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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 July 2016 - X January 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
A sensitiser is an agent that is able to cause an allergic response in susceptible individuals, leading to characteristic adverse health effects of allergic contact dermatitis or atopic dermatitis upon subsequent exposures. Skin sensitisation potential is related to its ability to react with proteins to form covalently linked conjugates and the subsequent recognition of these by the immune system. In the vast majority of cases, this is dependent on electrophilic reactivity of the skin sensitiser or a derivative produced (usually by oxidation) in vivo or abiotically. However, the mechanism by which metals induce the innate immune system are not completely understood. Consequently, skin sensitisation should be evaluated on a case-by-case basis depending on the metal and amount of available information.

According to REACH Annex VI, all existing available information should be evaluated prior to testing. Should these data be inadequate for hazard and risk assessment, including classification and labelling, further testing should be carried out in accordance with the requirements of Annex VII (≥1 tpa) to the REACH Regulation (Sections 8.3, 8.3.1 and 8.3.2 in Column 1 of Annex VII). Due to the biological complexity, skin sensitisation has historically been evaluated using the murine Local Lymph Node Assay, which measures the induction of lymphocyte proliferation.

However, the REACH Annex VII information requirements were revised in 2016, to endorse a battery of in vitro test method(s), recognised according to article 13(3) to address molecular interactions with skin proteins, inflammatory responses in keratinocytes and activation of dendritic cells. In vivo skin sensitisation studies initiated before 11 October 2016 and that meet the requirements set out in Article 13(3) and Article 13(4) are considered appropriate to address the standard information requirement. The LLNA (OECD 429) for bismuth silicate was initiated in July 2016 and is considered sufficient to fulfil the REACH Annex VIII information requirement for skin sensitisation.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
Adopted July 22, 2010
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
Council regulations (EC) No. 440/2008 method B.42. (amended in Commission Regulation (EU) No. 640/2012, adopted July 06, 2012).
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: sponsor lot# 231/08/15
- Expiration date of the lot/batch: March 2020
- Purity test date: 21 January 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Composition: Bismuth oxide (Bi2O3) 83.35%; Silicon oxide (SiO) 16.63%.
- Physical characteristics: Powder
- Storage condition of test material: At ambient temperature (10 - 25°), container kept tightly closed and stored in a dry and well-ventilated place.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: In a preliminary solubility assessment, the following vehicles were assessed: acetone / olive oil (4 : 1 v/v), N,N-dimethylformamide, methyl ethyl ketone, dimethyl sulfoxide and propylene glycol. Only a 50% concentration (w/w) of the test item in dimethyl sulfoxide gave a homogenous suspension suitable for application of the test item. It was decided to employ dimethyl sulfoxide as vehicle.
- Final dilution of a dissolved solid, stock liquid or gel: 10%, 25% and 50% Bismuth silicate (w/w)

FORM AS APPLIED IN THE TEST (if different from that of starting material): Suspended fine powder

In vivo test system

Test animals

Species:
mouse
Strain:
NMRI
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: NMRI / Crl:NMRI female mice
- Source: Charles River Deutschland GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Approximately 9 weeks
- Weight at study initiation: 31-40 g
- Housing: Type II Makrolon cages (surface area 360 cm2; height 14 cm)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C +/- 3°C
- Humidity (%): 55% +/- 15% (maximum range)
- Photoperiod (hrs dark / hrs light): 12 hour light:dark schedule with fluorescent lighting (150 Lux)

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Concentration:
- Test substance: Bismuth silicate suspended in dimethyl sulphoxide
- Amount(s) applied (volume or weight with unit): 25 µL of the test item suspension per ear
- Concentration (if solution): The test item was tested at concentrations of 10%, 25% or 50% (w/w)
No. of animals per dose:
6 animals per dose
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: Partially soluble, a 50% suspension was the highest feasible concentration of Bismuth silicate in dimethyl sulfoxide. Three concentrations of 10%, 25% and 50% suspended in dimethyl sulfoxide were examined.
- Irritation: In a preliminary experiment, concentrations of 10%, 25% and 50% of Bismuth silicate, employing 1 animal per concentration, were examined. No irritating properties were observed in this preliminary experiment,
- Systemic toxicity: No signs of local or systemic intolerance were recorded
- Ear thickness measurements: No differences in ear weight and ear thickness were noted
- Erythema scores: No erythema was reported

ANIMAL ASSIGNMENT AND TREATMENT
- Animal assignment: Six (6) groups of 6 female animals each were examined. At the start of the experiment the mice were weighed and assigned to each of the 6 groups by a randomisation program (block size n = 6).

