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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation, other
Remarks:
in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04.12.2013 - 10.12.2013
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
Test item: Tar wood CAS No.: 91722-33-7 Batch No.: 18092013 Appearance: viscous Colour: brown Purity: 100 % mixture Expiry date: December 31, 2015 Storage: room temperature

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
epicutaneous, open
Vehicle:
other: N,N-Dimethylformamide
Concentration / amount:
Tar wood was examined in the LLNA as 25 %, 10 %, 5 % or 2.5 % (w/v) formulations in DMF according to the relevant guidelines.
Challenge
Concentration / amount:
Tar wood was examined in the LLNA as 25 %, 10 %, 5 % or 2.5 % (w/v) formulations in DMF according to the relevant guidelines.

Study design: in vivo (LLNA)

Vehicle:
other: N,N-Dimethylformamide
Concentration:
Tar wood was examined in the LLNA as 25 %, 10 %, 5 % or 2.5 % (w/v) formulations in DMF according to the relevant guidelines.

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
other: Migrated information from in vivo LLNA study
Remarks on result:
other: Group: other: Vehicle control for the PC: AOO. Dose level: 0. No with. + reactions: 0.0. Total no. in groups: 4.0.
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
Each mouse was topically treated with 25 µL of the appropriate formulations of the test item, the positive control substance (positive control group) or the vehicles (DMF or AOO as negative control groups) using a pipette, on the dorsal surface of each ear.

Any other information on results incl. tables

DPM, DPN and Stimulation Index Values for all Groups in the Main Test:

Test Group 

Measured DPM/group

Group*

DPM

DPN (DPM/Node)

Stimulation

Name

Index Values

Vehicle control

7638

 

7612.0

 

951.5

 

1.0

for the positive control: AOO

 

Positive control:

118205

 

118179.0

 

14772.4

 

15.5

 

25 % HCA in AOO

Tar wood

65558

 

65532.0

 

8191.5

 

11.4

25 % in DMF

 

Tar wood

61656

 

61630.0

 

7703.8

 

10.8

10 % in DMF

 

Tar wood

26602

 

26576.0

 

3322.0

 

4.6

 

5 % in DMF

Tar wood

15367

 

15341.0

 

1917.6

 

2.7

2.5 % in DMF

 

Vehicle control

5759

 

5733.0

 

716.6

 

1.0

for the test item: DMF

 

Applicant's summary and conclusion

Interpretation of results:
other: a moderate skin sensitizer
Remarks:
Criteria used for interpretation of results: OECD GHS
Conclusions:
In conclusion, under the conditions of the present Local Lymph Node Assay, Tar wood tested at the maximum applicable (non-toxic, non-irritant) concentration of 25 % and at concentrations of 10 %, 5 % and 2.5 % (w/v) as formulations in an appropriate vehicle (DMF) was shown to have skin sensitization potential. Based on the EC3 value calculated using the dose-response curve Tar wood was classified as a moderate skin sensitizer in this LLNA according to the published data.
Executive summary:

The aim of this study was to evaluate the skin sensitization potential of Tar wood following dermal exposure in the Local Lymph Node Assay.

Selection of test item concentrations based on the results of a formulation evaluation and also result of a preliminary irritation/toxicity test according to the relevant guidelines [1-2]. The test item was a liquid hence applicability of the undiluted test item (100 %) was evaluated. Due to its high viscosity, the undiluted test item could not be applied on the ears of animals. The test item was formulated in the selected vehicle ofN,N-Dimethylformamide (DMF). In the preliminary test significant adverse effects (irritation and/or toxicity) were observed at higher test concentrations (75 % or 50 %, w/v) so Tar wood was examined in the LLNA as 25 %, 10 %, 5 % or 2.5 % (w/v) formulations in DMF according to the relevant guidelines.

In the main test 28 female CBA/Ca mice were allocated to 7 groups of four animals each:

-four groups received Tar wood at four different concentrations of 25 %, 10 %, 5 % and 2.5 % (w/v),

-the positive control group receivedα-Hexylcinnamaldehyde (HCA) at concentration of 25 % (w/v),

-one control group (used as negative control for the groups treated with the test item) received the vehicle of the test item (DMF) only,

-one control group (used as negative control for the group treated with the positive control substance) received the vehicle of the positive control (AOO) only.

Each substance was applied on the external surface of each ear (25µL/ear) of the animals for three consecutive days (Day 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6 animals were intravenously injected via the tail vein with tritiated methyl thymidine (3HTdR), than sacrificed approximately 5 hours after the injection. Auricular lymph nodes were removed and processed. The cell proliferation in the local lymph nodes was measured by incorporation of3HTdR and the obtained values were used to calculate stimulation indices (SI).

The positive control item (25 % HCA in AOO) induced the appropriate (SI3) stimulation over the control (SI value was 15.5), thus confirming the validity of the assay.

No mortality was observed during the study. No significant, treatment related effect on body weights or any other signs of systemic toxicity were observed in any treatment group during the test. No signs of significant irritation or any other significant local effect were observed at the treatment site (ears) in any treatment group. Although slight loss of hair (at the base of ears) was observed in the 25 % dose group the effect was considered not significant.

Visually larger lymph nodes than the relevant controls were observed in the positive control group and in the 25 % and 10 % (w/v) dose groups. Visual appearance of the lymph nodes was normal in the negative control groups (both DMF and AOO) and in the 5 % and 2.5 % (w/v) dose groups.

Significantly increased lymphoproliferation (indicated by an SI3) compared to the relevant control (DMF) was noted for Tar wood at concentrations of 25 %, 10 % and 5 % (w/v) No significantly increased lymphoproliferation was observed at test item concentration of 2.5 % (w/v). The stimulation index values were 11.4, 10.8, 4.6 and 2.7 at concentrations of 25 %, 10 %, 5 % and 2.5 % (w/v), respectively. Dose-related response was observed, although its linearity was not statistically significant (p = 0.17, r = 0.83, evaluated by linear regression using SI values).

Since the test was valid and no sign of systemic toxicity or significant irritation was observed during the main test, the proliferation values obtained are considered to reflect the real potential of the test item to cause/not cause lymphoproliferation in the Local Lymph Node Assay.

According to evaluation criteria of the relevant guidelines the increased lymphoproliferation observed at three test concentrations (25 %, 10 % and 5 %, w/v) and the dose related response are considered evidence that Tar wood has skin sensitization potential.

EC3calculation was conducted by linear interpolation using data points lying immediately above and below the SI value of 3 on the LLNA dose-response curve (based on published method [5]). The calculated EC3value of Tar wood based on the dose-response was 2.9 % in this LLNA. Using the EC3value Tar wood can be ranked among moderate skin sensitizers (1 ≤ EC3< 10) in this LLNA according to the published data for classification of contact allergens.