Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 13, 2010 - September 17, 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP study performed according to current guideline; however behaviour of vehicle was atypical.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Titanium tetraisopropanolate, referred in the study report 2-Propanol titanium (4+) salt batch number V0303339, was received on 01 April 2010 (100 ml). An expiry date of 07 April 2011 was arbitrarily allocated. The purity was reported to be ≥90% on documentation supplied by the Sponsor.
Subsequent to the receipt of the test article, further information was received from the Sponsor detailing the following purity profile for the batch: 2-propanol, titanium (4+) salt >99%, heptanes (naphtha) 0.56%, iso-propanol <0.5%.

When not in use the test article was stored in a sealed container, at room temperature in the dark. The test article was stored over a desiccant from 14 April 2010 in accordance with the definitive protocol. It was considered that storage without desiccant prior to this date did not have any adverse effect on the test article as the container was not opened during this period.
Although not specified in the protocol, the container was purged with nitrogen each time it was opened from 26 April 2010, to further protect the test article from moist air.

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
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.

Acclimatisation period and allocation to study: 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 the day prior to commencement of treatment.
Animals in the main study were in a body weight range of 17 to 20 g on the day before dosing commenced. Based on information from the supplier the mice were approximately 9 to 10 weeks old on Day 1.

Caging: The animals were group housed before the start of treatment and individually housed from Day -1 until the end of the study.

Diet, Water and Bedding: SQC(E) Rat and Mouse Maintenance Diet No 1, from Special Diets Services Ltd, Witham, UK was freely available to the animals at all times. Each batch of diet had been analysed for specific constituents and contaminants by the manufacturer.
Mains water was provided, ad libitum, via cage-mounted water bottles. The water had been periodically analysed for specific contaminants.
Certificates of analysis for contaminant levels in each batch of diet or in the water supply were consigned to central files at this laboratory.
Bedding was provided on a weekly basis to each cage by use of clean Aspen wood chips (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 diet, water or bedding at levels which might interfere with achieving the objective of the study.

Environment: The animal rooms were designed to permit 15 to 20 air changes per hour. The temperature and humidity ranges were 20 to 24°C and 45 to 65% respectively. The rooms were illuminated by fluorescent strip-lights for twelve hours daily.

Study design: in vivo (LLNA)

Vehicle:
methyl ethyl ketone
Remarks:
The vehicle was chosen by the Sponsor based on its moisture content and compatibility with TIPT which hydrolyses rapidly in the presence of water.
Concentration:
25, 50 and 100% v/v
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: Test item is a liquid. The vehicle was confirmed to be suitable for use in this study as a solution and a concentration of 50% was achieved.
- Irritation: one mouse was treated with undiluted test item by daily application of 25 µl of undiluted test item onto the back of the pinnae for 3 days. No signs of toxicity or irritation were noted.

MAIN STUDY
TREATMENT PREPARATION AND ADMINISTRATION:
Formulations were freshly prepared as required using methyl ethyl ketone 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 item were expressed volumetrically and in terms of test item received (without regard to purity or active content).
Tritiated 3H-methyl thymidine (3HTdR), batch 201003, was obtained as a 1000 μCi/mL preparation from PerkinElmer Life and Analytical Sciences, Boston, USA. An aliquot of 5.75 mL phosphate buffered saline was mixed with 0.5 mL 3HTdR shortly before intravenous dosing commenced on Day 6. The final product contained 3HTdR at 80 μCi/mL.

The test item was prepared for administration in the main study at 25 and 50% v/v in methyl ethyl ketone and was also used undiluted. Groups of four female CBA / CaCrl mice were subjected to topical applications of vehicle 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 nodes from mice subjected to the same treatment were pooled and suspensions of the cellular components of the lymph nodes were prepared in 5% w/v trichloroacetic acid and processed through a scintillation counter.
Test results are expressed in terms of Stimulation Indices, the ratios of the mean scintillation count per group obtained from the test groups relative to the corresponding mean scintillation count 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.

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 petri dishes 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 200 µm mesh 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 190 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 190 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 1 to 10 °C (nominal 4 °C).
On the following day the suspension was re-centrifuged at 190 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 an homogenous suspension. The suspension (1 mL) was transferred to a scintillation vial and scintillation fluid (ca 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 ca 10 mL of scintillation fluid and the other containing 1 mL of 5% w/v aqueous trichloroacetic acid and ca 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 using a 3H quench curve.
Incorporation of 3HTdR was measured by ß-scintillation counting as disintegrations per minute (DPM). In error, this was conducted for all groups over a five-minute period and not for ten minutes as specified in the protocol. This deviation was considered not to have affected the integrity or outcome of the study, as the five-minute counting period has been shown to work on a previous in-house positive control study (whereby the reduction in counting time did not impact on the positive control result).
Incorporation of 3HTdR is measured by ß-scintillation counting as disintegrations per minute (DPM). This value was corrected to account for the background containing 5% w/v aqueous trichloroacetic acid and scintillation fluid.

