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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:
20 Aug - 04 Nov 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2010
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
2003
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/Jcr
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 21.3 - 24.6 g
- Housing: 1 to 5 animals per cage in polycarbonate boxes, with bedding
- Diet: Formulab #5008 (PMI Feeds Inc.), ad libitum
- Water: municipal water supply, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 23
- Humidity (%): 48 - 97
- Air changes (per hr): minimum 10
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Test substance: 25, 50 and 100%
Positive control: 100%
No. of animals per dose:
5
Details on study design:
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 3H-methyl thymidine incorporation determined by ß-scintillation
- Criteria used to consider a positive response: A stimulation index (SI) was calculated for each group using the activity of each test group divided by the activity of the vehicle control group. The criterion for a positve response is that one or more concentrations of the test substance should elicit a 3-fold or greater increase in isotope incorporation relative to the vehicle control group.

TREATMENT PREPARATION AND ADMINISTRATION: The dorsal surface of both ears was topically treated (25 μL/ear) with the test substance on Day 1. The application was repeated on Day 2 and 3. Three days after the third application an injection of 250 µl phosphate buffered saline (PBS) containing 20 µCi of 3H-methyl thymidine (³HTdR) was made into the tail vein of each experimental mouse. Approximately five hours later, following injection of ³HTdR, the mice were sacrificed and draining auricular lymph nodes were excised and pooled for each individual animal. A single cell suspension was prepared by gentle separation through a 200 mesh stainless steel gauze. The cell suspensions were washed two times with an excess of PBS and precipitated with 5% trichloroacetic acid at 4 °C for 18 h. The pellets were resuspended in 1 mL of trichloroacetic acid and transferred to 10 mL of scintillation fluid.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
A one-way parametric analysis of variance (ANOVA) with Dunnett's Multiple Comparisons Test, using GraphPad InStat version 3.06, was performed on DPM counts.
If test groups showed a SI >3, then an extrapolated EC3 value was calculated from SI values at low% and either mid or high% concentrations.
If all three groups show SI > 3, then the formula is: extrapolated EC3 = 2 exp {log2 (c) + [(3 - d)/(b - d)] x [log2 (a) - log2 (c)]}
If at least one concentration shows SI < 3, then the formula is: EC3 = [(3-d)/(b-d)] x (a-c) + c

Results and discussion

Positive control results:
The SI value calculated for the positive control was 13.3.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
2.97
Test group / Remarks:
25% (v/v)
Parameter:
SI
Value:
1.84
Test group / Remarks:
50% (v/v)
Parameter:
SI
Value:
2.39
Test group / Remarks:
100% (v/v)
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA: The lymph nodes of each individual animal were pooled and DPM values were measured from the pooled lymph node cell suspensions. Treatment with test substance concentrations of 25 and 50% in acetone/olive oil (4:1) and undiluted test substance resulted in DPM values per mouse of 2020, 1252 and 1629, respectively. The DPM value per mouse of the vehicle control was 681.

EC3 CALCULATION: As the SI was < 3 in all test groups, an exprapolated EC3 was not calculated.

CLINICAL OBSERVATIONS: All test and vehicle animals appeared normal for the duration of the study. One animal of the positive control group exhibited activity decrease on Day 6.

BODY WEIGHTS: All test groups exhibited weight gain during the study.

Any other information on results incl. tables

Table 1: Body weights and DPM counts.

 

Animal

Body weight (g)

DPM count

Mean DPM ± SD

Day 1

Day 6

Vehicle Control Group

1

21.7

23.4

982

681 ± 329

2

21.8

22.8

362

3

24.0

25.7

322

4

23.8

25.1

1010

5

24.6

26.0

730

Test Group I - 25%

1

23.4

24.7

1176

2020 ± 696

2

22.1

25.0

2767

3

22.0

23.9

2609

4

22.3

24.8

1460

5

22.7

24.6

2090

Test Group II - 50%

1

21.8

22.9

1733

1252 ± 524

2

23.6

24.7

1882

3

22.6

23.7

774

4

24.0

24.8

791

5

21.7

23.6

1078

Test Group III - 100%

1

21.6

22.9

2406

1629 ± 549

2

22.8

23.8

922

3

22.5

24.0

1350

4

24.0

25.7

1781

5

23.7

24.3

1684

Positive Control Group

1

21.7

23.8

18488

9030 ± 7926

2

23.0

25.0

3142

3

21.3

23.8

16581

4

21.8

23.7

1429

5

23.7

24.9

5512

 

Applicant's summary and conclusion

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification according to Regulation (EC) No 1272/2008
Conclusions:
Under the conditions of the mouse Local Lymph Node Assay the test substance at concentrations of 25, 50 and 100% revealed no sensitising properties.
Executive summary:

The skin sensitising potential of the test substance was investigated in a Local Lymph Node Assay (LLNA) in mice according to OECD Guideline 429 and in compliance with GLP (2016). Five female CBA/Jcr mice per test group were treated with the test substance at concentrations of 25 or 50% (v/v) in acetone/olive oil 4:1 or with 100% test substance. The vehicle control group was treated with vehicle alone and the positive control group with 100% hexyl cinnamaldehyde. The treatment was performed for three consecutive days by open application on the ears (25 µL/ear). Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each individual animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. Treatment with test substance concentrations of 25 and 50% (v/v) in acetone/olive oil 4:1 and 100% resulted in DPM values per lymph node of 2020, 1252 and 1629, respectively. The SI values calculated for the substance concentrations 25, 50 and 100% were 2.97, 1.84 and 2.39, respectively. As all SI values were < 3, an extrapolated EC3 value was not calculated. The reliability check with hexyl cinnamaldehyde indicated that the LLNA is an appropriate model for testing for contact hypersensitivity. The SI value calculated for the positive control was 13.3. All animals appeared normal and exhibited weight gain during the study. One animal of the positive control group exhibited activity decrease on Day 6. Based on the results of this study, the test substance was not regarded as a skin sensitizer under the conditions of the test.