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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:
2017-02-22 to 2017-03-06
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 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)
Deviations:
no
Remarks:
No deviation occurred during the study
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA): BrdU-ELISA

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: Not specified

In vivo test system

Test animals

Species:
mouse
Strain:
CBA:JN
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 7 to 8 weeks old
- Weight at study initiation: 19 to 21 grams
- Housing: Up to 5 during acclimatisation; 1/cage during the study; Polysulphone solid bottomed cages measuring 35.5 × 23.5 × 19 cm with nesting material
- Diet (e.g. ad libitum): 4 RF 21 (Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI) Italy) ad libitum
- Water (e.g. ad libitum): drinking water supplied to each cage via a water bottle ad libitum
- Acclimation period: At least 5 days
- Indication of any skin lesions: Healthy animalswithout observable skin lesions were chosen

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C ± 2°C
- Humidity (%): 55% ± 15%
- Air changes (per hr): Approximately 15 to 20 air changes per hour
- Photoperiod (hrs dark / hrs light): Artificial (fluorescent tubes), daily light/dark cycle of 12/12 hours

- IN-LIFE DATES: From: 2017-02-16 To: 2017-03-06

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
The concentrations tested in the preliminary test were 25, 10, 5, 2.5 and 1 % (w/w).
The concentrations used in the main assay were 25, 10 and 5 % (w/w). Concentrations were calculated considering the test item as supplied. No correction factor was applied.
No. of animals per dose:
7/concentration - Main Assay
Details on study design:
PRE-SCREEN TESTS:
Preliminary Test: A preliminary test was carried out to select three concentrations to be used in a main assay,according to the criteria described in the relevant guideline for this test. A main assay wasthen carried out to fully evaluate lymph node cell reaction. In the preliminay test, the animals were treated for three consecutive days (Days 1, 2, 3) with the vehicle or test item formulations. A dose volume of 25 µL/ear/day of the appropriate concentration was applied to the dorsal surface of each ear (50 µL/animal/day).

Observations:
- Mortality and morbidity: Throughout the study, all animals were checked twice daily
- Clinical signs: Day 1: before and 1 hour after dosing; Day 2 to 6: daily (approximately 1 hour after daily dosing, when applicable)
- Irritation: The treated sites of all animals were examined daily (once before first dosing, before dosingon Days 2 and 3 and daily thereafter).Irritation to the skin was assigned a numerical value according to the Table 1.
- Ear thickness measurements: It was measured by a suitable micrometer on Day 1 (before dosing), on Day 3 (before dosing) and on Day 6.
- Erythema scores: The treated sites of all animals were examined daily (once before first dosing, before dosingon Days 2 and 3 and daily thereafter).Irritation to the skin was assigned a numerical value according to the Table 1.
- Body weight: The animals were weighed at allocation (Day 1) and on sacrifice (Day 6).

Termination: The animals were sacrificed on Day 6 by carbon dioxide narcosis. After sacrifice, regularly shaped biopsies were obtained from both ears and weighed together.No necropsy was performed on the animals.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA: BrdU-ELISA method
- Criteria used to consider a positive response: The test item is considered to induce sensitisation when the SI for any single treatmentdose group is≥1.6. It is not required that an increased response is observed at increasingdose levels, but dose-related activity and/or statistical significance may be taken as furtherevidence of a sensitisation effect (i.e.in case of borderline results with 1.6 ≤ SI ≤ 1.9).

TREATMENT PREPARATION AND ADMINISTRATION:
Main Test: Animals were treated for three consecutive days (Days 1, 2, 3) with the vehicle, test or control item formulations. A dose volume of 25μL/ear/day of each selected concentration and controls was applied tothe dorsal surface of each ear (50μL/animal/day), using a micropipette. Animals were treated intraperitoneally, on Day 5 (once only), with 0.5 mL/animal of asolution of BrdU at a concentration of 10 mg/mL in physiological saline (0.9% NaCl), using aplastic graded syringe.

