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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2012-02-01 to 2012-02-09
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP and guideline compliant. An experimental study was performed with a structural analogous read-across substance. Please refer to IUCLID section 13 for read-across justification.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
, adopted 2012
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
, adopted 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
, adopted 2003
Deviations:
no
GLP compliance:
yes (incl. certificate)
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
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: TOXI-COOP ZRT. H-1103, Budapest, Cserkesz u. 90.
- Age at study initiation: young adult mice, 9-10 weeks (at start of the experiment)
- Weight at study initiation: 16.8 - 21.5 g
- Housing: grouped caging (5 animals/cage)in type II. polypropylene/polycarbonate cages
- Diet: Animals received ssniff® SM R/M-Z+H complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany, ad libitum
- Water: tap water for human supply, ad libitum
- Acclimation period: 7 days (pre-test) and 21 days (main study)

ENVIRONMENTAL CONDITIONS
- Temperature: :22 ± 3 °C
- Humidity: 30 – 70 %
- Photoperiod: 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:
other: 1 % Pluronic®PE 9200
Concentration:
25 %, 10 %, 5 % and 2.5 %
No. of animals per dose:
5 animals per dose
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: Solubility of the test item was evaluated in different solvents recommended by the relevant guideline. Appropriate solubility was achieved with aqueous 1 % Pluronic®PE 9200 (1 % Pluronic) with a maximum concentration of 25 % (w/v). The formulation was a real solution and applicability on the ears of animals was acceptable.

- Irritation: A preliminary irritation/toxicity screen was conducted in a similar experimental manner to the exposure phase of the main assay except there was no assessment of lymph node proliferation and fewer animals were used per dose group. Two groups of 2 CBA/Ca mice were treated with the test item formulations of 25 % or 10 % (w/v) in the vehicle (1 % Pluronic) once daily for 3 consecutive days.
No mortality was observed during the preliminary test.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay (Individual Approach)
- Criteria used to consider a positive response: The test item is considered as a skin sensitizer, if the following criteria are fulfilled:
That exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index.


TREATMENT PREPARATION AND ADMINISTRATION:
Each mouse was topically treated with 25 µL of the appropriate formulations of the test item, of the positive control substance (positive control group) and of the vehicles (negative control groups). The formulations were applied, with a pipette, on the dorsal surface of each ear. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.
On Day 6 each mouse was intravenously injected via the tail vein with 250 µL of sterile PBS containing 20 µCi of
3H-methyl-thymidine using a hypodermic needle with 1 mL sterile syringe. Once injected, the mice were left for 5 hours
(± 30 minutes).
Five hours (± 30 minutes) after intravenous injection the mice were anesthetized by inhalation of Isofluran CP® and sacrificed. A single cell suspension was prepared and incorporated radioactivity was measured accordingly.

Clinical Observation:
During the main test (from Day 1 to Day 6) all animals were observed at least once a day for any clinical signs, including systemic toxicity and local irritation. Irritation was monitored by erythema scoring. Individual records were maintained.

Body Weight:
Individual body weights were recorded on Day 1 (beginning of the test) and on Day 6 (prior to 3HTdR injection) with a precision of ± 0.1 g.

Evaluation of the Results:
DPM was measured for each animal. The results were expressed as DPN (DPM divided by the number of lymph nodes). The mean DPM and DPN values were calculated for each treatment groups.
The stimulation index (SI = the mean DPN of a treated (positive control or test item) group divided by the DPN of the respective negative control group) for each treatment group was calculated. A stimulation index of 3 or greater is an indication of a positive result.

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Use of the individual approach enabled the performance of a statistical analysis of the data. Statistical analysis was performed by SPSS/PC+ (4.0.1) software package. The heterogeneity of variance between groups was checked by Bartlett's test for the measured DPM values corrected with the mean background value. Since no significant heterogeneity was detected, a one-way analysis of variance was carried out. Since the result was positive Duncan's Multiple Range test was used to assess the significance of inter-group differences. Significance of the positive control response was evaluated by T-test versus control. Significance of the dose-response was evaluated by linear regression made with Microsoft Excel Software.

