4,4'-diaminostilbene-2,2'-disulphonic acid

Skin sensitisation

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:

guideline study

Remarks:

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

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

DIAMINOSTILBEN FREE ACID

IUPAC Name:

DIAMINOSTILBEN FREE ACID

Constituent 2

Reference substance name:

4,4'-diaminostilbene-2,2'-disulphonic acid

EC Number:

201-325-2

EC Name:

4,4'-diaminostilbene-2,2'-disulphonic acid

Cas Number:

81-11-8

Molecular formula:

C14H14N2O6S2

IUPAC Name:

2,2'-ethene-1,2-diylbis(5-aminobenzenesulfonic acid)

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

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)

Results

Results 1

Parameter:

SI

Value:

1.26

Test group / Remarks:

50% in vehicle

Results 2

Parameter:

SI

Value:

1.24

Test group / Remarks:

10% in vehicle

Results 3

Parameter:

SI

Value:

1.47

Test group / Remarks:

25% in vehicle

Remarks on result:

other:

Results 4

Parameter:

other: disintegrations per minute (DPM)

Variability:

not specifed

Test group / Remarks:

not specified

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.

 

 

Disintegration per minute:

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 ##

Applicant's summary and conclusion

Interpretation of results:

other: non sensitising

Conclusions:

The test substance does not show a skin sensitizing effect in the Murine Local Lymph Node Assay under the test conditions chosen.

Executive summary:

The sensitising potential of the test substance was evaluated according to an experimental study according to OECD Guideline 429 using the radioactive Murine Local Lymph Node Assay.
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 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.

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).

However, 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.

Thus it is concluded that the test substance does not show a skin sensitizing
effect in the Murine Local Lymph Node Assay under the test conditions chosen.