Dilithium titanate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Start Date: 07 September 2020
Experimental Completion Date: 29 September 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

As an assessment of skin sensitisation potential could not be made using either existing information on lithium titanate, or by conduct of in chemico and in vitro testing according to OECD test guidelines 442C, D and E, it is considered that in vivo testing in accordance with Point 8.3.2 of Annex VII of the REACH regulation is justified, and that if possible, this should be a Local Lympf Node Assay in accordance with the OECD 429.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Dilithium titanate

EC Number:

234-759-6

EC Name:

Dilithium titanate

Cas Number:

12031-82-2

Molecular formula:

Li.1/2O3Ti

IUPAC Name:

dilithium(1+) oxotitaniumbis(olate)

Test material form:

solid: bulk

Details on test material:

- Purity: >98%
- Empirical Formula: Li2TiO3
- Molecular mass: 109.8 g/mol

Specific details on test material used for the study:

Lithium titanate, also known as Dilithium titanate, Lithium metatitanate and Lithium titanium oxide (CAS number 12031-82-2, EC number 234-759-6), batch number SLEA 7084, was a white powder. It was received on 13 August 2020 and stored at 15-25°C, protected from light. Purity was stated as >98% and the expiry date was given as 23 September 2021. The test article information and certificate of analysis provided by the Sponsor are considered an adequate description of the characterisation, purity and stability of the test article. Determinations of stability and characteristics of the test article were the responsibility of the Sponsor.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

Animal Specifications and Acclimatisation
Nulliparous, non-pregnant female CBA/CaCrl strain mice were obtained from Charles River (UK) Ltd., Margate. All animals were given a clinical inspection for ill health on arrival and a sample was weighed.

Mice were arbitrarily selected from the delivery boxes and allocated to the appropriate number of cages. The condition of the animals was assessed daily throughout the acclimatisation period of 8 to 15 days. A second inspection was performed prior to study commencement to ensure the animals were suitable for the study. Overtly healthy animals were arbitrarily allocated to the study groups on Day 1, prior to the commencement of treatment.

Animals in the main study were in a body weight range of 18 to 22 g on Day 1 of dosing. Individual body weights were within ±20% of the mean body weight for mice on the study. Based on information from the supplier the mice were approximately 9 to 10 weeks old on Day 1.

Environmental Conditions, Diet and Water
The animals were kept in the following conditions except for short periods of time where experimental procedures dictated otherwise.

Housing
The animals were housed in groups of up to five during acclimatisation, in cages that conformed to the 'Code of Practice for the Housing and Care of Animals Bred, Supplied or Used for Scientific Purposes’ (Home Office, London, 2014). From Day 1 the preliminary animal housed individually, while the main study animals were housed in pairs.

Bedding was provided on a weekly basis to each cage by use of clean European softwood bedding (Datesand Ltd., Manchester, UK). The bedding had been analysed for specific contaminants and the results retained on file at Covance.

No contaminants were present in bedding at levels which might have interfered with achieving the objective of the study.

Water
Mains water was provided, ad libitum, via cage-mounted water bottles. The water had been periodically analysed for specific contaminants.

No contaminants were present in water at levels which might have interfered with achieving the objective of the study. Results are retained on file at Covance.

Diet
5LF2 EU Rodent Diet 14% was freely available to the animals at all times. Each batch of diet had been analysed for specific constituents and contaminants by the manufacturer.

No contaminants were present in diet at levels which might have interfered with achieving the objective of the study. Results are retained on file at Covance.

Environment
The animal rooms were designed to permit a minimum of 15 air changes per hour. The temperature and humidity ranges were 19 to 25C and 40 to 70% respectively. Daily recordings of maximum and minimum temperature and humidity were made.

Fluorescent lighting was controlled automatically to give a cycle of 12 hours light and 12 hours dark.

