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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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Although the study is not a conventional guideline study, it is well-conducted and scientifically acceptable.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Principles of method if other than guideline:
The primary aim of the study was to evaluate the allergenic potential of the test substance and to determine its ability to cause allergic sensitization of the respiratory tract. Following analysis of lymphocyte proliferation using a standard local lymph node assay (LLNA) protocol cytokine fingerprinting was undertaken to explore respiratory sensitizing potential. Repeated topical exposure of BALB/c strain mice to chemical respiratory allergens, or contact allergens, has been shown to provoke characteristic cytokine secretion profiles consistent with the divergent activation of discreet T cell subpopulations. Cytokine profiles were compared with the recognised respiratory sensitizers Trimellitic anhydride (TMA) and Ammonium hexachloroplatinate (AHCP), as well as the known contact sensitizer 2,4-Dinitrochlorobenzene (DNCB) and the non-sensitizer Tetraammine platinum dichloride (TPC).
GLP compliance:
no
Remarks:
Non-standard guideline study conducted at academic institution
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid - liquid: suspension
Details on test material:
- Name of test material (as cited in study report): Hexahydroxyplatinic acid (HHPA)
- Analytical purity: records of test compound characterisation, including purity, were held by the sponsor
- Other: All test preparations were fine suspensions of HHPA in DMF after sonification for 15 mins

In vivo test system

Test animals

Species:
mouse
Strain:
Balb/c
Sex:
not specified

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
40% and 80% in LLNA; 80% for cytokine fingerprinting
No. of animals per dose:
4/group in LLNA; 3/group for cytokine fingerprinting
Details on study design:
Using a standard LLNA protocol, mice (4/group) received 25 ul of chemical [80% or 40% HHPA in DMF, or DMF alone] on the dorsum of both ears on days 0, 1 and 2. Five days after the initiation of treatment, draining lymph nodes were isolated, pooled on a treatment group basis and a single cell suspension prepared. Total cellularity per lymph node was recorded. Cells (in quintuplicate) were cultured at 107 cells/ml for 24 hr in the presence of radiolabelled thymidine (2 uCi). Culture was terminated by automated cell harvesting and thymidine incorporation measured by beta-scintillation counting.

Results and discussion

Positive control results:
Details on positive and negative control results for the cytokine fingerprinting assay are presented below under Any other information on results incl tables

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: See results, below
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Full details on results are presented below under Any other information on results incl. tables

Any other information on results incl. tables

HHPA displayed some activity in the modified LLNA, with 80% HHPA/DMF achieving a combined stimulation index (SI) of 5.7. Full results are given below:

Treatment

Cellularity (cells/lymph node x 106)

Thymidine incorporation (dpm)

Combined Stimulation Index (SI)

80% HHPA/DMF

5.5

8570

5.7

40% HHPA/DMF

7.3

6285

5.4

DMF

4.25

1953

1

In the 13 -day cytokine fingerprinting assay, total cellularity per lymph node was recorded as a measure of immune activation. Topical exposure to the positive control substances TMA and DNCB induced marked increases in lymph node cellularity. AHCP induced a more modest increase in lymph node cellularity, whereas TPC and HHPA were without marked effects on lymph node cell (LNC) numbers. Full results are given below:

Treatment

Cellularity (cells/lymph node x 106)

10% TMA/AOO

13.5

1% DNCB/AOO

14.3

5% AHCP/DMF

11.5

5% TPC/DMF

4

80% HHPA/DMF

4

Type 2 cytokine IL-4 results are presented below. LNCs isolated from the known skin sensitizer DNCB treated mice expressed very low levels of IL-4, whilst treatment with the respiratory sensitizer TMA resulted in relatively vigorous IL-4 expression. Exposure to AHCP (also known to be a respiratory sensitizer) also induced the production of IL-4, albeit at lower levels than that provoked by TMA. Treatment with 80% HHPA (or 5% of the non-sensitizer TPC), however, stimulated very low levels of IL-4 production:

Treatment

IL-4 (pg/ml)

10% TMA/AOO

668

1% DNCB/AOO

73

5% AHCP/DMF

213

5% TPC/DMF

11

80% HHPA/DMF

20

In the analysis of additional type 2 cytokines (IL-5, IL-10, IL-13) and type 1 cytokines (IL-12, IFN-gamma), LNCs derived from TPC-treated animals expressed relatively low levels of all cytokines with the exception of (type 1) IL-12. DNCB exhibited a selective type 1 cytokine profile with relatively vigorous IFN-gamma production and IL-12 expression, but relatively low levels of type 2 cytokines IL-5, IL-10 and IL-13. TMA-activated LNCs expressed a type 2 cytokine profile with relatively high levels of IL-5, IL-10 and IL-13, but low levels of the type 1 cytokines. A similar type 2 pattern of cytokine expression was recorded following topical exposure to AHCP. For HHPA-activated LNCs, levels of type 2 cytokines were below the limit of detection and the cytokine profile was similar to the non-sensitizing TPC.

