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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:
2007
Report date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Reference substance name:
polyfluorosulphonic acid
IUPAC Name:
polyfluorosulphonic acid
Test material form:
liquid
Details on test material:
- Purity: 35.6% wt

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/JHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague Dawley, Frederick, Maryland, U.S.A.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: ~ 9 weeks old
- Weight at study initiation: Mean weight of 5 mice - Control group: 22.0 g; 5% group: 21.9 g; 25% group: 22.0 g; 50% group: 21.9 g; 75% group: 22.1 g; and Positive control group: 22.0 g
- Housing: All animals were housed in stainless steel, wire-mesh cages suspended above cage boards. During quarantine, animals were housed in pairs. After assignment to groups, and during the dosing and resting phases of the study, animals were housed singly. After final weighing (test day 5) until sacrifice, animals were housed one group per plastic shoebox cage with appropriate bedding.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: A minimum of 6 days
- Indication of any skin lesions: No lesions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26ºC
- Humidity (%): 30-70%
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12-hour light/dark cycle

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
0, 5, 25, 50, and 75%
No. of animals per dose:
5
Details on study design:
Prior to study start, a quantity of the test substance was evaluated for solubility in a particular vehicle. The control and test substance concentrations and method of preparation were based on solubility information. All dose preparations were formulated fresh daily. Dose preparations were not analysed for homogeneity or accuracy of concentration. The dose preparation procedures were believed to provide homogeneous mixtures at the targeted concentrations. In the absence of visible change in colour or physical state, all dose preparations were assumed to be stable throughout the study. All dose preparations applied to the test site were assumed to be available for absorption by the test system unless otherwise indicated in the study records. All calculations and the evaluation of effects were based on the applied dose.
Twenty-five μL of vehicle control, test substance, or positive control were administered topically to the dorsum of each mouse ear for 3 consecutive days (test days 0-2). Test days 3-4 were days of rest followed by intravenous injection of 20 μCi of ³H-thymidine in PBS per mouse on test day 5. Approximately 5 hours after the injection, animals were sacrificed by carbon dioxide asphyxiation, draining auricular lymph nodes were removed, and single cell suspensions were prepared. The single cell suspensions were incubated at 2-8°C overnight. On test day 6, the single cell suspensions were counted on a beta counter and reported as disintegrations per minute (dpm). A stimulation index (SI) was derived for each experimental group by dividing the mean dpm of each experimental group by the mean dpm of the vehicle control group. The decision process in regard to a positive response includes an SI of greater than or equal to 3.0 together with consideration of dose response and, where appropriate, statistical significance.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
For body weight body weight gain, the preliminary tests used wereLevene’s test for homogeneity and the Shapiro-Wilk test for normality. If the preliminary tests were not significant, One-way analysis of variance followed by Dunnett's test was applied. If the preliminary tests were significant, the Kruskal-Wallis test followed by Dunn's test was applied.

For the lymph node dpm data, the preliminary test was a Test for lack of trend. If the preliminary test was not significant, sequential application of the Jonckheere-Terpstra trend test was applied. If the preliminary test was significant, preliminary tests for pairwise comparison were applied. Pairwise comparisons and associated preliminary tests were only conducted if the test for lack of trend was significant. If that was so, Levene’s test for homogeneity and the Shapiro-Wilk test for normality were applied. If these tests did not show significance, One-way analysis of variance followed by Dunnett's test were applied. If Levene’s test for homogeneity and the Shapiro-Wilk test for normality showed significance, the Kruskal-Wallis test followed by Dunn's test were applied.

Significance was judged at p < 0.05 except for dpm data that were judged at p < 0.01. Lymph node dpm data were transformed to Log to obtain normality or homogenous variances.

Results and discussion

Positive control results:
A 25% concentration of the positive control, HCA, produced a dermal sensitization response in mice. The mean dpm and standard deviation for the positive control was 3096.35 ± 1087.38 versus 234.95 ± 120.74 for the vehicle control resulting in a SI for the positive control of 13.18.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
EC3
Remarks on result:
not determinable
Remarks:
SIs of less than 3.0 were observed at all test concentrations
Key result
Parameter:
SI
Value:
1.84
Test group / Remarks:
5%
Key result
Parameter:
SI
Value:
2.04
Test group / Remarks:
25%
Key result
Parameter:
SI
Value:
2.6
Test group / Remarks:
50%
Key result
Parameter:
SI
Value:
2.13
Test group / Remarks:
75%
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA: Statistically significant increases in cell proliferation measurements compared to the vehicle control group(p < 0.01 by the Jonckheere-Terpstra trend test) were observed at the 25, 50, and 75% test concentrations. Mean dpm was 431.35±124.44, 478.15±206.60, 609.75±119.33, and 499.75±212.71 at 5, 25, 50, and 75%, repsectively.

DETAILS ON STIMULATION INDEX CALCULATION: SIs of less than 3.0 were observed at all test concentrations of test substance. SI's were 1.84, 2.04, 2.60, and 2.13 at 5, 25, 50, and 75%, respectively.

EC3 CALCULATION: the EC3 value (the estimated concentration required to induce a threshold positive response, i.e., SI = 3) for the test substance under the conditions of this study was not calculable.

CLINICAL OBSERVATIONS: No clinical signs of toxicity were observed in the study.

BODY WEIGHTS: No statistically significant differences in mean body weights and body weight gains compared to the vehicle control group were observed at any test concentration.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test substance was not a dermal sensitizer in the LLNA test system.
Executive summary:

The objective of this study was to evaluate the potential of the test substance to produce a dermal sensitization response in mice using the local lymph node assay (LLNA) in accordance with OECD Guideline 209. Five groups of 5 female CBA/JHsd mice were dosed for 3 consecutive days with 0 (vehicle control), 5, 25, 50, or 75% test substance on both ears. N,N-dimethylformamide (DMF) was used as the diluting vehicle. One group of 5 female mice was dosed for 3 consecutive days with 25% hexylcinnamaldehyde (HCA) in DMF as a positive control. On test day 5 of the assay, mice received ³H-thymidine by tail vein injection and were sacrificed approximately 5 hours later. The cell proliferation in the draining auricular lymph nodes of the ears from the test substance groups was then evaluated and compared to the vehicle control group.

No statistically significant differences in mean body weights and body weight gains compared to the vehicle control group were observed at any test concentration. No clinical signs of toxicity were observed in the study. Statistically significant increases in cell proliferation measurements compared to the vehicle control group were observed at the 25, 50, and 75% test concentrations. Stimulation indices (SIs) of less than 3.0 were observed at all test concentrations of the test substance. Therefore, the EC3 value (the estimated concentration required to induce a threshold positive response, i.e., SI = 3) for the test substance under the conditions of this study was not calculable. A 25% concentration of the positive control, HCA, produced a dermal sensitization response in mice. Therefore, the LLNA test system was valid for this study. Under the conditions of this study, the test substance did not produce a dermal sensitization response in mice. Based on these data, the test substance is not a dermal sensitizer in mice.