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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
2010-05-19 to 2010-07-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The basis for the read-across concept for this project is the equilibrium between sulfites, hydrogensulfites, and metabisulfites in aqueous solutions depending on pHvalue which is clearly described in published literature and summarised in the following equations:[1],[2]
           SO2+ H2O <->`H2SO3´         H2SO3<->H++ HSO3-<->2H++SO32-    2HSO3-<->H2O +S2O52 -
As the nature of the cation should make no significant difference in this case concerning toxicity and solubility (all compounds are very soluble in water), only the chemical and biological properties of the anion are considered relevant. Based on the described equilibrium correlations, we propose unrestricted read-across between the groups of sulfites, hydrogensulfites and metabisulfites.

Additionally, it is known that sodium dithionite disproportionates in water to form sodium hydrogen sulfite and sodium thiosulfate (equation II) so that this substance can also be added to the read-across concept.[2],[1]It is expected for this case that the substance is not stable enough under physiological conditions to fulfil the requirements of study guidelines and so the products of decomposition have to be considered.
       2 S2O42-+ H2O→2HSO3-+ S2O32 -
 
Not completely included in this read-across concept is the substance class of thiosulfates. Although thiosulfates may also disproportionate in aqueous solution to form polythionic acids and SO2(HSO3-), the required conditions are somewhat different (more acidic) and are therefore not strictly comparable with physiological conditions, except for the case of oral application where read-across should be considered unrestricted due to the strongly acidic conditions in the stomach:
       HS2O3-+ H2S2O3→HS3O3- + SO2 + H2O
Nevertheless, read-across for all other routes (dermal, inhalation) should also be considered.
The proposed read-across concept only applies to toxicological and ecotoxicological/environmental fate endpoints.
[1]Hollemann Wiberg, Lehrbuch der Anorganischen Chemie, 101.Auflage
[2]Handbook of Chemistry and Physics, Ed. Lide, DR, 88thedition, CRC Press

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2002-04-24
Deviations:
yes
Remarks:
modified OECD 429, method according to Ehlings et al. 2005
Principles of method if other than guideline:
The test was performed in accordance with the method according to Ehling et al (2005): An european inter-laboratory validation of alternative endpoints of the murine local lymph node assay: first round, Toxicology 212 (2005) 60-68 and Ehling et al (2005): An european inter-laboratory validation of alternative endpoints of the murine local lymph node assay: 2nd round, Toxicology 212 (2005) 69-79.

Threshold values of the stimulation indices of lymph node cell count and ear weight were calculated by dividing the average values per group of the test item treated animals by the vehicle treated ones. Values above 1.4 (cell count) or 1.1 (ear weight) are considered positive
(these values were fixed empirically during the inter-laboratory validation of this method). In addition, the lymph node weights were determined for concentration related properties.
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2009-11-12
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium sulphite
EC Number:
231-821-4
EC Name:
Sodium sulphite
Cas Number:
7757-83-7
Molecular formula:
NA2SO3
IUPAC Name:
disodium sulfite
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report): Sodium sulfite
- Molecular formula: Na2SO3
- Molecular weight: 126.04 g/mol
- Physical state: Solid, white powder
- Melting point: Decomposition ≥150 °C [MSDS]
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Cool and dry, well ventilated place

In vivo test system

Test animals

Species:
mouse
Strain:
NMRI
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: Approx. 9 weeks
- Weight at study initiation: 25 - 30 g
- Housing: The animals were kept singly in MAKROLON cages (type III) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 15 cm. Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany) was used as bedding material for the cages.
- Diet (ad libitum): Commercial diet ssniff® R/M-H V1534 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water (ad libitum): Tap water
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C +/- 3°C (maximum range)
- Relative humidity (%): 55% +/- 15% (maximum range)
- Air changes: 12 - 18 times per hour
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
other: aqua ad iniectabilia
Concentration:
10 % w/w, 25 % w/w, and 50 % w/w of sodium sulfite
No. of animals per dose:
6 female mice
Details on study design:
RANGE FINDING TESTS:
A preliminary experiment was carried out in 3 animals to examine the irritating potential and handling/application of the test item in order to select the appropriate concentrations. Three concentrations of 10%, 25% and 50% of sodium sulfite in aqua ad iniectabilia, w/w, were examined.
Results:
In a preliminary experiment, concentrations of 10%, 25% and 50%, employing 1 animal per concentration, were examined. No pronounced irritating properties were observed in this preliminary experiment at concentrations of 10%, 25% or 50%, no differences in ear weight and ear thickness were noted.

MAIN STUDY
The test item had to be dissolved in aqua ad iniectabilia (Batch no. 911728; Delta Select, 63303 Dreieich, Germany). Due to the chemical properties of sodium sulfite no suitable homogeneous dosage form could be obtained with less polar, aprotic solvents.

The test item solution was administered to the dorsum of both animal's ears at an application volume of 25 µL/ear.

