[No public or meaningful name is available]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Title:

Unnamed

Year:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

24. April 2002

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:

-

EC Number:

458-880-0

EC Name:

-

Cas Number:

85209-93-4

Molecular formula:

Hill formula: C29 H42 O4 P Li

IUPAC Name:

2,4,8,10-tetra(tert-butyl)-6-hydroxy-12H-dibenzo- [d,g][1,3,2]dioxaphosphocin 6-oxide, lithium salt

Test material form:

solid: particulate/powder

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Mouse (healthy females only), strain: CBA/Ca with appropriate range of bodyweight at study start.
- Source: Harlan UK.
- Age at treatment start (1st induction): Approx. eight to twelve weeks.
- Weight at treatment start (1st induction): Minimum 18.4 g, maximum 22.2 g.
- Housing: Individual housing in polycarbonate cages inside a barriered rodent facility.
- Bedding material: Woodflake bedding.
- Cage enrichment: Nestlets and untreated wood blocks
- Diet (ad libitum): Standard rodent diet (Special Diet Services RM1 (E) SQC)
- Water (ad libitum): Tap water
- Acclimation period: At least 7 days before treatment start under laboratory conditions.

Analysis of the batch of diet used and water did not provide evidence of contamination that might have prejudiced the study.

ENVIRONMENTAL CONDITIONS

Air conditioned room kept at positve pressure without re-circulation of the filtered fresh air supplied to the room.
Controlled environment, environmental conditions were set at:
- Temperature (°C): 21 ± 2°C
- Relative Humidity (%): 40 to 70%
- Photoperiod (artificial lighting): 12 hrs day / 12 hrs night
There was no mentioning of any deviations from these ranges, which compromised the integrity or validity of the study.

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

Main Study:
Induction on Days 1, 2 and 3 at the following concentrations (v/v):
0% (vehicle control, 4 females), 25% (4 females), 50% (4 females), undiluted test material (4 females).

No. of animals per dose:

Main Study: 4 female animals per dose

Details on study design:

The maximum practical concentration for pinna dosing wass 25 % w/v in acetone:olive oil (4:1) v/v.

Treatment Preparation and Administration:

On three consecutive days, groups of 4 female mice were treated by topical application to the entire dorsal surface of both ears with 25 μL/ear at the following concentrations (v/v) of test material in the vehicle:

Group 1 (Vehicle Control): 0%,
Group 2 (Low Dose): 5%,
Group 3 (Mid Dose): 10%,
Group 4 (High Dose): 25% test material

The test substance preparations foreseen for treatment of Groups 2, 3 and 4 were prepared on the day of dosing at the required concentrations.

Observations, Measurements and Endpoints (Pooled treatment group approach):

All animals were checked daily for signs of ill health or toxicity. The ears were also examined for signs of irritation. In addition, bodyweights were recorded on arrival (not reported), Days 1 (prior to treatment) and 6 (three days after the third induction administration). On Day 6, all animals were injected into the tail vein 3H-methyl thymidine diluted in sterile phosphate buffered saline at a nominal dose of 20 µCi per mouse, in order to measure lymphocyte proliferation by radioactive labelling. Five hours afterwards the draining (auricular) lymph nodes were excised and pooled for each experimental group. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed on Day 7. Radioactivity was expressed as the number of radioactive disintegrations per minute (dpm). The ratio of the proliferation (reflected by the magnitude of measured dpm/node) in treated groups to that in the vehicle control group, termed the stimulation index (SI), was subsequently calculated for each group.

Criteria Used to Consider a Positive Response:

The test substance is regarded as a sensitizer if at least one concentration of the test substance produces a stimulation index (SI) ≥ 3.

Positive control substance(s):

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

Results and discussion

Positive control results:

A stimulation index (SI) of 5.2, 9.1 and 11.4 was attained in a contemporaneous positive control assay with the same strain of mice (CBA/Ca) in response to 10, 25 and 50 % v/v hexyl cinnamic aldehyde in acetone:olive (4:1 v/v), thus demonstrating the reliability and sensitivity of this test system and assay to detect skin sensitization potential in this laboratory.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

2.3

Test group / Remarks:

5 %

Results 2

Key result

Parameter:

SI

Value:

1.5

Test group / Remarks:

10 %

Results 3

Key result

Parameter:

SI

Value:

1.5

Test group / Remarks:

25 %

Any other information on results incl. tables

Mortality / clinical signs: There were no deaths, no signs of ill health or toxicity and no signs of local irritation over the treated area.

Greasy fur was noted for all control and test animals post-dose from Day 1. This finding had resolved completely by Day 5 in all animals.

White test substance staining was noted in all animals in Groups 3 and 4 shortly after dosing from Day 1 or 2 and had resolved by Day 4. In addition, white test substance staining was also noted in all animals in Group 2 on Day 3 only.

Body weight: A loss in body weight was recorded for two females in Group 2, one female in Group 3 and two females in Group 4 during the study. All remaining animals gained weight during the study.

Applicant's summary and conclusion

Interpretation of results:

other: not sensitising according to Regulation (EC) 1272/2008.