Hexanoic acid, 3,5,5-trimethyl-, tin(2+) salt (2:1)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-03-08 to 2011-03-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 442B (Skin Sensitisation: Local Lymph Node Assay: BrdU-ELISA

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:

tin(2+) bis(3,5,5-trimethylhexanoate)

EC Number:

700-567-0

Cas Number:

1231728-34-9

Molecular formula:

C18H34O4Sn

IUPAC Name:

tin(2+) bis(3,5,5-trimethylhexanoate)

Test material form:

liquid: viscous

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, Germany
- Age at study initiation: approx. 12 weeks at start of the main experiment
- Weight at study initiation: 23.3 ± 1.6 g at start of the main experiment
- Housing: Single housed in Makrolon - cages Type III
- Diet (e.g. ad libitum): Altromin 1324 ad libitum
- Water (e.g. ad libitum): ad libitum, tap water
- Acclimation period: Before the main experiment was started, the animals had an acclimatisation period after delivery of 16 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 30 - 70
- Air changes (per hr): 8 TIMES/HOUR
- Photoperiod (hrs dark / hrs light): 2 hours dimmed Iight, 12 hours dark

IN-LIFE DATES: From: To: 2011-03-01 to 2011-03-23

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

test substance:
50 % (w/w); 75 % (w/w); 100 %
positive control:
HCA 25 % (v/v) in acetone/olive oil (3+1)
negative control: vehicle

No. of animals per dose:

4 animals per group

Details on study design:

RANGE FINDING TESTS:
- Compound solubility:
- Irritation: All mice were observed daily for any clinical sign of systemic toxicity or local irritation at the application site.
Both ears of each mouse were observed for erythema and scored. Ear thickness measurements were taken using an electronic external measuring gauge (Kroeplin GmbH) from day 1 (pre-dose) throughout to day 4 (prior to termination).
- Lymph node proliferation response: The LLNA: BrdU-ELISA is a nonradioactive modification to the traditional LLNA test method, which utilises non-radiolabelled bromo-deoxyuridine in an ELISA-based test system to measure lymphocyte proliferation.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: BrdU-ELISA
- Criteria used to consider a positive response: the test substance is regarded as positive when at least with one concentration the Sl is >/= 1.6.

TREATMENT PREPARATION AND ADMINISTRATION:
Main experiment:
For the main experiment three consecutive concentrations of the test substance were selected:
50 % (w/w), 75 % (w/w) and 100 % (undiluted).
In addition a concurrent negative control group (NC) treated only with the vehicle and a
positive control group (PC) treated with the reference substance (HCA 25 % (v/v) in acetone/olive oil (3+1))
was used for the reliability check.
Four animals per group were treated. Except for absence of treatment with the test substance, animals in the control groups were handled and
treated in a manner identical to that of animals in the test substance groups.

Test regime:
Day 1: the weight of each animal and any clinical observation were recorded prior to the application of the test or control substances.
25 µL of the selected test substance concentration, reference substance or vehicle was applied to the dorsal side of each ear.

Day 2: any clinical observation was recorded the application procedure carried out on day 1 was repeated.

Day 3: any clinical observation was recorded the application procedure carried out on day 1 was repeated.

Day 4: no treatment.

Day 5: 5-bromo-2-deoxyuridine (BrdU) was solved in 0.9 % saline solution in a concentration of 10 mg/mL and filter sterilised (0.22f.1m filter).
0.5 mL of the BrdU-solution was injected intraperitoneal to each mouse (= 5 mg/mouse).

Day 6: the weight of each animal was recorded.
Approx. 24 hours after BrdU injection the animals were sacrificed and the draining auricular lymph nodes from each mouse ear were exercised.
Both lymph nodes of one animal were pooled and further processed for each animal separately in phosphate buffered saline (PBS).
From each mouse a single-cell suspension of lymph node cells was prepared by gentle mechanical disaggregation by the use of a disposable plastic
pestle to crush the lymph nodes followed by passage through a 74 µm nylon mesh. Cell counts using C-Chip disposable hemocytometer with the
negative control group revealed a mean cell number of 5.6 x 10 E6 cells/ml.
The target volume of the cell suspensions was adjusted by 1:30 dilution with PBS to the optimised volume for the determination of cellular proliferation (= 1.9 x 10 E4 cells/100 µL). 100 µL of the lymph node cell suspension was added to the wells of a flat-bottom microplate in triplicate.
The microplate was centrifuged at 319g for 10 minutes to affix the cells to the bottom. The PBS was carefully removed by aspiration by pipetting and the cells were dried at approximately 60°C in an oven for 1 h. The dried cells were stored refrigerated overnight until cell proliferation measurement
on the next day.

