Dihydrogen hexahydroxyplatinate, compound with 2-aminoethanol (1:2)

Endpoint summary

Description of key information

In a GLP mouse local lymph node assay (LLNA): BrdU-ELISA, conducted accroding to OECD guideline 442B, dihydrogen hexahydroxyplatinate compound with 2-aminoethanol (1:2) was not sensitising (Haferkorn, 2016).

 

No respiratory tract sensitisation data are available.

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

Study period:

5 August - 15 September 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted according to GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EC method B.51. Skin Sensitisation: Local Lymph Node Assay: BrdU-ELISA

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: Approximately 8 weeks
- Weight at study initiation: 18 - 23 g
- Housing: Before application the animals were housed in groups of 5 animals in MAKROLON cages (type III) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 15 cm. After application the animals were housed singly in order to prevent them licking off the test item from the ears of the other animals. Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany) was used as bedding material for the cages.
- Diet (e.g. ad libitum): Commercial diet ssniff® R/M-H V1534 (sourced from ssniff Spezialdiäten GmbH, 59494 Soest, Germany) was offered ad libitum. Food residue was removed.
- Water (e.g. ad libitum): Tap water was offered ad libitum.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 3 (maximum range)
- Humidity (%): 55 ± 15 (maximum range)
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 10 September 2015 To: 15 September 2015

Vehicle:

propylene glycol

Concentration:

Pre-screening test: 0.5, 1, 2.5, 5, 10, 25, 50 or 100% (w/w)
Main study: 0, 10, 25 or 50% (w/w)

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS
- Compound solubility: In a preliminary solubility assessment, the following vehicles were assessed: acetone/olive oil (3+1 v/v), N,N-dimethylformamide, Methyl ethyl ketone, Dimethyl sulfoxide and propylene glycol. Propylene glycol gave homogenous suspensions suitable for application of the test item and was used as the vehicle for the LLNA. The test item was suspended in propylene glycol to the appropriate concentrations. The highest feasible concentration was 50%.
In the preliminary study, 0.5, 1, 2.5, 5, 10, 25, 50% and the undiluted test item (100%) were tested in 2 animals per concentration.
- Irritation: All mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Body weights were recorded pre-test and prior to termination (Day 6). Both ears of each mouse were observed for erythema and scored on a 0-4 scale (0: no erythema; 1: very slight erythema (barely perceptible); 2: well-defined erythema; 3: moderate to severe erythema; 4: severe erythema (beet redness) to eschar formation preventing grading of erythema). Ear thickness measurements were taken using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose), and Day 6. Additionally, on Day 6, ear thickness was determined by ear punch weight determinations, which was performed after the animals were humanely sacrificed. Excessive local irritation would be indicated by an erythema score ≥3 and/or ear thickness of ≥25% on any day of measurement. The highest dose selected for the main LLNA: BrdU-ELISA study was selected as the next lower dose in the pre-screen concentration series that did not induce systemic toxicity and/or excessive local skin irritation.
- Lymph node proliferation response: Not assessed

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT:
- Name of test method: Local lymph node assay BrdU-ELISA
- Criteria used to consider a positive response: Results of each treatment group are expressed as the mean SI. The SI is derived by dividing the mean BrdU labelling index/mouse within each test item group and the positive control group by the mean BrdU labelling index for the solvent/vehicle control group. The average SI for the vehicle control is then one. The BrdU labelling index is defined as:

BrdU labelling index = (ABSem – ABS blankem) – (ABSref – ABS blankref)

Where: em = emission wavelength; and ref = reference wavelength

The decision process regards a result as positive when SI ≥1.6. However, the strength of the dose-response relationship, the statistical significance and the consistency of the solvent/vehicle and positive control responses would also be used when determining whether a borderline result (i.e. SI value between 1.6 and 1.9) is declared positive.

For a borderline positive response between an SI of 1.6 and 1.9, it may be necessary to consider additional information such as dose-response relationship, evidence of systemic toxicity or excessive irritation, and where appropriate, statistical significance together with SI values to confirm that such results are positives. Consideration would be also given to various properties of the test item, including whether it has a structural relationship to known skin sensitizers, whether it causes excessive skin irritation in the mouse, and the nature of the dose-response observation.

In addition, the average ear weights per group and the average ear thickness per group were compared to the vehicle control group as an indication for possible irritating properties.

TREATMENT PREPARATION AND ADMINISTRATION: groups of mice were given topical applications to the dorsum of the ear of 25 µl of formulation on three consecutive days (1, 2 and 3). On day 5, animals were injected intraperitoneally with 0.5 mL (5 mg/mouse) of a 10 mg/mL solution of of 5-bromo-2-deoxyuridine (BrdU) and killed 24 hours later. The draining auricular lymph nodes from each mouse ear were excised and processed separately in phosphate buffered saline (PBS) for each animal.

Positive control substance(s):

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

Statistics:

As no concentration-related increased values were observed, no statistical evaluation was carried out. Comparison of positive with negative control was carried out using Student’s t-test.

