) Benzalconium chloride. Clear opacity and distinctive permeability of the cornea showed. The mean in vitro score was calculated as 155.94

Remarks on result:

no indication of irritation

Remarks:

The calculated mean in vitro score was -0.26. Therefore, non eye irritant.

Other effects / acceptance of results:

The test item Disodium titanate did not cause any opacity or permeability of the corneae compared with the results of the negative control. The calculated mean in vitro score was – 0.26 and therefore, the test item was classified as non eye irritant.

Results

Results after 240 Minutes Incubation Time

Test Group	Opacity value = Difference (t240-t0) of Opacity^*		Permeability at 490 nm (OD490)^*		In vitro Score	Mean in vitro Score	Proposed in vitro Irritation Scale
		Mean		Mean
Negative Control	1	1.00	0.059	0.056	1.89	1.84	Non eye irritant
	2		0.054		2.81
	0		0.054		0.81
Positive Control	153.00*		0.733*		164.00	155.94	Severe eye irritant
	160.00*		0.322*		164.84
	134.00*		0.332*		138.99
Disodium titanate	- 1.00*		- 0.008*		- 1.12	- 0.26	Non eye irritant
	- 1.00*		- 0.002*		- 1.03
	1.00*		0.024*		1.37

*corrected values

Historical Data

	Positive Control	Negative Control
Mean	137.19	2.22
Standard Deviation	34.87	0.47

Values of 36 studies with solid test items performed in 2008 and 2009

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item Disodium titanate is not considered to be an eye irritant.

Executive summary:

The test item Disodium titanate did not cause any opacity or permeability of the corneae compared with the results of the negative control.

The calculated mean in vitro score was – 0.26 and therefore, the test item was classified as non eye irritant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Disodium titanate substance (EC234-802-9) has the molecular formula Na2TiO3 and its composition is expressed as (Na2O)x(TiO2), where x is ranging from 0.1 to 6 according to the SIP.

This substance, Reaction mass of Disodium Hexatitanate and Sodium Metatitanate, has a value of x = 0.21, calculated from XRF results, has been identified as a mixture of two specific types of disodium titanate and is therefore within the scope of the disodium titanate SIP.

It is assessed therefore that disodium titanate is an acceptable read-across substance for Reaction mass of Disodium Hexatitanate and Sodium Metatitanate

Justification for classification or non-classification

Skin irritation

The test method based on the EPISKIN™ assay allows the prediction of both irritant and non-irritant substances and can thus be considered as a stand alone method to be used as replacement for the animal test. After treatment with the test item Disodium titanate the relative absorbance values were decreased to 93.8%. This value is well above the threshold for irritancy of <= 50%. Therefore, the test item is not considered to possess an irritant potential. Disodium titanate is considered not to have skin irritating properties and should not be classified and labelled according to regulation (EC) No.: 1272/2008.

 

Skin corrosion

The test method based on the Corrositex™ assay allows the discrimination between corrosive and non-corrosive chemical substances and mixtures. After treatment of the bio-barriers with Disodium titanate for more than 60 minutes the colour of the CDS reagent did not change. Therefore, Disodium titanate is considered as non-corrosive and should not be classified and labelled according to regulation (EC) No.: 1272/2008.

Eye irritation

Disodium titanate is not considered to be an eye irritant and should not be classified and labelled according to regulation (EC) No.: 1272/2008.