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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:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2010-07-22
Deviations:
yes
Remarks:
test modified according to Ehling.
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 (i.e. sensitising properties) and ear weight (i.e. irritating properties) were calculated by dividing the average values per group of the test item treated animals by the vehicle treated ones. Values above 1.4 (lymph node 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
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
N-[2-(4-oxo-4H-3,1-benzoxazin-2-yl)phenyl]naphthalene-2-sulphonamide
EC Number:
233-360-4
EC Name:
N-[2-(4-oxo-4H-3,1-benzoxazin-2-yl)phenyl]naphthalene-2-sulphonamide
Cas Number:
10128-55-9
Molecular formula:
C24H16N2O4S
IUPAC Name:
N-[2-(4-oxo-4H-3,1-benzoxazin-2-yl)phenyl]naphthalene-2-sulphonamide
Test material form:
solid: particulate/powder
Details on test material:
- State of aggregation: solid white
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, store in a tightly closed original container and in a cool, dry and well-ventilated place. Protected from light.

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 Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approx. 8 - 12 weeks
- Weight at study initiation: weight variation < 20 % of mean
- Housing: kept singly in MAKROLON cages (type II) with a basal surface of approx. 360 cm² and a height of approx. 14 cm; bedding material: granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany); animals are not group-housed to prevent contact of the application sites.
- Diet (ad libitum): ssniff® R/M-H V1534 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water (ad libitum): drinking water
- Acclimation period: at least 5 adaptation days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 °C ± 3 °C (maximum range)
- Relative humidity: 55 % ± 10 % (maximum range)
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
10 %, 25 % and 50 % (w/w) of the test item
No. of animals per dose:
6 females
Details on study design:
RANGE FINDING TEST
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. Doses were selected from the concentration series 100%, 50%, 25%, 10%, 5%, 2.5%, 1%, 0.5% etc.
A 50% suspension was the highest feasible concentration of N-[2-(4-oxo-4H-3,1-benzoxazin-2-yl)phenyl]naphthalene-2-sulphonamide in N,N-dimethylformamide.

MAIN STUDY
The experimental schedule of the assay was as follows:
Day 1:
The weight of each animal was individually identified. The weights and any clinical observation were 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 substances, the vehicle alone, or the positive control (as appropriate), to the dorsum of each ear.
Days 2 and 3:
The application procedure carried out on day 1 was repeated.
Day 4 (24 h after the last application):
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. 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:
- Clinical signs: animals were observed once daily for any clinical signs of local systemic irritation at the application site or of systemic toxicity. In addition, animals were checked regularly throughout the working day and on the weekend.
- 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, however, an increase in lymph node weight is an indication for possible irritating properties not sensitising properties.
In addition, the average ear weights per group and the average ear thickness per group were compared to the vehicle control group as an indication for possible irritaitng properties. 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 and lymph node weight (statistically significant at p ≤ 0.01). Therefore, the study
can be regarded as valid.
positive control: SI: 1.564 (lymph node weight); SI: 1.106 (ear thickness)
positive control (vehicle): SI: 1.000 (lymph node weight); SI: 1.000 (ear thickness)