Determination of the cellular proliferation was performed by the measurement of BrdU content in the DNA of the lymphocytes.
BrdU was measured colorimetric by ELISA using a commercial kit from Roche Diagnostics GmbH, Mannheim, Germany (Lot 12015100, Expiry March 2012) according to the procedure given in the instruction manual.
After fixation and denaturation anti-BrdU antibody was added to each well and allowed to react. Subsequently the anti-BrdU antibody excess was
removed by washing and the substrate solution was then added and allowed to produce chromogen. Absorbance at 360 nm with a reference
wave length of 485 nm was then measured.

Positive control substance(s):

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

Statistics:

no

Results and discussion

Positive control results:

For the reference substance a Stimulation Index of 3.09 was determined indicating the adequate function of the test system.

In vivo (LLNA)

Results

Parameter:

SI

Remarks on result:

other: Test substance: 50 % SI=3.37 75 % SI=6.44 100 % SI=7.11 Negative control: SI=1.0

Any other information on results incl. tables

Pilot Experiment

At the daily clinical observation the animals did not show any visible symptoms of systemic toxicity. Animals showed slightly decreased or nearly unchanged body weights. Only slight local irritation was observed in the highest concentration group but ear thickness values were unaffected by the treatment with the 100 %, 75 % and 50 % (w/w) concentrations of the test substance. As no excessive local irritation and no ear thickness increase was observed in the pilot experiment the same concentrations were selected for the main experiment.

Main Experiment

Visible symptoms of systemic toxicity caused by the test substance were not observed. No unusual finding occurred. The body weight of the animals was slightly decreased. Only slight symptoms of local irritation at the ears of some animals in the 75 % and 100 % test substance concentration groups and the positive control group were observed.

Test substance: 50 % SI=3.37; 75 % SI=6.44; 100 % SI=7.11

Negative control: SI=1.0

Positive control: SI=3.09

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: other: CLP, EU GHS (Regulation (EC) No 1272/2008)

Conclusions:

In this Local Lymph Node Assay the test substance is a dermal sensitizer.

Executive summary:

In a dermal sensitization study with the test substance hexanoic acid, 3,5,5-trimethyl-, tin (2+)salt (2:1) in acetone/olive oil (4:1, v/v), groups of 4 female CBA mice were tested using the LLNA method according to the OECD Guideline 442B, July 2010. BrdU was measured by ELISA using a commercial kit.

STIMULATION INDICES of 3.37, 6.44 and 7.11 were determined with the test substance at concentrations of 50 %, 75 % and 100 % (w/w) in acetone:olive oil, 4:1 (v/v), respectively.

 

Positive control substance was alpha hexyl cinnamic aldeyde, which gave a positive result (SI = 3.09 at a concentration of 25 % in acetone:olive oil, 4:1 (v/v).

A result is regards as positive when the SI (Stimulation Indes) is ≥ 1.6.

 

Based on these criteria, the test substance was found to be a sensitizer when tested from 50 % up to 100 %.

 

In this study, hexanoic acid, 3,5,5-trimethyl-, tin (2+)salt (2:1) is a dermal sensitizer.

According to the GHS Regulation (EC) No 1272/2008, 2nd ATP, (No 286/2011) sensitizers may be allocated to one of two sub-catogories; sub-category 1A, strong sensitisers, or subcategory 1B for other skin sensitisers. The EC3-values are used for evaluation of data from a standard LLNA, EC3-values of≤2 % are assigned to category 1A, and EC3-values of > 2 % to category 1B, respectively.

For hexanoic acid, 3,5,5-trimethyl-, tin (2+)salt (2:1) the reported SI values were 3.37, 6.44 and 7.11 for test concentrations of 50 %, 75 %, and 100%, respectively. However, no EC3-value was derived in the study report.

Hence, based on the observed dose response curve, it can be assumed that the EC3 will be well beyond the value of > 2 % and thus leading to a classification as moderate sensitizer (category 1B).