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Description of key information

A modified LLNA, combined with a detailed cytokine fingerprinting assay, was used to assess allergenic potential of dihydrogen hexahydroxyplatinate compared to a number of known respiratory-, contact-, and non-sensitisers. Although dihydrogen hexahydroxyplatinate at high concentrations (40% and 80% HHPA in DMF gave a Stimulation Index (SI) of 5.4 and 5.7, respectively) demonstrated a mild sensitisation response (based on SI>3), the more sophisticated cytokine fingerprinting assay indicated that it was not a significant skin (or respiratory) sensitiser (Dearman and Kimber, 2011).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
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.
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)
Species:
mouse
Strain:
Balb/c
Sex:
not specified
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.
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
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

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.

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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

No in vitro skin sensitisation studies were identified, or are required, as reliable in vivo studies are already available.

Repeated topical exposure of BALB/c strain mice to chemical contact allergens has been shown to provoke characteristic cytokine secretion profiles consistent with the divergent activation of discreet T cell subpopulations (Dearman et al., 1998; 2002).

 

Dihydrogen hexahydroxyplatinate was subjected to a modified LLNA, combined with a detailed cytokine fingerprinting assay, to evaluate its allergenic potential. 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 sensitiser. Type 1 and type 2 cytokine profiles were compared with those of the known respiratory sensitisers TMA and AHCP, the contact sensitiser 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 (Dearman and Kimber, 2011). The results of this scientifically valid approach fit with current industrial experience to date.

 

It is concluded, based on the available experimental data, and industrial experience to date, that dihydrogen hexahydroxyplatinate does not represent a significant skin sensitisation hazard.

 

Although it is not considered necessary to repeat the LLNA, it is recommended that a lack of skin sensitising potential be confirmed using a standard in vivo assay for skin sensitisation using guinea-pigs, following current international guidelines.

 

References

Dearman RJ et al. (2002). Cytokine fingerprinting of chemical allergens: species comparisons and statistical analyses. Food and Chemical Toxicology 40, 1881 -1892.

 

Dearman RJ et al. (1998). Selective induction of type 2 cytokines following topical exposures of mice to platinum salts. Food and Chemical Toxicology 36, 199 -207.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

At present there is no validated or widely accepted standard test protocol to detect respiratory allergenicity of low molecular weight compounds. No sensitisation data involving inhalation exposure of experimental animals are available for dihydrogen hexahydroxyplatinate. A new study was not conducted as it is not a REACH Standard Information Requirement. Nevertheless, a good-quality and scientifically-acceptable study involving dermal exposure of mice is available for dihydrogen hexahydroxyplatinate, providing results which are considered suitable and sufficiently valid to form the basis of a preliminary assessment of respiratory sensitisation.

 

Repeated topical exposure of BALB/c strain mice to chemical respiratory allergens has been shown to provoke characteristic cytokine secretion profiles consistent with the divergent activation of discreet T cell subpopulations (Dearman et al., 1998; 2002).

 

Dihydrogen hexahydroxyplatinate 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. The cytokine profiles were compared with the recognised respiratory sensitisers TMA and AHCP, as well as the platinum substance TPC, considered to be a non-sensitiser to the respiratory tract based on occupational study data. Detailed cytokine fingerprinting indicated that it was not a significant respiratory sensitiser, demonstrating a cytokine profile similar to that of TPC (Dearman and Kimber, 2011). The results of this scientifically valid approach fit with current industrial experience to date.

 

It is concluded, based on the available experimental data, and industrial experience to date, that dihydrogen hexahydroxyplatinate does not represent a significant respiratory sensitisation hazard.

 

References

Dearman RJ et al. (2002). Cytokine fingerprinting of chemical allergens: species comparisons and statistical analyses. Food and Chemical Toxicology 40, 1881 -1892.

 

Dearman RJ et al. (1998). Selective induction of type 2 cytokines following topical exposures of mice to platinum salts. Food and Chemical Toxicology 36, 199 -207.

Justification for classification or non-classification

Based on the results of the detailed cytokine profiling analysis on dihydrogen hexahydroxyplatinate in mice, and compared with the results for a number of known respiratory-, contact-, and non-sensitisers, it is concluded that dihydrogen hexahydroxyplatinate is not a significant skin or respiratory tract sensitiser and that non-classification is justified under EU CLP criteria (EU 1272/2008). The results of this assessment fit with current industrial experience of handling this substance.

 

It is proposed that a lack of significant skin sensitising potential could be confirmed using a standard in vivo assay for sensitisation using guinea-pigs. Results from such a study would add to the LLNA and cytokine fingerprinting data described above.