Observations:
- Mortality and morbidity: Throughout the study, all animals were checked twice daily.
- Clinical signs: The animals were observed for clinical signs before dosing commenced and daily up to sacrifice (approximately 1 hour after dosing on Days 2, 3 and 5).
- Body weight: The animals were weighed at allocation (Day 1) and on sacrifice (Day 6).

Termination: The animals were killed on Day 6, approximately 24 hours after BrdU injection, by carbondioxide narcosis. No necropsy was performed on the sacrificed animals. Shortly after sacrifice of each animal, the auricular lymph nodes were rapidly excised, pooled on individual basis and individuallycollected in a solution of 2 % BSA-PBS[2 % bovine serum albumine (BSA) in phosphate buffered saline, PBS]. Cell suspensions were prepared for the evaluation of proliferation at lymph node.

Determination of cellular proliferation:
- Preparation of single cell suspension: A single cell suspension of lymph node cells (LNC) was prepared from each animal by gentle mechanical disaggregation and passage through a 70μm nylon mesh. The suspensions thus obtained were centrifuged and each supernatant resuspended in 20 mL of 2% BSA-PBS.

- Measurement of BrdU content in lymphocytes DNA: BrdU was measured by ELISA using a commercial kit (Roche Applied Science, Mannheim,Germany, batch No. 17267000), according to manufacturer instructions. Briefly, 100μL ofthe LNC suspension were added to the wells of a flat-bottom 96-well microplate in triplicate.Two replicate microplates were prepared. After fixation and denaturation of the LNC, 100μLof anti-BrdU antibody labelled with peroxidase were added to each well and allowed to react. Subsequently the anti-BrdU antibody was removed by washing and 100μL of the substrate solution were then added and allowed to produce chromogen. The reaction was finally stopped by adding 25μL of stop solution (1 M H2SO4). Absorbance (OD) was detected at450 nm (with reference wavelength: 690 nm).The replicate plate was destroyed after the evaluation of the results obtained in the first plate, since the objective of the study had been achieved.
Positive control substance(s):
other: 25% (w/w) alpha-hexylcinnamaldehyde (Sigma Cat. W256900, batch no. MKBS3936V) in acetone:olive oil 4:1 v/v.
Statistics:
Differences between each treated group and the concurrent negative control group (individual BrdU labelling indices) were assessed by Dunnett’s test.
The homogeneity of the data was verified by Bartlett’s test before Dunnett’s test. If data were found to be inhomogeneous, a Modified t test (Cochran and Cox) was applied.

Results and discussion

Positive control results:
In the group treated with the positive control item, a Stimulation Index of 2.63 was calculated. As it was greater than 2, the study was regarded as valid.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Remarks:
Vehicle (0% w/w)
Value:
ca. 1
Test group / Remarks:
Group 1
Key result
Parameter:
SI
Remarks:
Low Dose Level (5%)
Value:
ca. 1.43
Test group / Remarks:
Group 2
Key result
Parameter:
SI
Remarks:
Mid Dose Level (10%)
Value:
ca. 1.51
Test group / Remarks:
Group 3
Key result
Parameter:
SI
Remarks:
High Dose Level (25%)
Value:
ca. 0.98
Test group / Remarks:
Group 4
Key result
Parameter:
SI
Remarks:
Positive Control
Value:
ca. 2.63
Test group / Remarks:
Group 5
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
No significant increase in cell proliferation of draining lymph nodes was observed in anytreatment group.

DETAILS ON STIMULATION INDEX CALCULATION
The calculated Stimulation Indices (SI) were 1.43, 1.51 and 0.98, respectively at low, mid- and high dose levels.

CLINICAL OBSERVATIONS:
A) Preliminary Test: No signs of toxicity (clinical signs) were observed at any of the tested concentrations. The evaluation of visible reactions showed no erythema at any of the concentrations investigated [25, 10, 5, 2.5 and 1 % (w/w)].