Environmental Enrichment
In order to enrich both the environment and the welfare of the animals, they were provided with wooden Aspen chew blocks and nesting materials. The nesting materials were removed from the cages on Day 1, prior to dosing.

Animal Identification and Assignment to Study
A unique number was inscribed onto the tail with indelible ink on Day 1, prior to dosing, to individually identify the mice. A colour-coded card on each cage gave information including study number, animal number and sex.

Mice were arbitrarily selected from the delivery boxes and allocated to the appropriate number of cages. Overtly healthy animals were arbitrarily allocated to the study groups on Day 1, prior to commencement of treatment.

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

Preliminary screening test: 25 µL of the test article at the maximum suitable concentration (50% w/v in propylene glycol)
Main study: 10% w/v, 25% w/v and 50% w/v

No. of animals per dose:

Four

Details on study design:

Test Article Formulation
Formulations were freshly prepared using propylene glycol on Days 1, 2 and 3. The formulations were stored at room temperature, in sealed, air-tight containers prior to dosing and were used within two hours of preparation.

The formulations were mixed by multiple inversion of the containers prior to administration to ensure homogeneity.

Concentrations of test article were expressed gravimetrically and in terms of test article received (without regard to purity or active content).

Preliminary Screening Test
According to two in vitro skin irritation/corrosion studies, the test substance was considered to have no skin irritation/corrosion potential (Warren, 2020; Lacey, 2020). Furthermore, no mortality was observed in rats during an acute oral toxicity study (LD50 > 2000 mg/kg bw; Ferreira, 2020). A preliminary screening test was conducted with one animal prior to the Main Study.

The mouse was treated by daily application of 25 µL of the test article at the maximum suitable concentration (50% w/v in propylene glycol) to the dorsal surface of each ear for three consecutive days (Days 1, 2 and 3). The mouse was observed daily for any signs of toxicity or irritation at the application site. The body weight was recorded prior to dosing on Day 1 and prior to termination on Day 6. Both ears were observed for erythema and scored

Ear thickness measurements were taken using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6.

Excessive local irritation is indicated by an erythema score ≥3 and/or an increase in ear thickness of ≥25% on any day of measurement.

Main Study
Groups of four female mice were assigned to study according to the table given below. Doses were selected from the concentration series 50%, 25%, 10%, 5%, 2.5%, 1.0%, 0.5% etc. Three consecutive concentrations were selected on the basis of the preliminary screening test so that the highest concentration maximised exposure whilst avoiding systemic toxicity and/or excessive local irritation. As the vehicle for the test article formulation was different to that used for the positive control formulation, a positive control vehicle group (acetone / olive oil in a ratio of 4:1 v/v) was also included in the study.

Inlife Procedures
Test Article Administration
Each mouse was manually restrained with both auditory pinnae left free. The outer aspect of both pinnae of each mouse was treated by direct application of the appropriate test or control formulation (0.025 mL/pinna) dispensed from an automatic micro pipette.

Treatment Regimen
The six groups of four female mice were subjected to application of the test article vehicle control, positive control vehicle, positive control or one of the test formulations to the outer aspect of the auditory pinnae, once daily on Days 1, 2 and 3.

On Day 6 the mice were placed in a thermacage in order to dilate the peripheral blood vasculature and thus facilitate intravenous dosing. Each mouse was transferred to a cylindrical restrainer. A plastic syringe and fine gauge hypodermic needle were used to administer 0.25 mL phosphate buffered saline incorporating 20 μCi (0.74 MBq) of 3HTdR into a tail vein of each mouse by slow bolus injection. After this treatment, the mice were returned to their cages.

Five hours after intravenous injection of the 3HTdR, all mice were killed by exsanguination under a deep plane of inhalation anesthesia. Killing was organised to minimise the interval between death and the recovery of the auricular lymph nodes to no more than fifteen minutes.

Clinical Signs
Treated mice were observed twice daily on Days 1 to 5 and once on Day 6 for clinical signs of reaction to treatment or for irritation or other changes at the sites of application of the test article.

