Iodonium, bis[4-(1,1-dimethylethyl)phenyl]-, hexafluorophosphate(1-) (1:1)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 - 22 Nov 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

bis(4-tert-butylphenyl)iodanium; hexafluoro-λ⁵-phosphanuide

EC Number:

620-341-4

Cas Number:

61358-25-6

Molecular formula:

C20H26F6IP

IUPAC Name:

bis(4-tert-butylphenyl)iodanium; hexafluoro-λ⁵-phosphanuide

Details on test material:

- Name of test material (as cited in study report): PF-6
- Analytical purity: > 98%
- Lot/batch No.: 2010 1020
- Expiration date of the lot/batch: 2012-04-12
- Storage condition of test material: at room temperature

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaCrl (pre-tests) and CBA/CaOlaHsd (main experiment)

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River UK, Margate, UK (pre-tests), Harlan Laboratories B.V., AD Horst, The Netherlands (main study)
- Age at study initiation: 1st pre-test: 10-11 weeks; 2nd pre-test: 8-9 weeks; main study: 10-11 weeks
- Weight at study initiation: 1st pre-test: 17.3 and 21.6 g; 2nd pre-test: 17.3 and 19.0 g; main study: 20.2 ± 1.1 g
- Housing: animals were housed in groups of 4 in Makrolon Type II / III, with wire mesh top (EHRET GmbH, Emmendingen, Germany) and granulated soft wood bedding (Rettenmaier & Söhne GmbH + Co. KG, Rosenberg, Germany)
- Diet: pelleted standard diet (Harlan Laboratories B.V., AD Horst, Netherlands), ad libitum
- Water: tap water (Gemeindewerke, Rossdorf, Germany), ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 32-65
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

methyl ethyl ketone

Concentration:

1, 2.5 and 5% (w/v)

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: the compound solubility was determined according to the recommendations given in OECD guideline 429. The highest test substance concentration which can be technically used was 25% in methyl ethyl ketone. Vortexing of the test item was necessary to formulate the test item. At higher concentrations, an applicable formulation of the test item was not achieved, neither by the use of other vehicles nor by using additional methods to formulate the test item (e.g. sonication and warming to 37°C).
- Irritation: to determine the highest non-irritant dose that does at the same time not induce signs of systemic toxicity, two mice were treated once daily on three consecutive days with the test substance at concentrations of 10 and 25% in methyl ethyl ketone by topical (epidermal) application to the dorsal surface of each ear. Clinical signs were recorded once daily during the study period. Signs of local skin irritation were documented and a score was used to grade a possible reddening of the ear skin. Prior to the first application of the test item (on Day 1), on Day 3 and before sacrifice (on Day 6) the ear thickness was determined using a micrometer (S0247 Kroeplin, 36381 Schlüchtern, Germany). In addition, the initial and terminal body weights of each animal were determined. Furthermore, ear punch weights of both animals were determined on Day 6. On Days 2 to 4, animals treated with the test item showed an erythema of the ear skin (Score 1). On Day 5, the animal treated with 10% test item concentration did not show any signs of local skin irritation, but loss of weight was observed (weight reduction of 18.5%). The animal treated with 25% still showed an erythema of the ear skin (Score 1) and additionally a reduction in spontaneous activity. Therefore, a second pre-test was performed using test item concentrations of 2.5 and 5%. No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the second pre-experiment. Thus, the test item in the main study was assayed at 1, 2.5, and 5%. The highest concentration tested was the highest level that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation in the pre-experiment.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: ³H-methyl thymidine (³HTdR) incorporation determined by β-scintillation
- Criteria used to consider a positive response: the test substance is considered a skin sensitiser in the LLNA if the following criteria were met:
(1) the exposure to at least one concentration of the test substance resulted in an incorporation of ³HTdR at least 3-fold greater than in controls (as indicated by the stimulation index) and
(2) the data were compatible with a conventional dose response, although allowance had to be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION: each animal was treated by topical application of 25 μL of the different test substance concentrations and the respective vehicle (methyl ethyl ketone as vehicle control) on the entire dorsal surface of each ear. The application was repeated on Days 2 and 3. On Day 6, each animal was treated with of phosphate-buffered saline (PBS) containing 19.8 µCi of ³HTdR (equivalent to 79.2 µCi/mL ³HTdR) by intravenous injection via the tail vein. Approximately 5 h after treatment with ³HTdR, mice were euthanised by intraperitoneal injection of sodium pentobarbital and the draining auricular lymph nodes were rapidly excised. The nodes of each animal were pooled (8 nodes per group) and single cell suspensions (SCS) in PBS were prepared by gentle mechanical disaggregation of each pooled lymph node cells (LNCs) through a stainless steel gauze (200 µm mesh size). LNCs were pelleted by centrifugation, and washed twice with PBS. After removal of the washing solution, pellets were resuspended in 5% trichloroacetic acid and incubated at 4 °C for at least 18 h for precipitation of macromolecules.The precipitates were resuspended in 5% trichloroacetic acid (1 mL), and transferred to plastic scintillation vials with 10 mL of ‘Ultima Gold’ scintillation liquid (Perkin Elmer (LAS) GmbH, Rodgau, Germany) and thoroughly mixed. The level of ³HTdR incorporation was measured on a scintillation counter (Tricarb 2900 TR, Perkin Elmer (LAS) GmbH, Rodgau, Germany). Similarly, background ³HTdR levels were also measured in two aliquots (1 mL) of 5% trichloroacetic acid.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Mean values and standard deviations of body weights and DPM values were calculated. Statistical analysis of the mean DPM values was performed between treated and control groups to assess a dose-response relationship.

