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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:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
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
Details on test material:
Chemical identity: Chrome antimony titanium buff rutile

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS B.V.,lnc., Postbus 6174, 5960 AD Horst, The Netherlands
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 15-25g
- Housing: single
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
propylene glycol
Concentration:
10, 25, 50%
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: insoluble, max. 50% suspension in propylenglycol
- Irritation: no
- Systemic toxicity: no
- Ear weight and Lymphnode weight determined (S.I. well below 1)


MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: OECD 429, murine local lymph node assay
- Criteria used to consider a positive response: A test item is regarded as a sensitizer in the LLNA if exposure to at least one concentration of
the test item results in an incorporation of 3H thymidine at least 3-fold or greater than that recorded in control mice as indicated by the stimulation index. However, the biological relevance of the obtained stimulation indices is considered in conjunction with the other assessed end points (i.e. cell counts, lymph node weights, ear weights).

TREATMENT PREPARATION AND ADMINISTRATION:
Randomization: Prior to first application, the animals will be distributed to the individual groups, will receive their animal numbers and will be allocated to the respective cages according to the randomization instructions of „Nijenhuis, A. and Wilf, H.S.: Combinatorial Algorithms, Academic Press, New York, San Francisco, London, 1978, pp. 62 - 64".
Route of application: Epicutaneously to the dorsum of both ears
Application volume: 25 μL per ear
Site of application: Dorsal surface of both ears
Frequency of application: 3 consecutive applications (day O - day 2) on the same application site
Treatment of control group 1 analogously to the test groups, but only with the vehicle without test substance.

3H thymidine injection
On study day five (about 66 to 72 hours after the last application of test substance to the ears), 20 μCi 3H thymidine1 in 250 μL sterile saline will be injected intravenously (i.v.) into a tail vein of the mice.

Determination of ear weight: lmmediately after sacrifice, a circular piece of tissue (diameter 0.8 cm) will be punched out of the apical part of each ear of all animals. The weight of the pooled punches will be determined for each animal by using an analytical balance. These measurements serve for detecting a potential inflammatory ear swelling.

Removal and weight determination of the lymph nodes: Subsequently, the auricular lymph nodes (right and left) will be dissected. lmmediately after the removal, the total weight of both lymph nodes of each animal will be determined by using an analytical balance.

Preparation of cell suspension and determination of cell counts: Both lymph nodes (per animal) will be carefully passed through an iron mesh (mesh size 200 μm) into phosphate-buffered physiological saline. lmmediately afterwards, the cell suspension will be further diluted with isotone and measured by a cell counter straight away

Measurement of 3H thymidine incorporation in lymph node cells: The cell suspensions will be washed twice with phosphate buffered saline (PBS) and precipitated with 5% trichloro-acetic acid (TCA). Each precipitate will be transferred to scintillation fluid and incorporation of 3H thymidine will be measured by a ß-scintillation counter
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Cell count, 3H thymidine incorporation, lymph node weight and ear weight: WILCOXON - Test

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
10%
Key result
Parameter:
SI
Value:
1.69
Test group / Remarks:
25%
Key result
Parameter:
SI
Value:
1.2
Test group / Remarks:
50%
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA: see table below

EC3 CALCULATION

CLINICAL OBSERVATIONS: neither systemic toxicity nor skin irritation was not observed at any concentration

BODY WEIGHTS: see table below

Any other information on results incl. tables

   ear weight [mg]  body weight day 5 [g]  lymph node weight [mg]  cell counts [count / LN pair]
 vehicle propylene glycol  30.2  19.4  5.1  9,009,360
 10% in propylene glycol  31.8  19.3  4.7  7,643,520
 25% in propylene glycol  33.3  20.4  6.0  10,070,400
 50% in propylene glycol  33.8  19.3  5.2  9,534,000

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of this murine local lymph node assay the test item is not considered to be a skin sensitizer