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1.413
Test group / Remarks:
10 % test item
Remarks on result:
other: lymph node cell count
Key result
Parameter:
SI
Value:
1.021
Test group / Remarks:
10 % test item
Remarks on result:
other: ear weight
Key result
Parameter:
SI
Value:
2.098
Test group / Remarks:
25 % test item
Remarks on result:
other: lymph node cell count
Key result
Parameter:
SI
Value:
1.117
Test group / Remarks:
25 % test item
Remarks on result:
other: ear weight
Key result
Parameter:
SI
Value:
1.754
Test group / Remarks:
50 % test item
Remarks on result:
other: lymph node cell count
Key result
Parameter:
SI
Value:
1.124
Test group / Remarks:
50 % test item
Remarks on result:
other: ear weight
Parameter:
SI
Value:
1
Test group / Remarks:
negative control group
Remarks on result:
other: lymph node cell count
Parameter:
SI
Value:
1
Test group / Remarks:
negative control
Remarks on result:
other: eat weight
Parameter:
SI
Value:
1.91
Test group / Remarks:
positive control group
Remarks on result:
other: lymph node cell count
Parameter:
SI
Value:
1.184
Test group / Remarks:
positive control group
Remarks on result:
other: ear weight
Parameter:
SI
Value:
1
Test group / Remarks:
positive control goup vehicle
Remarks on result:
other: lymph node cell count
Parameter:
SI
Value:
1
Test group / Remarks:
positive control group vehicle
Remarks on result:
other: 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
In the main study treatment with N-[2-(4-oxo-4H-3,1-benzoxazin-2-yl)phenyl]naphthalene-2-sulphonamide at concentrations of 10 %, 25 % or 50 % revealed increased values for the lymph node cell count (statistical significantly at 25% and 50% (p ≤ 0.01)). The stimulation index of the lymph node cell count of 1.4 was exceeded markedly at all tested concentrations.
In addition, increases were noted for lymph node weights for all concentrations. At the 25% and the 50% concentrations the stimulation indices of ear weight exceeded the threshold level of 1.1, the test item was considered to have irritating properties in this concentration range in this test system. However, as the 10% concentration did not exceed the threshold level of 1.1 the test item possesses sensitising potential at this concentration.

STIMULATION INDEX RESULTS OF LYMPH NODE WEIGHT AND EAR THICKNESS
10 % w/w: SI: 1.106 (lymph node weight); SI: 1.000 (ear thickness)
25 % w/w: SI: 1.340 (lymph node weight); SI: 1.050 (ear thickness)
50 % w/w: SI: 1.319 (lymph node weight); SI: 1.054 (ear thickness)
negative control: SI: 1.000 (lymph node weight); SI: 1.000 (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:
N-[2-(4-oxo-4H-3,1-benzoxazin-2-yl)phenyl]naphthalene-2-sulphonamide was tested with a modified LLNA test according to Ehling et al. (2005) in concentrations of 10, 25 and 50 % to determine the sensitising potential of the substance. During the assay the proliferation of the lymphocytes are measured using the parameters lymph node cell count and lymph node weight, whereas the former indicates a sensitizing property of the substance, if a stimulation index of 1.4 or greater is calculated. The latter parameter also provides information on sensitising properties, however, a statistical significant increase in lymph node weight is an indication of possible irritating not sensitizing properties.

In addition to the parameters, as stated above, ear weight and ear thickness are also recorded during testing. These parameters measure the irritating potential of a substance. If the ear weight results in a stimulation index of 1.1 or greater the substance is considered to be irritating to the skin.

Considering the results of the current study, all tested concentrations gave a stimulation index of 1.4 or greater for lymph node cell count, which obviously demonstrates skin sensitising potential of the substance. However, the 25 % and 50 % concentrations of the substance afforded a stimulation index of 1.1 for ear weight, which indicates skin irritating property of the substance within this test system. Furthermore, lymph node weight showed a statistical significant increase for all tested concentrations. Hence, the substance is considered to have skin irritating properties within this test system rather than a skin sensitising potential.

In order to gain more information on a possible skin sensitising property of the substance, a Guinea pig maximization test according to the OECD 406 (1992) or an adequate alternate test system could be conducted to provide more conclusive information.

Nevertheless, the REACH regulation requires, according to Annex VII point 8.3 column 2, the performance of an LLNA study as the first choice method for in vivo sensitisation testing, at the same time suggesting that only in exceptional circumstances another test should be used (e.g. Magnusson and Kligman study (OECD 406)), for animal welfare reasons.

One argument for initiating the GMPT: Several investigations have generated evidence that irritancy increases the induction response in the LLNA and may thereby lead to non-specific proliferation responses (Basketter et al. 2007a,b,c; Montelius et al. 1998; Woolhiser et al. 1998). For example, methyl salicylate and nonanoic acid showed a dose-response relationship and clearly positive results in the LLNA studies by Montelius et al. (1998) and would normally be classified as potential sensitisers according to the current criteria for positive assay results. However, both irritants are well known to be non-sensitisers in older studies but induced false positive results in the LLNA. Hence, N-[2-(4-oxo-4H-3,1-benzoxazin-2-yl)phenyl]naphthalene-2-sulphonamide may induce skin irritation in mice the initiation of an GMPT should be considered.

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