Results and discussion

Positive control results:
The positive control group animals were treated with 25 % (w/v) Hexyl cinnamic aldehyde (HCA) solution concurrent to the test item treated groups. No confounding effects of irritation or systemic toxicity were observed in the positive control group. Significant lymphoproliferative response (SI 3) was noted for HCA with stimulation index value of 11.82. The result of the positive control item demonstrated the appropriate performance of the test in accordance with the Guidelines and confirmed sensitivity and validity of the assay.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: The Stimulation index values per treatment group were as follows: negative control: 1.0 SI positive control: 11.82 SI Test item: 25 %: 0.91 SI 10 %: 0.72 SI 5 %: 0.97 SI 2.5 %: 1.05 SI
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: The mean DPM values per treatment group were as follows: negative control: 818.4.0 DPM positive control: 18398.6 DPM Test item: 25 %: 743.0 DPM 10 %: 588.0 DPM 5 %: 795.0 DPM 2.5 %: 858.6 DPM

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
No study on skin sensitisation with cesium sulphate is available. Consequently, read-across with cesium nitrate was performed. Cesium nitrate tested at 25 %, 10 %, 5% and 2.5 % as a solution in 1 % Pluronic was shown to have no sensitisation potential. Accordingly, cesium sulphate is considered to have no sensitisation potential.
Executive summary:

No study on skin sensitisation with cesium sulphate is available. Consequently, read-across with cesium nitrate was performed.

The aim of this study was to determine the skin sensitisation potential of cesium nitrate in the Local Lymph Node Assay (LLNA) according to the OECD Guideline 429 and EU Method B.42. The individual approach was used in this test whereby the lymph nodes of each animal were evaluated individually (in comparison to a pooled test group approach). The maximum attainable test item concentration, resulting in a homogenous formulation in an appropriate vehicle, was examined.

A preliminary solubility test was performed to select a suitable vehicle. The maximum attainable test concentration based on solubility was 25 % (w/v) in aqueous 1 % Pluronic. No higher test concentration was available in this test.

Based on results of a preliminary irritation/toxicity test 25 % was selected as the highest test concentration in the main test.

In the main test, 35 female CBA/Ca mice were allocated to seven groups of five animals each:

-four groups received cesium nitrate at 25 %, 10 %, 5 % or 2.5 %,

-the negative control group received the vehicle (1 % Pluronic) only,

-the positive control group received 25 % alpha-Hexylcinnamaldehyde

-the negative control group for the positive control group received Acetone: Olive oil 4:1 mixture (v/v/) (AOO) only.

No study on skin sensitisation with cesium sulphate is available. Consequently, read-across with cesium nitrate was performed.

Each substance was applied on the external surface of each ear (25 µL/ear) of the animals for three consecutive days (Day 1, 2 and 3).

There was no treatment on Days 4, 5 and 6. On Day 6, the cell proliferation in the local lymph nodes was measured by determining incorporation of tritiated methyl thymidine (3HTdR) and the obtained values were used to calculate stimulation indices (SI). No mortality was observed during the test. No local effects at the application sites (ears) were observed in any treatment group. Larger lymph nodes than the control were observed in the positive control group only. No statistically significant lymphoproliferation was observed in any group treated with the test item. The obtained SI values for the test item were 0.91, 0.72, 0.97 and 1.05 at concentrations of 25 %, 10 %, 5 % and 2.5 %, respectively. No dose-related response was observed. The positive control item induced the appropriate stimulation (SI = 11.82), thus confirming the validity of the assay. Since the mean SI value was below 3 at the maximum attainable test concentration of 25 % and at concentrations of 10 %, 5 % and 2.5 % and no dose-related response was observed, the test item was considered to be a non-sensitiser in the LLNA. Accordingly, cesium sulphate is considered to have no sensitisation potential.