Positive control results:

Treatment with the positive control item caused the expected increases in the BrdU labelling index (see Table 1). Therefore, the study can be regarded as valid.

Parameter:

SI

Remarks on result:

other: The stimulation indices were 1.040, 0.835 and 0.815 at concentrations of 10, 25 and 50% w/w, respectively.

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Not applicable

Table 1: Stimulation indices (SI)

Parameter	Negative control	10% TM	25% TM	50% TM	Positive control
BrdU labelling index	1.000	1.040	0.835	0.815	1.861
Ear weight	1.000	0.965	1.006	1.047	1.281
Difference of ear thickness (TD 3)	1.000	0.990	1.029	1.029	1.181
Difference of ear thickness (TD 6)	1.000	1.009	1.047	1.047	1.519

Notes:

TM (Test material dihydrogen hexahydroxyplatinate/2-aminoethanol (1:2)

Bold: significantly increased compared to control at p<=0.01

Treatment with the test item at concentrations of 10%, 25% or 50% did not reveal increases in the BrdU labelling index. The stimulation indices of the test item treated groups calculated for the BrdU labelling index did not exceed the threshold value of 1.6. Hence, the test item is classified as not sensitising.

The ear weight (punch biopsies) and the difference of ear thickness on test day (TD) 3 and 6 were not increased, i.e. no irritating properties were noted.

No signs of systemic intolerance were recorded. The animal body weight was not affected by the treatment. Very slight erythema (score 1) was observed in 3 of 5 animals treated with the 50% concentration of the test item and well-defined erythema (score 2) was observed in 5 of 5 animals of the positive control group.

Interpretation of results:

GHS criteria not met

Conclusions:

In a GLP mouse LLNA: BrdU-ELISA, conducted accroding to OECD guideline 442B, dihydrogen hexahydroxyplatinate compound with 2-aminoethanol (1:2) was not sensitising.

Executive summary:

The skin sensitising potential of dihydrogen hexahydroxyplatinate compound with 2-aminoethanol (1:2) has been assessed in a GLP mouse LLNA: BrdU-ELISA, conducted according to OECD Test Guideline 442B. Following a preliminary range-finding study to assess irritancy, female CBA mice (5/group) were treated topically with 0, 10, 25 or 50% dihydrogen hexahydroxyplatinate/2-aminoethanol (1:2) (in propylene glycol) on three consecutive days (1, 2 and 3). On day 6, cell proliferation in the local lymph nodes was measured by incorporation of injected 5-bromo-2-deoxyuridine (BrdU) using ELISA.

 

No mortality or clinical signs of toxicity were observed, and there were no signs of local irritation, as measured by ear weight or thickness differences, at the application site. Very slight erythema was observed in 3 animals at the highest tested concentration, compared to well-defined erythema in all positive control animals. Observed stimulation index values were 1.040, 0.835 and 0.815 at concentrations of 10, 25 and 50% w/w, respectively. Hence, the stimulation indices of the test item treated groups calculated for the BrdU labelling index did not exceed the threshold value of 1.6. Positive and vehicle controls performed as expected.

Under the conditions of this study, dihydrogen hexahydroxyplatinate/2-aminoethanol (1:2) does not require classification for skin sensitisation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

No relevant human skin sensitisation data were identified. No in vitro skin sensitisation studies were identified, or are required, as a reliable in vivo study is already available.

 

The skin sensitising potential of dihydrogen hexahydroxyplatinate compound with 2-aminoethanol (1:2) has been assessed in a GLP mouse LLNA: BrdU-ELISA, conducted according to OECD Test Guideline 442B. Following a preliminary range-finding study to assess irritancy, female CBA mice (5/group) were treated topically with 0, 10, 25 or 50% dihydrogen hexahydroxyplatinate/2-aminoethanol (1:2) (in propylene glycol) on three consecutive days (1, 2 and 3). On day 6, cell proliferation in the local lymph nodes was measured by incorporation of injected 5-bromo-2-deoxyuridine (BrdU) using ELISA. No mortality or clinical signs of toxicity were observed, and there were no signs of local irritation, as measured by ear weight or thickness differences, at the application site. Very slight erythema was observed in 3 animals at the highest tested concentration, compared to well-defined erythema in all positive control animals. Observed stimulation index values were 1.040, 0.835 and 0.815 at concentrations of 10, 25 and 50% w/w, respectively. Hence, the stimulation indices of the test item treated groups calculated for the BrdU labelling index did not exceed the threshold value of 1.6. Positive and vehicle controls performed as expected. Therefore, under the conditions of this study, dihydrogen hexahydroxyplatinate/2-aminoethanol (1:2) does not require classification for skin sensitisation (Haferkorn, 2016).

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

No respiratory tract sensitisation data are available. A new study was not conducted as no standard and validated test method is available and it is not a REACH Standard Information Requirement.

Justification for classification or non-classification

Based on the results of the available and reliable murine LLNA assay, dihydrogen hexahydroxyplatinate compound with 2-aminoethanol (1:2) does not warrant classification for skin sensitisation, according to EU CLP criteria (EC 1272/2008).