The experimental schedule of the assay was as follows:
Day 1:
The weight of each animal was individually identified and recorded. In addition, ear swelling measurements were carried out at the helical edge of both ears using an Oditest micrometer.
Open application of 25 µL of the appropriate dilution of the test item, the vehicle alone or the positive control (as appropriate) were administered to the dorsum of each ear.
Days 2 and 3:
The application procedure carried out on day 1 was repeated.
Day 4 (24 hours after the last application the animals were sacrificed under ether anaesthesia by cutting the aorta abdominalis):
Ear swelling measurements (immediately before sacrificing the mice) were carried out at the helical edge of both ears using an Oditest micrometer.
Punch biopsies of 8 mm in diameter of the apical area of both ears were prepared and immediately weighed on an analytical balance.
Lateral pairs of auricular lymph nodes draining the ear tissue were excised, carefully separated from remaining fatty tissue and weighed on an analytical balance immediately following preparation. The lymph nodes were then stored on ice in PBS/0.5% BSA and subjected to the preparation of single cell suspensions by mechanical tissue disaggregation. The cells were counted automatically in a cell counter.

OBSERVATIONS:
The following observations were made during the course of the study:
- Clinical signs: Animals were observed once daily for any clinical signs of local systemic irritation at the application site or of systemic toxicity. Observations were recorded for each individual animal. Cageside observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns. The onset, intensity and duration of any signs observed were recorded.
In addition, animals were checked regularly throughout the working day from 7:30 a.m. to 4:30 p.m. On Saturdays and Sundays animals were checked regularly from 8:00 a.m. to 12:00 noon with a final check performed at approximately 4:00 p.m., if applicable.
- Body weight: The weight of each mouse was recorded at the time of allocation of animals to groups (test day 1) and at the time of necropsy (test day 4).

ANALYSIS OF RESULTS:
The so-called stimulation (or LLN-) indices to determine the sensitising potential were calculated by dividing the average absolute lymph node weight or lymph node cell counts per group of the test item treated animals by the vehicle treated ones.
Thus, in case of no stimulating effect the index for the lymph node cell count is always below 1.4 (cut-off value). An index above 1.4 is considered positive.
For lymph node weight significance at p ≤ 0.01 is considered positive (U-test according to MANN and WHITNEY). A possible concentration-response-relationship for the lymph node weight in order to determine a possible sensitising potential was examined by linear regression analysis employing PEARSON's correlation coefficient. Outliers were determined according to the NALIMOV test.
In addition, the acute inflammatory skin reaction (irritating potential) was measured by ear weight determination of circular biopsies of the ears and ear thickness measurements on test day 1 and test day 4 to identify skin irritation properties of the test item employing the U-test according to MANN and WHITNEY by comparing the test groups to the vehicle control.
The stimulation indices were calculated by dividing the average ear weight and average ear thickness on test day 4 per group of the test item treated animals by the vehicle treated ones. The cut-off threshold value for ear weight was set at 1.1.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Please refer to "Details on study design".

Results and discussion

Positive control results:
The positive control group caused the expected increases in lymph node cell count (S.I.: 2.106) (statistically significant at p ≤ 0.01). Therefore, the study is regarded as valid. The stimulation index for lymph node weight was 2.160 and for ear weight 1.040.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
0.825
Test group / Remarks:
10 % w/w test item
Remarks on result:
other: stimulation index of lymph node cell count
Key result
Parameter:
SI
Value:
1.02
Test group / Remarks:
10 % w/w test item
Remarks on result:
other: stimulation index of ear weight
Key result
Parameter:
SI
Value:
1.014
Test group / Remarks:
25 % w/w test item
Remarks on result:
other: stimulation index of lymph node cell count
Key result
Parameter:
SI
Value:
1.093
Test group / Remarks:
25 % w/w test item
Remarks on result:
other: stimulation index of ear weight
Key result
Parameter:
SI
Value:
1.003
Test group / Remarks:
50 % w/w test item
Remarks on result:
other: stimulation index of lymph node cell count
Key result
Parameter:
SI
Value:
1.073
Test group / Remarks:
50 % w/w test item
Remarks on result:
other: stimulation index of ear weight
Cellular proliferation data / Observations:
DETAILS ON STIMULATION INDEX CALCULATION
Threshold values of the stimulation indices of lymph node cell count and ear weight were calculated by dividing the average values per group of the test item treated animals by the vehicle treated ones. Values above 1.4 (cell count) or 1.1 (ear weight) are considered positive (these values were fixed empirically during the inter-laboratory validation of this method).

RESULTS ON SKIN SENSITISATION
Treatment with sodium sulfite at concentrations of 10%, 25% or 50% did not reveal statistical significantly increased values for lymph node cell count, all stimulation indices for the lymph node cell count were beneath the threshold value of 1.4. In addition, the lymph node weight was not increased. Hence, the test item is classified as not sensitising.
The threshold level for the ear weight of 1.1 was not exceeded, i.e. no irritating properties were noted.
(Please refer to "Attached background material" for the raw data on stimulation indices.)

STIMULATION INDEX RESULTS OF LYMPH NODE WEIGHT AND EAR THICKNESS
10 % w/w: SI: 1.029 (lymph node weight); SI: 0.980 (ear thickness)
25 % w/w: SI: 1.257 (lymph node weight); SI: 0.984 (ear thickness)
50 % w/w: SI: 1.314 (lymph node weight); SI: 0.996 (ear thickness)

CLINICAL OBSERVATIONS
No signs of local or systemic intolerance were recorded.

BODY WEIGHTS
The animal body weight was not affected by the treatment.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The substance is not a skin sensitiser.
According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance does not require classification as skin sensitiser.