Registration Dossier

Administrative data

Description of key information

Skin sensitisation (OECD 429): skin sensitising

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 Sep - 09 Dec 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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 lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Remarks:
/Jcr
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Females nulliparous and non-pregnant: yes
- Weight at study initiation: 18.8 - 24.0 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): 20-23
- Humidity (%): 30-94
- Air changes (per hr): minimum 10
- Photoperiod (hrs dark / hrs light): 12/12
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:
PRE-SCREEN TEST:
No range-finding test was conducted.

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. Five hours after 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 at least one concentration shows SI < 3, then the formula is: EC3 = [(3-d)/(b-d)] x (a-c) + c
a = the dose concentration with higher SI
b = the higher SI value
c = the dose cocentration with lower SI
d = the lower SI value using SI values closest to 3, one above and one below
Positive control results:
The SI value calculated for the positive control was 5.2.
Parameter:
SI
Value:
1.9
Test group / Remarks:
25%
Parameter:
SI
Value:
3.5
Test group / Remarks:
50%
Parameter:
SI
Value:
3.4
Test group / Remarks:
100%
Parameter:
EC3
Value:
42.3
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA: The lymph nodes of each individual animal were pooled and disintegration per minute (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 4649, 8368 and 8274, respectively. The DPM value per mouse of the vehicle control was 2405. Compared to the vehicle control, the 50%, 100% and positive control groups had significantly higher DPM counts (p > 0.1 per ANOVA).

EC3 CALCULATION: The EC3 was 42.3% using the 25% and 50% concentrations.

CLINICAL OBSERVATIONS: All animals appeared normal for the duration of the study. However, one positive control animal was found dead following Day 6 injection.

BODY WEIGHTS: Four animals each of the 50 and 100% concentration test group failed to gain weight during the study. Some animals in both control groups also lost weight.

Table 1: Body weights and DPM counts.

 

Animal

Body weight (g)

DPM count

Mean DPM ± SD

Day 1

Day 6

Vehicle Control Group

1

24.0

22.9

3583

2405 ± 817

2

21.7

21.2

2231

3

20.9

21.6

1288

4

22.1

21.5

2468

5

20.9

19.6

2456

Test Group I - 25%

1

21.9

22.0

2371

4649 ± 2127

2

18.8

19.7

3271

3

20.8

21.3

3992

4

20.3

20.8

7604

5

21.2

22.0

6006

Test Group II - 50%

1

23.4

21.7

11118

8368 ± 2866

2

21.2

21.1

11854

3

21.8

21.8

5965

4

19.7

20.0

6526

5

20.9

20.8

6375

Test Group III - 100%

1

20.1

20.0

7400

8274 ± 2794

2

22.0

22.3

6121

3

22.1

21.3

5709

4

23.1

22.4

12412

5

21.8

22.0

9728

Positive Control Group

1

20.9

21.1

7950

12508 ± 3323

2

20.4

20.0

15924

3

21.2

21.3

13209

4

23.2

22.1

12949

5

22.1

16.1

*

* Animal was found dead following Day 6 injection

Interpretation of results:
other: Skin Sens. 1B according to Regulation (EC) No 1272/2008
Conclusions:
Under the conditions of the mouse Local Lymph Node Assay the test substance revealed a SI ≥ 3 at concentrations of 50 and 100%. The EC3 value was calculated to be 42.3%. Therefore, the test substance is considered as weak sensitiser.
Executive summary:

The skin sensitisation potential of the test substance was determined by a LLNA according to OECD Guideline 429 in compliance with GLP (2016). After treatment of CBA/Jcr mice with the test substance, test substance produced stimulation indices of 1.9, 3.5 and 3.4 at concentrations of 25, 50 and 100%, respectively. The EC3 value was calculated as 42.3%. In conclusion, based on the available data the test substance is considered to be a weak skin sensitiser.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

The skin sensitisation potential of the test substance was determined by a LLNA according to OECD Guideline 429 in compliance with GLP (2016). After treatment of CBA/Jcr mice with the test substance, test substance produced stimulation indices of 1.9, 3.5 and 3.4 at concentrations of 25, 50 and 100%, respectively. The EC3 value was calculated as 42.3%. In conclusion, based on the available data the test substance is considered to be a weak skin sensitiser.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The available data on skin sensitisation of the test substance meet the criteria for classification as Skin Sens. 1B (H317) according to Regulation (EC) 1272/2008.