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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2005-01-19 to 2005-01-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study reliable without restrictions (GLP certificate was not attached to the study report)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report date:
2005

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
, adopted 2002-04-24
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
Diantimony pentoxide
EC Number:
215-237-7
EC Name:
Diantimony pentoxide
Cas Number:
1314-60-9
Molecular formula:
O5Sb2
IUPAC Name:
diantimony pentoxide

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd, Margate
- Age at study initiation: approx. 8 to 10 weeks old
- Weight at study initiation: 16 to 19 g
- Housing: The animals were group housed during acclimatisation and individually housed from the day prior to dosing (Day -1) in cages that conform with the ' Code of Practice for Housing and Care of Animals Used in Scientific Procedures' (Home Office, London, 1989). Bedding was provided on a weekly basis to each cage by use of clean Aspen wood chips (Datesand Ltd, Manchester).
- Diet (ad libitum): SQC(E) Rat and Mouse Maintenance Diet No 1, from Special Diets Services Ltd, Witham
- Water (ad libitum): Mains water
- Acclimation period: 8 to 11 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 25 °C
- Relative humidity: 40 to 70 %
- Air changes: At least 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12
No further information on the test animals was stated.

Study design: in vivo (LLNA)

Vehicle:
propylene glycol
Concentration:
Preliminary screening test: 10 % w/v concentration
Main test: 2.5 % w/v concentration, 5 % w/v concentration, 10 % w/v concentration
No. of animals per dose:
Preliminary screening test: one mouse
Main study: 5 female mice
Details on study design:
RANGE FINDING TESTS:
As no toxicological information was available regarding the systemic toxicity/irritancy potential of the test article, a preliminary screening test was performed with one mouse. The mouse was treated by daily application of 25 µL of the test article at the maximum suitable concentration (10 % 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 five days from the initiation of treatment for any signs of toxicity or irritation during this period; none were recorded. The body weight was recorded on the day prior to dosing (Day -1). the aimal was killed by exposure to a rising concentration of carbon dioxide at the end of the observation period.
- Compound solubility: The vehicle for the test article was propylene glycol.
- Irritation/ Systemic toxicity: The preliminary screening test animal survived treatment with EFR-6N and showed no signs of systemic toxicity or local irritation.
Based in this information the dose levels for the main test were selected.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay (Individual method)
- Criteria used to consider a positive response: The scintillation counter printed data including the DPM value (disintegrations per minute during a ten minute period) for each individual animal. The DPM value was tranformed into a mean DPM value for each group. The mean DPM value for each test group was divided by the mean DPM for the control group to provide the Stimulation Index (SI) value for each test group.
Criteria for assessment of test results:
- The test result is not valid for those groups producing an SI value of 3.0 or more when the sites of application have shown excessive irritation and for those groups that have shown indications of systemic toxicosis.
- The test article is regarded as a sensitiser when the maximum value of the SI is 3.0 or above and there is evidence of direct dose response.
- The test article is classified as a non-sensitiser when the maximum value of the SI is less than 3.0 (This result is unchanged by observations of irritation at sites of application of the test formulation).

TREATMENT PREPARATION AND ADMINISTRATION:
- Formulation of test article: Doses are selected fromt eh concentration series 100 %, 50 %, 25 %, 10%, 5 %, 2.5%, 1.0%, 0.5% ect. Three consecutive concentrations were selected on the basis of the prelininary screening test so that the highest concentration maximised expousre whiles avoiding systemic toxicity and excessive local irritation. Formulations were freshly prepared as required, using propylene glycol, on Days 1, 2 and 3. The formaultaions were stored at room temperature in sealed, air-tight containers prior to dosing. The formulations wre mixed by multiple inversion of the container prior to administration to ensure homogeneity.
- 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 appropriate test or control formulation (0.025 mL/pinna) dispensed from an automatic micro pipette.
- Treatment regimen: The five groups of five females mice were subjected to application of the vehicle control, 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 under an infra-red heat lamp. This was intended to cause dilation of 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 salin incorporating 20 µCi of 3HTdR into a tail vein of each mouse by slow bolus injection.
Approximately five hours after intravenous injection of the 3HTdR, all mice were killed by exposure to a rising concentration of carbon dioxide. Killing was organised to minimise the interval between death and the recovery of the auricular lymph nodes to no more than fifteen minutes.
The auricular lymph nodeswere located and removed using curved end forceps. Any connective tissue was removed from the capsule of the nodes. The auricular lymph nodes of each mouse were placed into individual code-labelled 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 balde. The rsultant liquor was transferred into code-identified conical tubes. The petri dishes were rinsed with an additional 5 mL phosphate buffered saline and the second liqour was added to the first liquor. At each transfer debris, such as connective tissue or fragments of capsule, was retained in the petri dish wherever possible.
After 5 minutes the pooled liquor was filtered into a second concial tube by transferring the liquor into a 10 mL syringe and passing it through a 200 µm mesh stainless 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 lquor 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: The vials, including background samples (one sample of ca 10 ml of scintillation fluid and one sample of 1 mL of 5% w/v aqueous trichloroacetic acid and ca 10 ml scintillation fluid), were submitted for liquid scintillation counting for 10 minutes, using a 3H quench curve. incorporation of 3HTdR is measured by β-scintillation counting as disintegrations per minute (DPM) overa 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)
Statistics:
The proliferation response of lymph node cells in each animal was expressed as the number of radioactive disintegrations per minute (DPM).
The data were analysed using one-way analysis of variance (ANOVA). Levene's test for equality of variances among the groups was performed. Where this showed no evidence of heterogeneity (P≥ 0.01), pairwise comparisons with vehicle control were made using Dunnett's test. A linear contrast was used to determine whether there was a relationship between dose and response. A significant trend (P< 0.05) was only reported where none of the pariwise comparisons was significant.
All tests were performed with one-sided risk for increased incidence with increasing dose.

Results and discussion

Positive control results:
The stimulation Index for the positive control goup was 22.74.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
Test results are expressed in terms of Stimulation Indices; the ratio of the mean scintillation count per animal obtained from a test group relative to the corresponding mean scintillation count from controls. The stimulation Indices for the groups treated with EFR-6N (Diantimony pentoxide) were as follows: 2.5 % (w/v) concentration: 1.79 5 % (w/v)concentration: 1.31 10 % (w/v) concentration: 1.12
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: There were no significant increases in DPM between the test groups and vehicel control and no significant increasing dose response across the groups (P≥ 0.05 for all tests). The results are from the individual animals.

Any other information on results incl. tables

Mortality:

All animals surivived treatment with EFR-6N (Diantimony pentoxide).

Clincial signs:

There were no clinical signs indicative of a systemic effect or treatment among mice treated with the vehicle or with 2.5, 5, or 10 % w/v formulations of EFR-6N (Diantimony pentoxide).

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

The heads and necks of positive control animals treated with α-Hexylcinnamaldehyde (25 % in acetone/olive oil in a ratio of 4:1 v/v) showed reddening after dosing on Day 2.

body weights:

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

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
The Local lymph Node Assay demonstrated that EFR-6N (Diantimony pentoxide) does not have a potential to cause skin sensitisation.
According to the criteria specified by Directive 67/548/EEC and subsequent regulations, the test item is not a skin sensitiser.
According to the EC Regulation No. 1272/2008 and subsequent regulations, the test item is not a skin sensitiser.