DATA EVALUATION
The scintillation counter printed data including the DPM value (disintegrations per minute during a five minute period). The DPM value was transformed into a DLM value (disintegrations per minute per lymph node) by dividing by the number of sites yielding lymph nodes. The DLM value for each test group was divided by the DLM for the control group to provide the Stimulation Index (SI) value for each test group.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
mercaptobenzothiazole (CAS No 149-30-4)

Results and discussion

Positive control results:
The sensitivity and reliability of the test system are checked at least every six months. Preferred substances are α hexylcinnamaldehyde (CAS Number 101-86-0) and mercaptobenzothiazole (CAS Number 149-30-4). A Stimulation Index of 3.0 or greater is expected from these substances. The positive control results are tabulated in the section 'any other results including tables'.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
Stimulation index = Test group disintegrations per minute per node (DLM) / Control group DLM value Titanium tetraisopropanolate 25% v/v - 2.5 Titanium tetraisopropanolate 50% v/v - 2.5 Titanium tetraisopropanolate 100% v/v - 5.4 (see overall remarks section, result is skewed by atypically low MEK values achieved in this study)
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
Scintillation counts Scintillation fluid with 5% w/v trichloroacetic acid - 47 disintegrations per minute (DPM) Vehicle control (MEK) - 255 DPM (8 sites yielding lymph nodes) = 32 disintegrations per minute per node (DLM) Note this result is atypical as published MEK vehicle DPM/lymph node results are typically in the range 216 to 360. TIPT, 25% v/v - 632 DPM (8 sites yielding lymph nodes) = 79 DLM TIPT, 50% v/v - 647 DPM (8 sites yielding lymph nodes) = 81 DLM TIPT, 100% v/v - 1385 DPM (8 sites yielding lymph nodes) = 173 DLM

Any other information on results incl. tables

Preliminary screening test:

Death or signs of systemic toxicity/excessive irritation were not noted. On this basis 25, 50 and 100% concentrations were selected for the main test.

Main Test:

There were no clinical signs indicative of a systemic effect of treatment among mice treated with the vehicle or test article.

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

A white residue was noted on the backs of the ears of intermediate and high dose group animals on Day 2 and in all test groups from Day 3 to Day 6.

No effect on bodyweight was noted.

Positive control results

Study
number

Date
started

Date
finished

Positive
control
material

Concentrations

Vehicle control dpm
value per animal

Test group
dpm values per animal

Stimulation
indices

0000/643

December 2006

December 2006

a‑Hexylcinnamaldehyde

5%, 10% and 25% in AOO

572

3021, 4206, 9222

3.5, 6.3, 10.8

0000/650

March 2007

March 2007

α‑Hexylcinnamaldehyde

5%, 10%, and 25% in AOO

550

888, 2173, 2478

1.61, 3.95, 4.51

0000/696

September 2007

September 2007

α‑Hexylcinnamaldehyde

5%, 10%, and 25% in AOO

496

478, 992, 2586

0.96, 2.00, 5.22

0000/697

October 2007

October 2007

2-Mercaptobenzothiazole

5%, 10 % and 25% in DMF

323

377, 1103, 995

1.17, 3.42, 3.08

0000/741

March 2008

April 2008

α‑Hexylcinnamaldehyde

5%, 10%, and 25% in AOO

336

317, 652, 1825

0.9, 1.9, 5.4

0000/790

September 2008

September 2008

α‑Hexylcinnamaldehyde

5%, 10%, and 25% in AOO

891

727, 1270, 3527

0.8, 1.4, 4.0

8202569

April 2009

April 2009

α‑Hexylcinnamaldehyde

5%, 10%, and 25% in AOO

1160

1462, 1900, 4121

1.3, 1.6, 3.6

8215020

October 2009

October 2009

α‑Hexylcinnamaldehyde

5%, 10%, and 25% in AOO

596

1151, 1539, 3322

1.9, 2.6, 5.6

8225623

April 2010

April 2010

α‑Hexylcinnamaldehyde

5%, 10%, and 25% in AOO

222

294, 308, 1124

1.3, 1.4, 5.1

Key: AOO = 80% v/v acetone in olive oil DMF = dimethylformamide

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: expert judgment
Conclusions:
The potential of titanium tetraisopropanolate to cause skin sensitisation was assessed in the mouse. Based on the study results the test substance is not demonstrating evidence of skin sensitisation.
Executive summary:

The test item was prepared for administration at 25 and 50% v/v in methyl ethyl ketone and was also used undiluted. Groups of four mice were subjected to topical applications of vehicle 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 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 nodes from mice subjected to the same treatment were pooled and suspensions of the cellular components of the lymph nodes were prepared in 5% w/v trichloroacetic acid and processed through a scintillation counter.

Due to the abnormally low DPM/lymph node count achieved in the vehicle control group (MEK) an artificially high SI index was calculated for the undiluted test article group. There was no dose response in the 25 and 50% titanium tetraisoprpanolate groups. When the DPM/lymph node value obtained for the control MEK group in this study was compared to values obtained for other MEK control groups in studies performed according to the same guideline it was found to be very much lower. The value obtained in this study was 32 DPM/lymph node whilst published values were in the range 216 to 360 DPM/lymph node. Dr. Basketter was requested to write an expert review of this study and comment on the artificially low vehicle control values (Chapter 7.4.1 skin sensitisation_supporting study.002). He stated that titanium tetraisopropanolate should not be classified as a skin sensitiser on the basis of this LLNA study.

Based on the study results, conclusions and expert opinion, it was therefore concluded that the test item was not demonstrating any real evidence of skin sensitivity.

This study was regarded reliable with restrictions since the study was conducted in compliance with GLP and performed according to current guideline; however behaviour of negative control vehicle was atypical. The result of this study is used as a key study in hazard assessment.