Routine Health Checks
All animals were examined at the beginning and end of the working day throughout the acclimatisation and study periods to ensure they were in good health.

Body Weights
Mice were weighed on Day 1 (prior to administration) and on Day 6 prior to intravenous administration of 3HTdR.

Terminal Procedures
Recovery of Lymph Nodes
Once death had been confirmed each mouse was placed on a bench lined with paper with its ventral aspect uppermost. A mid-line incision from the lower jaw to the sternum was made and the cervical structures were exposed by reflecting the skin. The auricular lymph nodes were located and removed using curved end forceps. Any connective tissue was removed from the capsule of the nodes. The auricular lymph nodes of all mice from each dose group were placed into a petri dish containing 5 mL phosphate buffered saline.

Preparation for Scintillation Count
The lymph nodes collected into each petri dish were cut open and disaggregated by squashing the fragments with a sharp blade. The resultant liquor was transferred into code-identified conical tubes. The petri dishes were rinsed with an additional 5 mL phosphate buffered saline and the second liquor was added to the first liquor. At each transfer, debris such as fragments of capsule, were retained in the petri dish wherever possible.

After 5 minutes, the pooled liquor was filtered into a second conical tube by transferring the liquor into a 10 mL syringe and passing it through a stainless steel gauze containing a fabric filter, cut to size (Clarcor UK, Lockertex Filtration Products, Warrington, UK). Any visible sediment remaining prior to filtering was left in the conical tube. The liquor was centrifuged at 200 g for 10 minutes. Following centrifugation, the supernatant was discarded and the pellet was resuspended in 5 mL phosphate buffered saline. This was centrifuged at 200 g for 10 minutes. The supernatant was discarded and the pellet resuspended in 3 mL of 5% w/v aqueous trichloroacetic acid. The suspension was stored for 18 hours at 2 to 8C (nominal 4C).

On the following day, the suspension was re-centrifuged at 200 g for 10 minutes and the supernatant was drawn off and discarded. The pellet was resuspended in 1 mL 5% w/v aqueous trichloroacetic acid then subjected to ultrasonic dispersion for 25 minutes to ensure a homogenous suspension. The suspension (1 mL) was transferred to a scintillation vial and scintillation fluid (10 mL) was added.

Scintillation Counting
Once prepared, the scintillation vials were placed in the appropriate carrier racks. Two background vials were prepared, one containing 10 mL of scintillation fluid and the other containing 1 mL of 5% w/v aqueous trichloroacetic acid and 10 mL scintillation fluid. The carrier rack was passed into the scintillation counter. All vials, including the background samples, were submitted for liquid scintillation counting for 10 minutes, using a 3H quench curve.
Incorporation of 3HTdR is measured by ß-scintillation counting as disintegrations per minute (DPM) over a ten-minute period. This value was corrected to account for the background containing 5% w/v aqueous trichloroacetic acid and scintillation fluid.

Positive control substance(s):

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

Results and discussion

Positive control results:

The positive control article produced a Stimulation Index of 4.94, demonstrating adequate performance of the assay.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.13

Test group / Remarks:

50%

Results 2

Key result

Parameter:

SI

Value:

0.93

Test group / Remarks:

25%

Results 3

Key result

Parameter:

SI

Value:

1.27

Test group / Remarks:

10%

Any other information on results incl. tables

Preliminary Screening Test

Death or signs of systemic toxicity/excessive irritation were not noted.

Based on this information, the dose levels selected for the main test were 10%, 25% and 50% w/v in propylene glycol.

