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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:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline-compliant study (OECD 429) and GLP. No deficiencies noted.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (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
Type:
Constituent

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
Animals were from Charles RIver, Strain CBA/J. The animals were housed in fully air-conditioned rooms. Central air-conditioning guaranteed a range of 20 – 24°C for temperature and of 30 – 70% for relative humidity. There were no deviations from these ranges, which influenced the results of the study.

Study design: in vivo (LLNA)

Vehicle:
other: 1% Pluronic® L 92 Surfactant in highly de-ionized water
Concentration:
0, 10, 25, 50%
No. of animals per dose:
5
Details on study design:
Groups of 5 female CBA/J mice each were treated with 10%, 25% and 50% w/w preparations of the test substance in 1% aqueous Pluronic® or with the vehicle alone. The 50% preparation was the maximum technically applicable concentration. The study used 3 test groups and 1 control group. Each test animal was applied with 25 μL per ear of the respective test-substance preparation to the dorsum of both ears for three consecutive days. The control group was treated with 25 μL per ear of the vehicle alone. Three days after the last application the mice were injected intravenously with 20 μCi of 3Hthymidine in 250 μL of sterile saline into a tail vein. About 5 hours after the 3H-thymidine injection, the mice were sacrificed and the auricular lymph nodes were removed. Lymph node response was evaluated by measuring the cellular content and 3H-thymidine incorporation (indicator of cell proliferation) as well as the weight of each animal’s pooled lymph nodes. Moreover, a defined area with a diameter of 0.8 cm was punched out of the apical part of each ear and for each animal the weight of the pooled punches was determined in order to obtain an indication of possible skin irritation.
Statistics:
The statistical evaluations were performed using the WILCOXON-test ( # for p ≤ 0.05, ## for p ≤ 0.01 )

Results and discussion

Positive control results:
A concurrent positive control (reliability check) with a known sensitizer was not included into this study. Studies using the positive control substance Alpha-Hexylcinnamaldehyde, techn. 85% are performed twice a year in the laboratory in order to show that the test system is able to detect sensitizing compounds under the test conditions chosen. The most recent test from March 2010 with Alpha-Hexylcinnamaldehyde (techn. 85%) applied in concentrations of 5%, 10%, 25% in acetone + olive oil (4+1 v/v) gave stimulation factors of 0.98, 1.21, 1.91 for cell counts and 2.22, 3.47, 11.86 for 3H-thymidine incorporation. The test result was found positive.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
Cell Counts [Counts/Lymph Node Pair] Group Treatment Counts S.D. Stimulation Index Significance 1 vehicle 7242000 +/- 1607401 1.00 2 10% in vehicle 8980800 +/- 1160045 1.24 # 3 25% in vehicle 10653600 +/- 1295337 1.47 ## 4 50% in vehicle 9099600 +/- 1298266 1.26 #
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
³H-thymidine incorporation [DPM/Lymph Node Pair] Group Treatment Counts S.D. Stimulation Index Significance 1 vehicle 331.2 +/- 54.1 1.00 2 10% in vehicle 895.4 +/- 464.5 2.70 ## 3 25% in vehicle 1,085.2 +/- 398.8 3.28 ## 4 50% in vehicle 729.6 +/- 129.8 2.20 ##

Any other information on results incl. tables

Lymph Node Weight [mg/Lymph Node Pair]

Group

Treatment

Counts

S.D.

Stimulation Index

1

vehicle

4.9

+/- 0.5

1.00

2

10% in vehicle

5.5

+/- 0.3

1.12

3

25% in vehicle

6.1

+/- 0.1

1.24

4

50% in vehicle

5.8

+/- 0.6

1.18

 

Ear Weight [mg/animal]

Group

Treatment

Counts

S.D.

Stimulation Index

1

vehicle

31.0

+/- 1.1

1.00

2

10% in vehicle

31.5

+/- 2.2

1.02

3

25% in vehicle

31.6

+/- 2.5

1.02

4

50% in vehicle

33.4

+/- 1.2

1.08

No signs of systemic toxicity were noticed.

When applied as 10%, 25% and 50% preparations in 1% aqueous Pluronic®, the test substance induced an increase in the auricular lymph node cell counts below 1.5 fold of the control value (= stimulation index (SI) ≥ 1.5) without concentration dependence. There was a slight increase in lymph node weights without concentration dependence, as well. Concomitantly, the 25% concentration caused a borderline increase of 3H-thymidine incorporation into the cells above the cut off stimulation index of 3. The 50% and 10% concentration failed to reach the cut-off value (no increase above the stimulation index of 3).

Increases in cell counts, 3H-thymidine incorporation and lymph node weights were statistically significant at all concentrations without concentration dependence. The 50% test-substance preparation caused a statistically significant increase in ear weights. No relevant increases in ear weights were observed at the concentrations 10% and 25%. As the SI for 3H-thymidine incorporation at the 25% concentration lies at the border of biological relevance, whereas the increase of cell counts was not biologically relevant and because there was no concentration response relation, the 3H-thymidine incorporation is not considered to indicate a skin sensitization reaction.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
It is concluded that DIAMINOSTILBEN FREE ACID does not show a skin sensitizing effect in the Murine Local Lymph Node Assay under the test conditions chosen
Executive summary:

It is concluded that diaminostilben free acid does not show a skin sensitizing effect in the murine Local Lymph Node Assay under the test conditions chosen.