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Skin sensitisation

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skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-08-12 until 2015-08-18
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: per guideline study (GLP)

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
These differences of the environmental parameters were considered not to adversely affect the results of integrity of the study.
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
3,9-dimethyl-2,4,8,10-tetraoxa-3,9-diphosphaspiro[5.5]undecane 3,9-dioxide
EC Number:
EC Name:
3,9-dimethyl-2,4,8,10-tetraoxa-3,9-diphosphaspiro[5.5]undecane 3,9-dioxide
Cas Number:
Molecular formula:
Test material form:
solid: particulate/powder
Details on test material:

In vivo test system

Test animals

other: CBA/CaOlaHsd mice
Details on test animals and environmental conditions:
Female, nulliparous, non pregnant
10 weeks old (age-matched, within one week)
20.2-21.6 grams (The weight variation in animals involved in the study did not exceed ± 20 % of the mean weight.)
21 days acclimatization time.
Due to technical reasons, temperature and relative humidity values (18.0-27.5oC and 24-86%, respectively) outside the expected range of 19-25oC and 30-70% were recorded during the study.

Study design: in vivo (LLNA)

other: 1% Pluronic
Based on a preliminary solubility test and OECD 429 (2010), the test item was formulated in 1% aqueous Pluronic® PE9200 (1% Pluronic). Due to the physico-chemical characteristics of the test item, the highest achievable concentration was 50% (w/v).
0, 50, 25, 10,
No. of animals per dose:
Details on study design:
The Preliminary Irritation/Toxicity Test was started according to the Study Plan on
CBA/CaOlaHsd mice using two doses (2 animals/dose) at test item concentrations of 50 and
25% (w/v) in 1% Pluronic. The preliminary experiment was conducted in a similar
experimental manner to the main study, but was terminated on Day 6 and the radioactive
proliferation assay was not performed.
The maximum concentration of test item in an acceptable vehicle (solvent) was established
according to OECD guideline 429. Based on the observation of the solubility test, the
maximum achievable concentration was 50% (w/v).
In the Preliminary Irritation / Toxicity Test, no mortality or signs of systemic toxicity were
observed. No marked body weight loss (>5%) was detected for any experimental animals
(Table 6 of Appendix 4).
Minimal amount of test item precipitate was detected on the ears of the animals in the 50%
(w/v) dose group on Days 1-4. Clinical observations are summarized in Table 8 of
Appendix 4.
Ear thickness of the animals was measured using by a thickness gauge on Days 1, 3 and 6, and
by ear punch weight determination after the euthanasia of the experimental animals on Day 6.
The ear thickness values and the weights of the ear punches (2 per animal) are summarized in
Table 7 of Appendix 4. Increased ear thickness values were detected in some cases; but the
resulted data did not exceed the limit of positivity in any of those cases Slightly increased ear
thickness values were detected in some cases on Days 3 and/or Day 6; but all those results
were well below the limit of excessive local irritation (≥25% increase). The revealing ear punch
weights were in the acceptable range (for more details see Table 7 of Appendix 4). No
erythema was observed for any experimental animals in this preliminary experiment.
The draining auricular lymph nodes of the animals were visually examined: they were
considered to be normal in both dose groups (subjective judgement by analogy with
observations of former experiments).
Based on these results, the 50% (w/v) dose was selected as top dose for the main test.
Treatments in the main assay are shown in Table 2.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
The proliferative response of lymph node cells from the lymph nodes of each individual animal
was measured as radioactive disintegrations per minute (DPM) per animal. The average of the
two measured DPM values of 5% (w/v) TCA solutions was used as background DPM value.
The results were expressed as disintegrations per node (DPN = DPM divided by the number of
lymph nodes) for each animal following the industry standard for data presentation.
The stimulation index (SI = mean DPN of treated group divided by mean DPN of the
appropriate control group) for each treatment group was also calculated. A stimulation index
of 3 or greater is an indication of a positive result.
The use of the individual approach to calculate the SI made the use of a statistical analysis
available. The statistical analysis was performed using the SPSS/PC+ software package. The
heterogeneity of variance between groups was checked by Bartlett's test for the measured
DPM values.
Where no significant heterogeneity was detected, a one-way analysis of variance was carried
out. If the result was positive, then Duncan's Multiple Range test was used to assess the
significance of inter-group differences. Where significant heterogeneity was found, the normal
distribution of data was examined by the Kolmogorow-Smirnow test. In the case of not normal
distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was
applied. If a positive result was detected, the inter-group comparisons were performed using
the Mann-Whitney U-test.

Results and discussion

Positive control results:
The positive control group animals were treated with 25% (w/v) HCA solution in a relevant
vehicle (1% Pluronic) concurrent to the test item groups. The positive control substance was
chosen according to the OECD guideline [1].
No mortality, cutaneous reactions or signs of toxicity were observed in the positive control
group. A significant lymphoproliferative response (stimulation index value of 14.3) was noted
for α-Hexylcinnamaldehyde in this experiment. The results of the positive control group
demonstrated the appropriate performance of the assay.
The observed mean DPN values for the negative and positive control were within the historical
control range. Historical control data for the positive and negative control substances are
shown in Appendix 7.

In vivo (LLNA)

Resultsopen allclose all
Test group / Remarks:
test item concentration of 10%w/v
Remarks on result:
other: significant different versus negative control (SI=1.0)
Test group / Remarks:
test item concentration of 25%w/v
Key result
Test group / Remarks:
test item concentration of 50%w/v
Test group / Remarks:
Positive control HCA
Remarks on result:
other: significant different versus negative control

Any other information on results incl. tables

The test item was a white powder, which was formulated in 1% Pluronic. Since there were no confounding effects of irritation or systemic toxicity at the applied concentrations, the proliferation values obtained are considered to reflect the real potential of the test item to cause lymphoproliferation in the Local Lymph Node Assay. The resulted stimulation indices observed under these exaggerated test conditions was considered to be good evidence that the test substance is not a skin sensitizer (Figure 1). The size of lymph nodes was in good correlation with this conclusion.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
In conclusion, under the conditions of the present assay the test substance was shown to have no sensitization potential (non-sensitizer) in the Local Lymph Node Assay.
The following classification/labelling is triggered: Regulation (EC) No 1272/2008 (CLP) / GHS (rev. 5) 2013: none