Preliminary Screening Test - Observations, Body Weights and Mortality Data

Concentration (% w/v in propylene glycol)		Animal Number	Body Weight (g) on Day:		Observations (Days)
			1	6	1	2	3	4	5	6
50	527		20	21	ü	ü	ü	ü	ü	ü

 

Key

ü             No clinical signs seen

 

Table 10.2      Preliminary Screening Test - Erythema

Concentration (% w/v in propylene glycol)	Animal Number	Grade of Erythema on Day:
		1	2	3	4	5	6
50	527	0	0	0	0	0	0

 

Key

0              No erythema

 

Table 10.3      Preliminary Screening Test - Ear Thickness Measurements

Concentration (% w/v in propylene glycol)	Animal Number	Ear	Thickness (mm) on Day:
			1	3	6
50	527	Left	0.21	0.21	0.25

 

Main Test

Mortality

All animals survived treatment with Lithium titanate.

Clinical Signs

There were no clinical signs indicative of a systemic effect of treatment among mice treated with the test article vehicle, positive control vehicle or with 10, 25 or 50% w/v formulations of the test article.

The vehicle and test formulation application sites remained free of irritation.

Greasy fur behind the ears and on the back of the neck was noted in all Group 2 and Group 6 animals on Days 1 to 5.

Body Weights

There was no indication of a treatment related effect on body weight.

 

Group DPMs and Stimulation Index (SI)

Concentration (% w/v) in propylene glycol	Group Number	Group DPM	Stimulation Index (SI) ^a
Test Article Vehicle	1	981	NA
10	3	1241	1.27
25	4	917	0.93
50	5	1105	1.13
Positive Control Vehicle	2	1250	NA
Positive control	6	6180	4.94

 

^a = Stimulation Index of 3.0 or greater indicates a positive result

 

Body Weights

Concentration (% w/v) in propylene glycol	Group Number	Animal Number	Body Weights (g)
			Day 1	Day 6
Test Article Vehicle	1	528 529 530 531	21 22 20 20	22 24 21 20
Positive Control Vehicle	2	532 533 534 535	18 21 21 20	19 22 21 22
10	3	536 537 538 539	19 22 20 22	19 23 21 23
25	4	540 541 542 543	22 20 21 19	24 21 21 21
50	5	544 545 546 547	18 18 18 20	19 19 20 21
Positive control	6	548 549 550 551	19 20 19 19	20 23 20 21

 

NA = Not applicable

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The Local Lymph Node Assay demonstrated that Lithium titanate does not have the potential to cause skin sensitisation and therefore should not be classified and labelled for skin sensitisation according to Regulation (EC) No. 1272/2008 and its subsequent regulations.

Executive summary:

SUMMARY

This study was conducted to assess the potential of the test article, Lithium titanate, to cause skin sensitisation in the mouse.

Following a preliminary screening test using a 50% w/v formulation, the test article was prepared for administration at 10, 25 and 50% w/v in propylene glycol.

Groups of four female CBA / CaCrl mice were subjected to topical applications of test article vehicle control, positive control vehicle, positive control or of one of the test formulations to the outer aspect of the auditory pinnae once daily on Days 1, 2 and 3. On Day 6, a 20 μCi dose of tritiated ³H-methyl thymidine was injected intravenously into each mouse. Five hours later the auricular lymph nodes were recovered from each animal. The pairs of nodes from each animal of each group were pooled and suspensions of the cellular components of the lymph nodes were prepared in 5% w/v aqueous trichloroacetic acid and processed through a scintillation counter.

Test results are expressed in terms of Stimulation Indices, the ratios of the mean scintillation counts obtained from the test groups relative to the corresponding mean scintillation count obtained from controls. The threshold level for the Stimulation Index to be considered a positive indicator of the potential to cause skin sensitisation is 3.0.

	Concentration of Test Article in Applied Formulation (% w/v)
	10%	25%	50%
Stimulation Index	1.27	0.93	1.13

 

The Local Lymph Node Assay demonstrated that Lithium titanate does not have the potential to cause skin sensitisation and therefore should not be classified and labelled for skin sensitisation according to Regulation (EC) No. 1272/2008 and its subsequent regulations.

The positive control article produced a Stimulation Index of 4.94, demonstrating adequate performance of the assay.