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 µL of the test item suspension per ear
- Concentration (if solution): The test item was tested at concentrations of 10%, 25% or 50% (w/w)

NEGATIVE CONTROL
- Negative control: Dimethyl sulphoxide
- Source and lot/batch number: Sigma-Aldrich Chemie GmbH batch no# SHBG8313V
- Amount(s) applied (volume or weight): 25 µL/ear

POSITIVE CONTROL
- Positive control: α-Hexyl cinnamic aldehyde
- Source and lot/batch number: Sigma-Aldrich Chemie GmbH batch no# MKBJ8846V
- Amount(s) applied (volume or weight): 25 µL/ear
- Concentration (if solution): 20% (v/v)

MAIN STUDY
- Name of test method: Local Lymph Node Assay (LLNA)
In the absence of radioactive labelling to measure cell proliferation, the OECD 429 guideline permits the assessment of proliferation via alternative methods, such as the lymph node counts and weights validated by European inter-laboratory testing, (Ehling et al.,2005a; 2005b). In addition, the acute inflammatory skin reaction is 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 (Wohr and Ahr, 2005). 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): Ear swelling measurements (immediately before sacrificing the mice) were carried out at the helical edge of both ears using an Oditest micrometer. The animals were euthanized by carbon dioxide (CO2) inhalation and laparotomised. 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
- Clinical signs: Animals were observed once daily for any clinical signs of local systemic irritation at the application site or of systemic toxicity. Cage-side 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.
- 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).

ASSESSMENT CRITERIA
- Criteria used to consider a positive response: Stimulation indices were calculated for the lymph node cell count, lymph node weight, ear weight and ear thickness 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 to identify sensitisation) or 1.1 (ear weight to identify irritation) are considered positive (these values were fixed empirically during the interlaboratory validation of this method (Ehling et al. 2005a and 2005b)).

REFERENCES
- Ehling, G., M. Hecht, A. Heusener, J. Huesler, A. O. Gamer, H. van Loveren, T. Maurer, K. Riecke, L. Ullmann, P. Ulrich, R. Vandebriel, H.-W. Vohr: An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay: First round; Toxicology 212, 60-68 (2005a);
- Ehling, G., M. Hecht, A. Heusener, J. Huesler, A. O. Gamer, H. van Loveren, T. Maurer, K. Riecke, L. Ullmann, P. Ulrich, R. Vandebriel, H.-W. Vohr: An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay: 2nd round; Toxicology 212, 69-79 (2005b).
- Vohr, H.-W. and Ahr, H.-J.: The local lymph node assay too sensitive? Arch. Toxicol. 79: 721-728 (2005)
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
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 was examined by linear regression analysis employing PEARSON's correlation coefficient. U-test was performed for cell count, too. Outliers were determined according to the Nalimov test.

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 ≤ 0.05 or at p ≤ 0.01). Therefore, the study can be regarded as valid.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
1.047
Test group / Remarks:
10% test item
Remarks on result:
other: Lymph node cell counts
Parameter:
SI
Value:
1.413
Test group / Remarks:
10% tet item
Remarks on result:
other: Lymph node weight
Parameter:
SI
Value:
0.823
Test group / Remarks:
25% test item
Remarks on result:
other: Lymph node cell count
Parameter:
SI
Value:
1.196
Test group / Remarks:
25% test item
Remarks on result:
other: Lymph node weight
Parameter:
SI
Value:
1.007
Test group / Remarks:
50% test item
Remarks on result:
other: Lymph node cell count
Parameter:
SI
Value:
1.283
Test group / Remarks:
50% test item
Remarks on result:
other: Lymph node weight
Parameter:
SI
Value:
1.995
Test group / Remarks:
Positive control
Remarks on result:
other: Lymph node cell count
Parameter:
SI
Value:
1.674
Test group / Remarks:
Positive control
Remarks on result:
other: Lymph node weight
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
Treatment with Bismuth silicate at concentrations of 10%, 25% or 50% did not reveal any statistical significantly increased values for the lymph node cell count. The stimulation indices of the lymph node cell count did not exceed the threshold level of 1.4. The threshold level for the ear weight of 1.1 was not exceeded and no increase of ear thickness was observed, i.e. no irritating properties were noted.