Type 2 cytokines

IL-5 (pg/ml)

IL-10 (pg/ml)

IL-13 (pg/ml)

Treatment

96 hr

120 hr

144 hr

96 hr

120 hr

144 hr

96 hr

120 hr

144 hr

10% TMA/AOO

289

335

576

594

822

1019

872

1043

1423

1% DNCB/AOO

209

165

65

47

55

22

164

136

27

5% AHCP/DMF

98

175

98

101

129

187

75

142

99

5% TPC/DMF

ND*

<10

<10

ND*

<10

<10

ND*

<10

<10

80% HHPA/DMF

ND*

<10

<10

ND*

<10

<10

ND*

<10

<10

*not done - insufficient cells

Type 1 cytokines

IL-12 (pg/ml)

IFN-gamma (pg/ml)

Treatment

96 hr

120 hr

144 hr

96 hr

120 hr

144 hr

10% TMA/AOO

108

110

104

389

321

398

1% DNCB/AOO

187

201

209

1225

1241

1130

5% AHCP/DMF

97

86

86

93

126

116

5% TPC/DMF

ND*

154

152

ND*

16

<10

80% HHPA/DMF

ND*

132

138

ND*

<10

<10

*not done - insufficient cells

Total serum IgE concentrations are presented below:

 

IgE concentration (ng/ml)

Treatment

No 1

No 2

No 3

10% TMA/AOO

1864

1230

966

5% AHCP/DMF

654

1354

2937

5% TPC/DMF

49

36

14

80% HHPA/DMF

59

47

ND*

*not done - mouse terminated early due to lymphoproliferative disorder, not compound related.

TMA and AHCP resulted in marked increases in total serum IgE concentration compared with the negative control platinum TPC. There was no marked elevation in total serum IgE following topical exposure to HHPA.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: expert judgment
Conclusions:
Hexahydroxyplatinic acid (HHPA) at high concentrations demonstrated a mild sensitisation response using a conventional Local Lymph Node Assay (LLNA) (40% and 80% HHPA in DMF gave a Stimulation Index (SI) of 5.4 and 5.7, respectively). However, the more sophisticated detailed cytokine fingerprinting assay indicated that it was not a significant skin or respiratory sensitiser, demonstrating a cytokine profile similar to that of the recognised non-sensitiser tetraammineplatinum dichloride.
Executive summary:

Hexahydroxyplatinic acid (HHPA) was subjected to a modified LLNA, combined with a detailed cytokine fingerprinting assay, to evaluate its allergenic potential, particularly with regard to its ability to cause sensitisation of the respiratory tract.

 

Mice (4/group) received a 25 μL application of HHPA (concentrations of 40% or 80% HHPA in DMF, or DMF alone) on the dorsum of both ears on three consecutive days. Five days after the initiation of treatment, cell proliferation in the local lymph nodes was measured by incorporation of radiolabelled thymidine.

 

Subsequently, a standard 13-day cytokine fingerprinting assay was undertaken, involving mice (3/group) exposed to 80% HHPA in DMF. Additional groups of mice were treated with ammonium hexachloroplatinate (AHCP) (a respiratory sensitising platinum salt; positive control), tetraammineplatinum dichloride (TPC) (a non-sensitising platinum salt; negative control), as well as the reference respiratory allergen trimellitic anhydride (TMA) or the reference contact allergen 2,4 -dinitrochlorobenzene (DNCB). Animals received 50 μL of test chemical on both shaved flanks on day 0 and day 5, followed by a further dose of 25 μL of chemical on the dorsum of both ears on days 10, 11 and 12.

 

In the LLNA, the observed Stimulation Index (SI) values were 5.4 and 5.7 at 40 and 80%, respectively, demonstrating a mild sensitisation response. However, the more sophisticated detailed cytokine fingerprinting assay indicated that the test material was not a significant skin or respiratory sensitiser. Type 1 and type 2 cytokine profiles were compared with those of the known respiratory sensitisers TMA and AHCP, the contact sensitizer DNCB, and the known non-sensitiser TPC. HHPA failed to stimulate vigorous type 2 cytokine production, indicating a lack of respiratory sensitising potential, and demonstrated a cytokine profile similar to that of the non-sensitiser TPC. Type 1 cytokine IFN-gamma was below the limit of detection, and IL-12 was not elevated above normal background values, based on historical vehicle controls.

 

Overall, the results suggest that HHPA is not a significant skin or respiratory tract sensitiser. It is recommended that a lack of skin sensitising potential be confirmed using a standard in vivo assay for skin sensitisation using guinea-pigs.