Reaction mass of disodium hexatitanium tridecaoxide and disodium trititanium heptaoxide

Endpoint summary

Description of key information

Disodium titanate has been tested in one in vivo skin sensitisation study (Local lymph node assay (LLNA) in mice). This study shows a negative response, thus disodium titanate does not require classification as sensitiser.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-05-04 to 2010-05-19

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 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

, 2002-04-24

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2009-03-30

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source:Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 18 g - 24.5 g
- Housing: Single caging; Cage type: Makrolon Type II, with wire mesh top (EHRET GmbH, 79302 Emmendingen, Germany); Bedding: Granulated soft wood bedding (Rettenmaier & Söhne GmbH + Co. KG, 73494 Rosenberg, Germany)
- Diet (ad libitum): Pelleted standard diet (Harlan Laboratories B.V. 5960 AD Horst / Netherlands)
- Water (ad libitum): Tap water (Gemeindewerke, 64380 Rossdorf, Germany)
- Acclimation period: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 + 2°C
- Relative humidity: Approx. 45-65%
- Photoperiod (hrs dark / hrs light): 12/12
No further information on test animals was stated.

Vehicle:

acetone/olive oil (4:1 v/v)

Remarks:

Acetone: Batch no. K385 02214-812; Purity: 99.6%; Supplier: VWR International GmbH (64295 Darmstadt, Germany); Olive oil: Batch no. 058K0684; Supplier: Sigma/Aldrich Chemicals (82024 Taufkirchen, Germany)

Concentration:

5 %, 10 %, and 25 % concentrations

No. of animals per dose:

5 females

Details on study design:

A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which can be technically used was a 25 % suspension in acetone:olive oil (4 + 1). All other tested vehicles (dimethylformamide, methyl ethyl ketone, propylene glycol, dimethylsulfoxide, and ethanol:sterile water (at ratios of 7 + 3 and 3 + 7)) could also not achieve higher concentrations.

RANGE FINDING TESTS:
To determine the highest non irritant test concentration, pre-test was performed in two animals. Two mice were treated with concentrations of 10 and 25 % each on three consecutive days.
In the pre-test clinical signs were recorded with approx. 1 hour and 24 +/- 4 hours after each application as well as on day 7. Furthermore, prior to the first application of the test item and before sacrifice the body weight was determined. At the tested concentrations the animals did not show any signs of irritation or systemic toxicity.
The test item in the main study was assayed at 5, 10, and 25 %. the top dose is the highest technically achievable concentration whilst avoiding systemic toxicity and excessive local irritation. No severe irritant effects were tolerated choosing the test concentrations.

MAIN STUDY
The test item was placed into a volumetric flask on a tared balance and acetone:olive oil (4+1) was quantitatively added. The different test item concentrations were prepared individually. Homogeneity of the test item in vehicle was maintained during treatment using a magnetic stirrer.
The preparations were made freshly before each dosing occasion.

Topical application:
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear (left and right) with different test item concentrations of 5, 10, and 25% (w/v) in acetone:olive oil (4+1). The application volume, 25 µl, was spread over the entire dorsal surface (8 mm) of each ear once daily for three consecutive days. A further group of mice (5 control animals) was treated with an equivalent volume of the relevant
vehicle alone.

Administration of 3H-Methyl Thymidine:
Five days after the first topical application all mice were administered with 250 µL phosphate-buffered saline solution (PBS) containing 20.2 µCi 3HTdR (80.9 µCi/ml 3HTdR; 3HtdR was purchased from Hartmann Analytic, 38124 Braunschweig, Germany (specific activity, 2 Ci/mmol; concentration, 1 mCi/ml)) by intravenous injection via a tail vein.

Determination of incorporated 3HTdR:
Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Pentobarbital-Natrium (Release®, WDT, 30827 Garbsen, Germany).
The draining lymph nodes were rapidly excised and pooled per animal (2 nodes per animal) in phosphate buffered saline (PBS). Single cell suspensions of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing two times with phosphate buffered saline (approx. 10 ml) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 ml) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 ml) and transferred to plastic scintillation vials with 10 ml of ‘Ultima Gold’ scintillation liquid (Perkin Elmer (LAS) GmbH, 63110 Rodgau, Germany) and thoroughly mixed.
The level of 3HTdR incorporation was then measured on a β-scintillation counter (Tricarb 2900 TR, Perkin Elmer (LAS) GmbH, 63110 Rodgau, Germany). Similarly, background 3HTdR levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid. The β-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

Interpretation of raw data:
The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/node) and as the ratio of 3HTdR incorporated into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes (stimulation index). Before DPM/node values were determined, mean scintillation-background DPM was subtracted from test and control raw data.
A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
- First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index.
- Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

OBSERVATIONS
Mortality / Viability: Once daily (week day) from experimental start to necropsy.
Body weights: In the main experiment: prior to the first application and prior to treatment with 3HTdR.
Clinical signs (local/systemic): In the main experiment clinical signs were recorded within 1 hour after each application and 24 ± 4 hours after the first and second application as well as on the day of preparation. Especially the treatment sites were observed carefully.
No further information on the study design was stated.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

The mean values and standard deviations were calculated in the body weight tables.

Positive control results:

Positive control substance: α-Hexylcinnamaldehyde
5 % (w/v): SI: 2.04
10 % (w/v): SI: 3.41
25 % (w/v). SI: 6.14
EC3 (estimated concentration for a S.I. of 3) = 8.5 %

Key result

Parameter:

SI

Remarks on result:

other: 5 % disodium titanate: SI: 1.84 10 % disodium titanate: SI: 2.13 25 % disodium titanate : SI: 1.52 The EC3 value could not be calculated, since none of the tested concentrations induced an S.I. greater than 3.

Key result

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

5 % disodium titanate: DPM: 704.3 10 % disodium titanate: DPM: 813.3 25 % disodium titanate : DPM. 582.1 DPM/node was determined by dividing the sum of the measured values from all lymph nodes within a group by the number of lymph nodes taken from that group

- Mortality / viability: No deaths occurred during the study period.

- Clinical signs: No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

- Body weight: The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

Interpretation of results:

GHS criteria not met

Remarks:

not sensitising

Conclusions:

The test item disodium titanate was not a skin sensitiser under the described conditions.
According to the criteria specified by Directive 67/548/EEC and subsequent regulations, the test item is not classified as skin sensitiser.
According to regulation (EC) No.: 1272/2008 and subsequent regulations, the test item is not classified as skin sensitiser.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

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

Respiratory sensitisation

Endpoint conclusion

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

Sensitisation

The reference Vogel (2010) is considered as the key studies on skin sensitisation and will be used for classification. The sensitisation results are as follows:

SIs of less than 3.0 were observed at all test concentrations of disodium titanate (5, 10, 25%). Therefore, the EC3 value could not be

calculated, since none of the tested concentrations induced an SI greater than 3.

Thus, the classification criteria acc. to regulation (EC) 1272/2008 as skin sensitizer are not met since 0% of the test animals responded.