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Administrative data

Description of key information

No skin sensitizing potential of the test material was observed in a Local Lymph Node Assay in mice performed according to OECD TG 429 under GLP regulation. This in vivo test was the only test performed with regard to skin sensitization.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2007-03-19 - 2007-07-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted 24 April 2002
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 7-12 weeks
- Weight at study initiation: 19.5 +/- 1.8 g
- Housing: single (Makrolon Type I, with wire mesh top)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 1 week
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 3°C
- Humidity: 30-70%
- Photoperiod: 12 hrs dark / 12 hrs light
- IN-LIFE DATES: From: 2007-03-21 To: 2007-03-27
Vehicle:
other: Ethanol/water (30/70, v/v)
Remarks:
The vehicle was selected based on pretests: Ethanol/water (30/70, v/v) resulted in the the highest test item concentration which could be achieved technically. This concentration could not be prepared using other vehicles as recommended by OECD TG 429.
Concentration:
12.5, 25, or 50 %
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: up to 50% in Ethanol/water (30/70, v/v)
- Concentrations tested in the pretest: 6.25, 12.5, 25 and 50%
- Irritation: no signs of irritation
- Systemic toxicity: no systemic toxicity
- Ear thickness measurements: no relevant increased ear thickness observed

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay (LLNA)
- Criteria used to consider a positive response: A test item is regarded as a sensitizer in the LLNA if the following criteria are fulfilled:
First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indictaed by the SI (stimulation index)
Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was placed into a volumetric flask glass beaker on a tared balance and the vehicle was quantitatively added. The test item concentrations were prepared serially.
Three groups each of 5 mice were treated with 12.5, 25, or 50 % Dihydroxyacetone by topical application at the dorsum of each ear lobe (left and right) on three consecutive days. A control group of 5 mice was treated with the vehicle only. The application volume (25 µL) was spread over the entire dorsal surface of each ear lobe.

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated in the body weight tables.
The Dunnett-Test (ANOVA) was conducted to assess whether the difference of DPM per animal is statistically significant between test item groups and negative control (vehicle) group. A statistical analysis of the earthickness before and after treatment with the test item was also conducted.
Positive control results:
The validation-(positive control experiment was performed with a-Hexylcinnamaldehyde in acetone:olive oil (4+1) using CBA/CaOIaHsd mice.
Disintegrations per minute (DPM) of 1005.4, 3108.1, and 6130.7 were determined with the positive control at concentrations of 5, 10, and 25 %, respectively.
Key result
Parameter:
SI
Value:
1.04
Variability:
0.5 - 1.3
Test group / Remarks:
Low Dose (12.5%)
Key result
Parameter:
SI
Value:
1.16
Variability:
0.7 - 1.4
Test group / Remarks:
Mid Dose (25%)
Key result
Parameter:
SI
Value:
0.77
Variability:
0.3 - 1.3
Test group / Remarks:
High Dose (50%)
Cellular proliferation data / Observations:
DETAILS ON STIMULATION INDEX CALCULATION
see table

EC3 CALCULATION
All SI values were < 3, i.e. EC3 values could not be calculated.

CLINICAL OBSERVATIONS
No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

BODY WEIGHTS
The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR was within the range commonly recorded for animals of this strain and age.
Interpretation of results:
GHS criteria not met
Conclusions:
The test item is not a skin sensitiser based on the results of this study.
Executive summary:

In order to study a possible skin sensitizing potential of the test material, three groups of five female mice each were treated daily with the test item at concentrations of 12.5%, 25% and 50% (w/v) in ethanol/water (30/70 v/v) by topical application to the dorsum of each ear lobe for three consecutive days. A control group of five female mice was treated with the vehicle only. The vehicle ethanol/water (30/70 v/v) was selected based on results of a pretest as it enables the highest concentration of the dissolved test item.

Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine, 3HTdR). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes were excised and pooled per animal (2 nodes per animal). Single cell suspensions of pooled lymph node cells were prepared, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3HTdR measured in a β-scintillation counter.

All treated animals survived the scheduled study period. Neither clinical signs on the ears of the animals nor systemic findings were observed during the study period. The development of the body weight was inconspicuous.

The results obtained [Stimulation Index (S.I.)] are summarized in the following table.

 Test item concentration  SI value
 12.5%  1.04
 25.0%

 1.16

 50.0%  0.77

All SI values were </= 3, i.e. the test was negative with regard to a skin sensitizing potential.

Based on the results of this study, the test material is considered to be not a skin sensitizer.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The test material must not be classified with regard to skin sensitization.