Results and discussion

Positive control results:

A significant increase in the stimulation indices (SI ≥ 3) was noted for the positive control substance hexyl cinnamic aldehyde in acetone:olive oil (4+1) at concentrations of 25% (SI = 8.8), respectively.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: 1% (w/v) test substance: 3.70 2% (w/v) test substance: 2.16 5% (w/v) test substance: 6.02 The EC3 value could not be calculated, due to the unusual dose response.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

The lymph nodes of each group were pooled and group DPM values were measured from the pooled lymph node cell suspensions. The measured group DPM values were corrected by subtracting the background DPM value (average of the two measured DPM values of 5% (w/v) trichloroacetic acid solutions). Treatment with 1, 2.5 and 5% (w/v) test substance in methyl ethyl ketone resulted in group DPM/lymph node of 1005.8, 587.7 and 1636.8, respectively (see Table 1 under "Any other information on results incl. table"). The DPM/lymph node value for the control group was 271.9.

Any other information on results incl. tables

No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

Table1. Calculation of group DPM and SI values

Test item concentration % (v/w)	Group	Measurement DPM	Calculation			Result
			DPM-BG*	Number of lymph nodes	DPM per lymph node**	SI
-	BG I	17	-	-	-	-
-	BG II	18	-	-	-	-
0	1	2193	2176	8	271.9	1.00
1	2	8064	8047	8	1005.8	3.70
2.5	3	4719	4702	8	587.7	2.16
5	4	13112	13095	8	1636.8	6.02

BG = background (1 mL 5% trichloroacetic acid)

1 = control group; 2-4: test groups

SI = stimulation index relative to the mean of the control group (group 1)

* values corrected for mean background value (BG I and BG II)

** Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled.

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

CLP: Skin Sens 1A, H317
DSD: Xi, R43

Executive summary:

The skin sensitising potential of PF-6 was investigated in a Local Lymph Node Assay (LLNA) in mice according to OECD guideline 429 and in compliance with GLP. In a preliminary ranger-finder study, two CBA/CaCrl mice were treated once daily on three consecutive days with the test substance at concentrations of 10 and 25% in methyl ethyl ketone by topical application to the dorsal surface of each ear. On Day 5 of the study, a decreased bodyweight was noted in the animal treated with 10% of the test substance, whereas exposure to 25% test substance concentration resulted in reduced spontaneous activity and skin erythema on the ears of the other animal. Therefore, a second pre-test was performed using test item concentrations of 2.5 and 5% in methyl ethyl ketone. No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the second pre-experiment. Based on these results, the test item was assayed at 1, 2.5, and 5% in methyl ethyl ketone in the main study. In this assay, the test substance formulations or the vehicle alone were applied topically to the dorsal surface of each ear lobe (25 µL/ear) of 4 female CBA/CaOlaHsd mice per test group for three consecutive days. Five days after the first topical application, animals were sacrificed and the cell proliferation of pooled lymph nodes from all animals per group was measured by incorporation of ³H-methyl thymidine and expressed as the amount of radioactive disintegration per minute (DPM). The mean DPM/lymph node for each test group was 1005.8, 587.7 and 1636.8 at concentrations of 1, 2.5, and 5% of the test substance, respectively. Thus, treatment with test substance resulted in increased DPM/lymph node values compared to controls (DPM/node = 271.9). Based on these results, stimulation indices of 3.70, 2.16 and 6.02 were calculated for the treatment concentrations of 1, 2.5, and 5%, respectively. No clear dose-response relationship was observed. The EC3 value could not be calculated, since the obtained SI at the lowest concentration already was above the threshold value of 3. For classification purposes, the EC3 value was considered to be ≤ 2% in a worst case approach. The historical positive control hexyl cinnamic aldehyde at concentrations of 25% in acetone:olive oil (4+1) confirmed the sensitivity and reliability of the experimental technique (SI ≥ 3). Under the above mentioned conditions, the test substance was found to be a sensitiser in the LLNA.