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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:
22 October 2007 to 19 December 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OECD test guidlines in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified
Details on test material:
Identification: Dusantox 86, product of Duslo, a.s. Sal'aLot number: The lot No. 005/07Stability: Stable for 24 months (Certificate of Safety Data, Annex No 2)Storage Conditions: The test substance was stored at room temperature, protected against sunlight in laboratory no.468 of the pharmacology dept.Safety: According to the sponsor provided safety information known about the test article - Certificate of Safety Data from 1 st June 2007 (Annex No 2)

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
Species: Mice 18-20g (Delivery protocol no. 29/2007)Strain: CBN/JSource: AnLab s.r.o., Videiiska 1083, 142 20 PrahaNumber and Sex: 12 females - test substanceIdentification: The animals (8-12 weeks old, 18-20 g) were housed individually in plastic cages T II(Velaz Praha). The animals (4 per group) were marked by serial numbers 1 - 12. These numbers were placed on the cage together with the marking of group.Housing: The mice were housed according to SOP 002/53204/07 Mice.Diet: The standard diet MP (TOP DOVO) was served. The supplier of the diet is approved by State Veterinary and Food Administration SRWater: Ad libitum.Environment: Environmental controls for the animal room were set to maintain 22 ± 2°C, a relative humidity of 55 ± 10 % a minimum of 10 air changes/hour, and a regulated light regime- 12/12 (SOP 013/53204/07 Climatic condition in exp. animal house).

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
The test substance in concentrations of 75, 50 and 25% was applicated (25μl)
No. of animals per dose:
4 animals per dose.
Details on study design:
DOSING PROCEDURESDose Selection: The doses were estimated from available information about the test substance.Dose Administration: The test substance in concentrations of 75, 50 and 25% was applicated (25 μl) to the dorsum of each ear. As positive control a.-Hexylcinnamaldehyde in concentration 25%, the same volume was used. The vehiculum as control in volume 25 μl was applicated.Vehiculum: Acetone/olive oil (4:1 v/v)METHODETest procedureDay 1: Each animal was identified and the body weight was recorded (SOP G 008/52000/07). To the dorsum of each ear was applicated 25μl of the appropriate dilution of the test substance, or the vehicle alone.Days 2 and 3: The application procedure carried out on day 1 was repeated.Days 4 and 5: No treatment.Day6: The body weight of each animal was recorded. 250 μl of phosphate-buffered saline (PBS) containing 20 μCi of 3H-methyl thymidine into all test and control mice via the tail vein was injected.Five hours later, the animals were killed. The draining auricular lymph nodes from each ear were excised and pooled in PBS for each experimental group (pooled treatment group approach).Clinical observations: Animals were carefully observed once daily for any clinical signs, either of local irritation at the application site or of systemic toxicity. All observations were systematically recorded with individual records being maintained for each animal.The clinical observation were scored as 0 (no effect), + (weak effect), ++ (moderate effect),+++ (strong effect).Preparation of cell suspensions: Cell suspension of lymph node cells from pooled treatment groups was prepared by gentle mechanical desagregation by glass homogenizer. Lymph node cells were washed with an excess of PBS and centrifuged (SOP 056/211 /03) by 600 g at 4°C for 10 min. Suspension of cells were precipitated with 5% trichloroacetic acid (TCA) at 4°C for 18-20h. Pellets were centrifuged by 2000g at 4°C for 5 min., re-suspended in 1 ml TCA and transferred to scintillation vials containing 10 ml of scintillation fluid for 3H -counting.Determination of cell proliferation (incorporated radioactivity): Incorporation of 3H-methyl thymidine into the lymph node cells was measured by β-scintillation counting on Liquid scintillation analyzer TRI-CARB, 2000CA, Packard (SOP 023/53202/07) as disintegrations per minute (DPM). The incorporation was expressed as DPM/treatment group.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Calculation of SI and EC3 values: The SI values was obtained by dividing the pooled radioactive incorporation for each treatment group by the incorporation of the pooled vehicle control group.The concentration eliciting a SI of 3 is identified as the EC3 value and is calculated by linear interpolation of points on the dose-response curve, immediately above and below threefold the threshold having the coordinates (a,b) and (c,d), according to the equation (11,12): EC3 = c+[(3-d)/b-d)]x(a-c)

