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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:
weight of evidence
Study period:
18 Aug - 23 Dec 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2010
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: 15.7 - 25.8 g
- Housing: 1 to 5 animals per cage in polycarbonate boxes with bedding
- Diet: Formulab #5008 (PMI Feeds Inc.), ad libitum
- Water: municipal water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 23
- Humidity (%): 27 - 98
- 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:
Experiment 1
Test substance: 25, 50 and 100%
Positive control: 100%

Experiment 2
Test substance: 2.5, 5, 10 and 25%
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 at least one concentration 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 on Day 6 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 in 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 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 11.0.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
EC3
Value:
2.7
Parameter:
SI
Value:
10.3
Test group / Remarks:
100% test substance
Remarks on result:
other: experiment 1
Parameter:
SI
Value:
6.9
Test group / Remarks:
50% test substance
Remarks on result:
other: experiment 1
Parameter:
SI
Value:
5.7
Test group / Remarks:
25% test substance
Remarks on result:
other: experiment 1
Parameter:
SI
Value:
6.2
Test group / Remarks:
25% test substance
Remarks on result:
other: experiment 2
Parameter:
SI
Value:
4.7
Test group / Remarks:
10% test substance
Remarks on result:
other: experiment 2
Parameter:
SI
Value:
3.9
Test group / Remarks:
5% test substance
Remarks on result:
other: experiment 2
Parameter:
SI
Value:
2.9
Test group / Remarks:
2.5% test substance
Remarks on result:
other: experiment 2
Cellular proliferation data / Observations:
EC3 CALCULATION
The EC3 value was calculated based on the results from Experiment 2 using the SI values at 2.5 and 5% test substance concentration. The EC3 value was calculated to be 2.7%.

CLINICAL OBSERVATIONS
All animals appeared normal for the duration of the study.

BODY WEIGHTS
Two to three animals each of the treatment groups in Experiment 2 lost or failed to gain weight 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

Experiment 1

Vehicle Control Group

1

15.7

22.5

128

616 ± 327

2

17.2

23.8

500

3

15.7

22.5

702

4

16.0

24.8

743

5

16.4

23.9

1006

Test Group I - 25%

1

21.5

21.9

4715

3846 ± 978

2

22.7

22.8

3820

3

24.8

24.9

2477

4

23.7

24.3

3382

5

22.9

23.6

4837

Test Group II - 50%

1

21.8

22.6

5357

4271 ± 1087

2

21.8

22.6

2871

3

22.5

23.7

3448

4

24.0

24.9

4474

5

22.5

23.1

5205

Test Group III - 100%

1

21.4

23.3

7715

6322 ± 1932

2

23.5

23.7

6937

3

23.7

23.8

4944

4

21.3

21.8

3713

5

22.2

22.7

8299

Positive Control Group

1

21.8

22.6

3734

6767 ± 4685

2

21.0

22.1

6230

3

23.6

24.4

3838

4

23.3

23.9

5086

5

21.4

22.4

14947

Experiment 2

Vehicle Control Group

1

19.7

16.2

254

639 ± 416

2

19.1

18.9

351

3

23.9

24.3

511

4

22.9

23.3

791

5

23.4

24.4

1288

Test Group IV – 2.5%

1

24.1

23.2

1968

1863 ± 292

2

22.7

22.7

2283

3

24.0

24.9

1520

4

23.8

24.1

1870

5

22.2

22.4

1673

Test Group V - 5%

1

22.9

23.4

1995

2502 ± 641

2

23.9

22.5

2935

3

23.1

22.7

1707

4

24.2

24.5

3245

5

23.1

22.2

2630

Test Group VI - 10%

1

25.1

23.7

2294

3019 ± 799

2

22.7

23.7

2805

3

24.4

24.0

2305

4

24.2

23.1

3611

5

23.7

21.3

4079

Test Group VII – 25%

 1

20.8

21.0

3318

3635 ± 1169

 2

24.9

23.0

5577

 3

25.8

24.7

3067

 4

22.5

22.3

3701

 5

24.0

24.4

2510

Statistics

The treatment groups I - III (25, 50 and 100%) and IV – VII (2.5, 5, 10 and 25%) were compared to the vehicle control group of Experiment 1 and 2, respectively. In Experiment 1, statistically significant mean DPM counts (P < 0.01, ANOVA) were observed at 100% test substance concentration and for the positive control group. In Experiment 2, statistically significant mean DPM counts were observed at 5, 10 and 25% test substance concentration.

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Under the conditions of the test the test substance revealed sensitising properties. The EC3 value was calculated to be 2.7%.