The evaluation of ear thickness indicated that no increase was induced by treatment (values of Day 6 compared to Day 1). The evaluation of ear punch weight indicated that no increase was found at any dose level investigated.

Based on the results described above, the highest concentration selected for the main assay was 25 % (w/w)

B) Main Study: Neither mortality nor clinical signs were recorded in animals treated at all dose levels investigated [25, 10 and 5 % (w/w)].

BODY WEIGHTS
A) Preliminary Test: No signs of toxicity (toxicologically relevant body weight losses) were observed at any of the tested concentrations.

B) Main Study: Changes in body weight observed during the study were within the expected range for this strain and age of animals.

Any other information on results incl. tables

Table 2 . EVALUATION OF LYMPHOCYTE PROLIFERATION - BrdU LABELLING INDEX AND STIMULATION INDEX

Animal

No.

Group

Concentration

(% w/w)

BrdU labelling index/Mouse

BrdU Labelling index/group

SI/

Group

Replicate A

Replicate B

Replicate C

Mean

Mean

SD

CV%

 

13

1

0

0.085

0.081

0.071

0.079

0.091

0.029

31.40

1

15

0.114

0.131

0.138

0.127

17

0.064

0.068

0.049

0.060

19

0.088

0.113

0.093

0.098

 

21

2

5

0.103

0.109

0.102

0.104

0.130

0.030

23.40

1.43

23

0.155

0.177

0.130

0.154

25

0.205

0.160

0.109

0.158

27

0.082

0.105

0.124

0.103

 

29

3

10

0.111

0.140

0.114

0.121

0.137

0.037

26.79

1.51

31

0.137

0.112

0.107

0.118

33

0.175

0.189

0.214

0.192

35

0.139

0.119

0.094

0.117

 

37

4

25

0.075

0.086

0.066

0.075

0.089

0.027

30.56

0.98

39

0.157

0.108

0.101

0.122

41

0.090

0.110

0.095

0.098

43

0.066

0.050

0.065

0.060

 

45

5

25

0.239

0.233

0.182

0.218

0.239

0.022

9.19

2.63

47

0.258

0.243

0.305

0.268

49

0.229

0.225

0.229

0.227

51

0.267

0.242

0.223

0.244

 

SI = Stimulation index                 

Applicant's summary and conclusion

Interpretation of results:
other: Not classified according to the CLP Regulation
Conclusions:
The test material did not elicit any sensitisation response/s in mice following dermal exposure and is therefore considered to be not sensitizer.
Executive summary:

In a key Guideline (OECD 442b) skin sensitisation study, the potential of the test material, to cause skin sensitisation reactions was evaluated following topical application to the skin of CBA/JN mice using the LLNA: BrdU-ELISA method.

 

A preliminary test was conducted where five concentrations of the test material [25 (maximum feasible concentration), 10, 5, 2.5 and 1 % w/w in acetone:olive oil 4:1 (v/v)] were in order to identify a nontoxic and minimally irritant concentration and to avoid false positive results. No signs of toxicity (significant clinical signs or body weight losses) were observed at the tested concentrations. According to the results of the irritation screening, the concentration of 25 % w/w was judged to be not irritant.

 

In the main assay, the test material was topically administered at concentrations of 25, 10 and 5% (w/w), in acetone:olive oil 4:1 (v/v). No mortality or clinical signs were observed in any animal. Changes in body weight observed during the study were within the expected range for this strain and age of animals. No increase in cell proliferation of draining lymph nodes was observed in any treatment group. The calculated Stimulation Indices (SI) were 1.43, 1.51, and 0.98 for the low, mid-and high dose levels [5, 10, and 25% w/w], respectively. No correlation with the doses or statistical significance was observed.

 

The above results indicate that the test material did not elicit any sensitisation response/s in mice following dermal exposure and is therefore considered to be not sensitizer.