Results and discussion

Positive control results:
Results reported in table form, details under Any other information.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: The positive response, with SI value 3.05, was registered only after application of 50% concentration of tested substance.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
After application of Dusantox 86 the dose dependent increase of the lymph node weights was registered, but in comparison with control group the lymph node weights were lower. The lymph node weights of treated groups were 0.0171g, 0.0192g and 0.0265g (control- 0.0280g). The DPM values for treated groups were 390.4, 1096.0 and 888.4. The calculated EC3 value was 49.36%.

Any other information on results incl. tables

Table 1: Individual and mean body weights (g) at start of dosing and scheduled kill

 

Initial

Terminal

Control*

1

22.51

23.90

2

22.55

23.27

3

22.47

23.33

4

22.58

24.46

Mean

22.52

23.74

SD

0.048

0.056

Pos. Control*

1

24.85

25.81

2

21.15

25.24

3

21.35

22.48

4

24.63

22.77

Mean

22.99

24.08

SD

2.019

1.695

Dusantox 85 25%

1

23.39

23.92

2

23.61

24.65

3

23.18

24.39

4

23.87

24.68

Mean

23.51

24.41

SD

0.296

0.352

Dusantox 86 50%

1

22.78

23.01

2

22.81

23.57

3

22.51

23.59

4

21.75

21.25

Mean

22.46

22.85

SD

0.494

1.103

Dusantox 86 75%

1

21.10

21.01

2

21.92

20.74

3

21.13

20.55

4

21.73

21.16

Mean

21.47

20.86

SD

0.417

0.272

* The values of control and positive control were used from study no. 600008930

 

Table 2: Lymph node weight, DPM, SI EC3 values

 

Lymph node weight (g)

DPM

SI

EC3

Control*

0.0280

359.9

-

 

Pos. Control*

0.0497

1136.7

3.16

 

Dusantox 86 25%

0.0171

390.4

1.08

49.36

Dusantox 86 50%

0.0192

1096.0

3.05

 

Dusantox 85 75%

0.0265

888.4

2.47

 

* The values of control and positive control were used from study no. 600008930.

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated informationrare allergenCriteria used for interpretation of results: EU
Conclusions:
Based on the results, EC3 value and human potency estimation classification (12), Dusantox 86 was classified as a rare allergen.
Executive summary:

The study Dusantox 86. Skin sensitisation: Local Lymph Node Assay was performed in compliance with the OECD Guideline for Testing of Chemicals- Method No 429 Skin Sensitisation: Local Lymph Node Assay. The study was conducted in compliance with the Good Laboratory Practice Regulations.

In the presented study was evaluated the sensitization potential of Dusantox 86 when subjected to the local lymph node assay (LLNA). Test substance, including positive control α-Hexylcinnamaldehyde, were prepared in solvent acetone:olive oil (4:1). Female mice (CBA/J) were topically exposed (dorsum of both ears) to the test solution at several concentrations. Lymphocyte proliferation was measured using incorporation of radioactive thymidine into of the draining lymph nodes. Data were collected as dpm/pooled treatment group and evaluated as stimulation index (SI), the ratio of the dpm/treatment group against the dpm/solvent treated control group. The substance with the SI of 3 or greater is considered as potential skin sensitizer.

Application of three concentrations of Dusantox 86 (25%, 50% and 75%) resulted in positive response at concentration 50% with SI value 3.05. Calculated EC3 value was 49.36%. Based on the results, EC3 value and human potency estimation classification, Dusantox 86 was classified as a rare allergen.

The substance requires classification as a Skin sensitiser Cat 1- H317: May cause an allergic skin reaction applies