DETAILS ON STIMULATION INDEX CALCULATION
The so-called stimulation (or LLN-) indices to determine the sensitising potential (this value was fixed empirically during the interlaboratory validation of this method, 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.

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

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

ASSAY VALIDITY
The positive control group caused the expected increases in lymph node cell count and lymph node weight (statistically significant at p ≤ 0.05 or at p ≤ 0.01). Therefore, the study can be regarded as valid.

Any other information on results incl. tables

Main study results: stimulation indices (SI):

 Parameter DMSO  10% Test item  25% Test item  50% Test item Positive control (HCA)  Acetone/oilive oil (4:1)
 Lymph node cell count

 1.000

 1.047  0.823 1.007   1.995* 1.000 
 Lymph node weight  1.000  1.413*  1.196 1.283   1.674** 1.000 
 Ear weight 1.000   1.023  0.972  1.000  1.035 1.000
 Ear thickness, TD4  1.000  1.042  1.000  1.114  1.133 1.000 

* significantly increased relative to controls at p ≤ 0.05

** significantly increased relative to controls at p≤ 0.01

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The use of lymph node counts and weights as a measure of proliferation in the LLNA (OECD 429) has been extensively validated and is an accepted in vivo test method to detect skin sensitisation (Category 1) and/or the absence of effects (not classified under CLP). Whilst, the REACH Annex VII information requirements have been amended to incorporate a battery of in vitro test method(s), initiated prior to 11 October 2016, the LLNA is considered sufficient to fulfil Annex VII and Annex VIII requirements for skin sensitisation potential.

Under the test conditions, the lymph node cell count and ear weight Stimulation Indices (SI) for Bismuth silicate did not exceed the threshold levels of 1.4. and 1.1, respectively. A slight increase in lymph node weight was reported, which was attributed to non-specific cell activation as a result of inflammatory processes in the skin (irritation). At concentrations of 10%, 25% or 50% (w/w) Bismuth silicate did not present any skin sensitising properties in the local lymph node assay.Conducted according to the aforementioned guidelines and GLP, the LLNA passed all validity criteria and was considered to be reliable without restriction (Klimisch 1).
Executive summary:

The LLNA is designed to detect the potential of substances to induce sensitisation as a function of lymphocyte proliferative responses induced in regional lymph nodes (induction phase). In the OECD TG 429 compliant study, three concentrations of Bismuth silicate (10%, 25% and 50%) in dimethyl sulfoxide were tested in six female NMRI mice per group and compared to a vehicle control group. Presenting limited solubility, the highest feasible concentration of Bismuth silicate was 50%. In addition, a positive control group (20% solution (v/v) ofa-hexyl cinnamic aldehyde in acetone / olive oil (4:1, v/v)) and one group with the vehicle of the positive control were employed. The positive control group caused the expected increases in lymph node cell count and lymph node weight (statistically significant at p ≤ 0.05 or at p ≤ 0.01). Therefore, the study can be regarded as valid.

Treatment with Bismuth silicate at concentrations of 10%, 25% or 50% did not reveal any statistical significantly increased values for the lymph node cell count. The stimulation indices of the lymph node cell count did not exceed the threshold level of 1.4. The threshold level for the ear weight of 1.1 was not exceeded and no increase of ear thickness was observed, i.e. no irritating properties were noted. A slight increase in lymph node weight was reported (significant at p ≤ 0.05 for the 25% concentration), which was attributed to non-specific cell activation as a result of inflammatory processes in the skin (irritation). Under the present test conditions, Bismuth silicate at concentrations of 10%, 25% or 50% (w/w) indimethyl sulfoxide did not reveal any sensitising properties in the local lymph node assay.

The LLNA (EU B.42; OECD 429) is an intentionally accepted in vivo test method to detect skin sensitisation (Category 1, 1A or 1B under CLP) and/or absence of effects requiring classification for skin sensitisation (i.e. not classified under CLP), as described in the Annex to the EU Test Methods (TM) Regulation (Council Regulation (EC) No 440/2008). Conducted according to the aforementioned guidelines and GLP, the LLNA passed all validity criteria and was considered to be reliable without restriction (Klimisch 1).