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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2002
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan UK Ltd, Bicester, Oxon
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 18.9 - 23.0g
- Housing: individually housed in polycarbonate cages with woodflake bedding. Nestlets and untreated wooden blocks for environmental enrichment
- Diet (e.g. ad libitum): standard laboratory rodent diet, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21+/- 3°C
- Humidity (%): 40 - 70%
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12h dark/ 12h light

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
5, 10 and 25%
No. of animals per dose:
4 animals per dose
Details on study design:
MAIN STUDY

ADMINISTRATION OF THE TEST SUBSTANCE
Mice were treated by daily application of 25 uL of the appropriate test concentration for the test substance to the dorsal suface of each ear for 3 consecutive days (Day 1-3). Test substance was applied using an automatic micropipette and spread over surface of the ar using the tip of the pipette. Four further mice were treated with the vehicle in the same way as described above.

ADMINSTRATION OF 3H-METHYL-THYMIDINE (3HTdR)
5 days following the first topical application of the test substance (Day 6) all mice were injected via the tail vein with 250 uL phosphate buffered saline (PBS) containing 3H-methyl Thymidine giving a nominal 20uCi to each mouse.

OBSERVATIONS
Clinical signs: All animal observed daily, ears also examined for signs of irritation
Body weights recorded on Day 1 and prior to termination (Day 6)

TERMINATION
5 hours following the administration of 3HTdR on Day 6 all mice were killed by carbon dioxide asphyxiation and the draining auricular lymph nodes excised and pooled for each experimental group. 1mL of PBS was added to the pooled lymph nodes for each group.

PREPARATION OF SIGNLE CELL SUSPENSIONS
A single cell suspension of lymph node cells (LNC) was prepared by gentle mechanical disaggregation through a stainless stell gauze (200 mesh size). The pooled LNC were then washed by adding 10 mL PBS, pelleted at 190g for 10 minutes and resuspended. The cells were washed twice again and resuspended in 1mL trichloroacetic acid (TCA: 5%) following the final wash.

DETERMINATION OF INCORPORATED 3H-METHYL-THYMIDINE
After overnight incubation with 5% TCA at 4°C, the precipate was recoved by centrifuged and resuspended in 1mL of 5% TCA and transferred to Ultima gold scintillation fluid on Day 7. 3HTdR incorporation was measured by beta-scintillation counting. The proliferative response of LNC was expressed as radioactive disintegrations per minute per lymph node (dpm/node) and as a ratio of 3HTdR incorporation into LNC of test nodes relative to that of the controol nodes (test/control ratio)

INTERPRETATION OF RESULTS
The test substance is regarded as a sensitiser if at least one test concentration results in a 3-fold greater increase in 3HTdR incorporation compared to the control values.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
None

Results and discussion

Positive control results:
Study conducted between 31 October and 12 November 2002.
Substance was positive for skin sensitisation : test control ratio: 10% = 4; 25% = 9.1; 50% = 37.6

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
2.5
Test group / Remarks:
5%
Parameter:
SI
Value:
2.6
Test group / Remarks:
10%
Parameter:
SI
Value:
2.6
Test group / Remarks:
25%
Cellular proliferation data / Observations:
CLINICAL OBSERVATIONS:
There were no deaths or signs of ill health or toxicity observed during this study

BODY WEIGHTS
Loss of body weight was recorded in one control animal. All remaining mice gained weight duing the study

Any other information on results incl. tables

Table 1: Group dpm/node and test/control ratios

 Group  Concentration (% w/v)  dpm No. of lymph nodes  dpm/node  test/control ratio  Result   
 1  Control 1267.7   8  158.5 n/a   n/a
 2  5  3153.3  8  394.2 2.5   -
 3  10 3254.6   8  406.8 2.6   -
 4  25 3318.8   414.9 2.6 

 -

+ = positive

- = negative     

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The substance is not regarded as a potential skin sensitiser