Registration Dossier

Diss Factsheets

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 July 2016 - 22 September 2016
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 and VIII 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 Uverithe 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:
2016
Report date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
Adopted July 22, 2010
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
Council regulations (EC) No. 440/2008 method B.42. (amended in Commision Regulation (EU) No. 640/2012, published in the Official Journal of the European Union L193, dated July 20, 2012).
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Reference substance name:
antimony trioxide
IUPAC Name:
antimony trioxide
Constituent 2
Chemical structure
Reference substance name:
Titanium dioxide
EC Number:
236-675-5
EC Name:
Titanium dioxide
Cas Number:
13463-67-7
Molecular formula:
O2Ti
IUPAC Name:
Titanium(IV) oxide
Constituent 3
Chemical structure
Reference substance name:
Calcium oxide
EC Number:
215-138-9
EC Name:
Calcium oxide
Cas Number:
1305-78-8
Molecular formula:
CaO
IUPAC Name:
oxocalcium
Constituent 4
Chemical structure
Reference substance name:
Silicon dioxide
EC Number:
231-545-4
EC Name:
Silicon dioxide
Cas Number:
7631-86-9
Molecular formula:
O2Si
IUPAC Name:
dioxosilane
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: sponsor batch# 434/08/15
- Purity test date: 21 January 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Composition: Antimony oxide (Sb2O3) 38.60%; Titanium oxide (TiO) 31.15%; Calcium oxide (CaO) 26.67%; Silicon oxide (SiO2): 4.40%; and Fluorine (F-): 1.53%.
- 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), dimethylformamide, methyl ethyl ketone, dimethyl sulfoxide and propylene glycol. Acetone/olive oil (4:1, v/v) was selected as it provided a suitable suspension of the test item both for administration and adherence to the mouse ear of such high concentrations.
- Final dilution of a dissolved solid, stock liquid or gel: 5, 10 and 25% Uverithe (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 11 weeks
- Weight at study initiation: 27 - 35 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:
acetone/olive oil (4:1 v/v)
Concentration:
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 5%, 10% and 25% (w/w)

NEGATIVE CONTROL
- Negative control: Acetone / Olive oil (4:1 v/v)
- Acetone source and lot/batch number: Sigma-Aldrich Chemie GmbH batch no# STBF1414V
- Olive oil source and lot/batch number: Caeser & Loretz GmbH batch no# 15112703
- 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)
No. of animals per dose:
6 animals per dose
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: An insoluble inorganic compound, a 25% suspension was the highest feasible concentration of Uverithe in the vehicle. Three concentrations of 5%, 10% and 25% suspended in acetone / olive oil (4:1 v/v) were examined.
- Irritation: In a preliminary experiment, concentrations of 5%, 10% and 25% of Uverithe, 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: Five groups of 6 female animals each were examined. At the start of the experiment the mice were weighed and assigned to each of the 5 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 5%, 10% and 25% (w/w)

NEGATIVE CONTROL
- Negative control: Acetone / Olive oil (4:1 v/v)
- Acetone source and lot/batch number: Sigma-Aldrich Chemie GmbH batch no# STBF1414V
- Olive oil source and lot/batch number: Caeser & Loretz GmbH batch no# 15112703
- 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, 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)
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
Remarks:
Lymph node cell count
Value:
0.9
Test group / Remarks:
5% concentration
Parameter:
SI
Remarks:
Lymph node cell count
Value:
1.227
Test group / Remarks:
10% concentration
Key result
Parameter:
SI
Remarks:
Lymph node cell count
Value:
1.217
Test group / Remarks:
25% concentration
Parameter:
SI
Remarks:
Ear weight
Value:
1.046
Test group / Remarks:
5% concentration
Parameter:
SI
Remarks:
Ear weight
Value:
1.029
Test group / Remarks:
10% concentration
Parameter:
SI
Remarks:
Ear weight
Value:
1.057
Test group / Remarks:
25% concentration
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
Treatment with Uverithe at concentrations of 5%, 10% or 25% 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. Hence, the test item is classified as not skin sensitising. 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: Simulation Indices (SI):

Parameter   Negative control  5% Test Item  10% Test Item  25% Test Item  Positive control
 Lymph node cell count  1.000  0.900  1.227  1.217   1.834*
 Lymph node weight  1.000  1.184  1.132  1.342*   1.658*
 Ear weight  1.000  1.046  1.029  1.057  1.040
 Ear thickness, TD4  1.000  1.036  1.071  1.089  1.170

* Significantly different from control 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 uverithe did not exceed the threshold levels of 1.4. and 1.1, respectively. At concentrations of 5%, 10% and 25% (w/w) Uverithe 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 Uverithe (5%, 10% and 25% in acetone / olive oil (4:1, v/v) were tested in six female NMRI mice per group and compared to a vehicle control group. Insoluble in all solvents tested, the highest feasible concentration of Uverithe was a 25% suspension. 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 Uverithe at concentrations of 5%, 10% and 25% 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. No signs of local or systemic toxicity were reported, and the animal body weight was not affected by treatment. Under the present test conditions,Uveritheat concentrations of 5%, 10% or 25% (w/w) 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).