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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:
30 Aug - 12 Sep 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted in 2010
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Slovenska Narodna Akreditacna Sluzba, Bratislava, Slovak Republic
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Velaz Prague, Czech Republic
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 8 - 10 weeks
- Weight at study initiation: range: 18 - 22 g (prior to first treatment)
- Housing: 5 animals per cage in polypropylene cages suspended on stainless steel racks, on Lignocel S3/4 bedding (Lufa - ITL GmbH, Germany)
- Diet: ssniff (Ssniff Spezialdiäten GmbH, Germany), ad libitum
- Water: public tap water, ad libitum; analysis was performed
- Acclimation period: 5 days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 - 60
- Photoperiod (hrs dark / hrs light): 12/12

- IN-LIFE DATES: From: 24 Aug To: 12 Sep 2017

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Remarks:
(AOO)
Concentration:
25, 50 and 100%
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TESTS:
In the pre-screen test, 25 μL of the undiluted test item were applied to the dorsum of both ears of two animals, once a day for 3 consecutive days. Both animals were observed daily for clinical signs. The body weights were recorded prior to dosing and on the day of necropsy. Furthermore, the treated skin area was scored for erythema daily and ear thickness was measured on Day 1 (pre-dose) and Day 3 and 6. No necropsy was performed on the animals.

- Compound solubility: The test item was soluble in the vehicle, for the concentration of 50%, a homogeneous solution was obtained.
- Irritation: No irritation was observed (i.e. an erythema score ≥ 3 or an ear thickness increase ≥ 25%).)
- Systemic toxicity: No signs of toxicity were observed.
- Ear thickness measurements: No relevant increase was induced by treatment.
- Erythema scores: No erythema was observed (scores = 0).

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA test
- Criteria used to consider a positive response: A substance is regarded as sensitizer in the LLNA test if it induces a Stimulation Index (SI) ≥ 3.

TREATMENT PREPARATION AND ADMINISTRATION: Based on the lack of skin irritationsystemic toxicity and changes in the ear thickness in the pre-screen test, the test item was used at 25, 50 and 100% concentration in the main study. 25 μl of each dose formulation were applied to the dorsum of both ears of each animal once a day for 3 consecutive days. On Day 6, 250 µL of phosphate-buffered saline (PBS) containing 2 μCi (7.4 x 104 Bq) of 125 I-iododeoxyuridine and 10^-5 M fluorodeoxyuridine was injected into all test and control mice via the tail vein. 5 h later the local lymph nodes were collected from the sacrificed mice, then pooled and minced. The lymph node cells (LNC) were treated with 5% trichloroacetic acid (TCA) for 18 - 20 h before determination of the amount of 125 I-iododeoxyuridine incorporation (expressed as disintegrations per minute (DPM)/ animal, measured by the automatic Gamma Counter Wizard2 2470 (Perkin Elmer, USA).

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
T-test (MS Excel) for body weight

Results and discussion

Positive control results:
The positive control substance (25% HCA) induced a positive reaction with a SI of 6.79. The control group demonstrates the validity of the study.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Remarks:
mean of 5 animals
Value:
1.76
Test group / Remarks:
25% test group
Key result
Parameter:
SI
Remarks:
mean of 5 animals
Value:
5.43
Test group / Remarks:
50% test group
Key result
Parameter:
SI
Remarks:
mean of 5 animals
Value:
4.77
Test group / Remarks:
100% test group
Key result
Parameter:
EC3
Value:
33.5
Test group / Remarks:
test group
Parameter:
SI
Remarks:
mean of 5 animals
Value:
6.79
Test group / Remarks:
positive control group
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA : An increase of the pooled lymph node weights was observed at all used concentrations, but without being dose dependent. Increased but not dose dependent lymphoproliferation (SI > 3) was observed at the test substance concentrations of 50% (SI =5.43) and 100% (SI = 4.77). At 25% of the test substance, only a slight increase in the lymphoproliferation was observed (SI = 1.76).


DETAILS ON STIMULATION INDEX CALCULATION : DPM = CPM/ absolute detector efficiency

DPM = desintegrations per minute
CPM = counts per minute
absolute detector efficiency (Gamma Counter Wizard2 2470) = 66.73%

SI = pooled DPM test group/ pooled DPM vehicle control group


EC3 CALCULATION : EC3 = c + [(3 - d)/ (b - d)] x (a - c)

a = higher concentration
b = SI of higher concentration
c = lower concentration
d = SI of lower concentration

An EC3 of 33.5% was obtained.


CLINICAL OBSERVATIONS: Neither mortality nor clinical signs were recorded in animals treated at all dose levels investigated.

BODY WEIGHTS: Minor, but not significant, increases in body weight were observed during the study.

Any other information on results incl. tables

Table 1: Mean Stimulation Indices in mice

 

Concentration
[%]

Lymph node weight (of 10 lymph nodes) [g]

DPM

Stimulation index

EC3 [%]

Vehicle control (AOO)

100

0.0347

1423

-

33.5

Test substance

25

0.0450

2503

1.76

50

0.0740

7724

5.43

100

0.0676

6785

3.1

Positive control (HCA)

25

0.0619

9659

6.79

AOO = acetone/olive oil (4:1 v/v)

DPM = desintegrations per minute

HCA = α-Hexylcinnamaldehyde

 

Applicant's summary and conclusion

Interpretation of results:
other: CLP/EU GHS Category 1B (H317) according to Regulation (EC) No 1272/2008
Conclusions:
CLP: Skin Sens. 1B