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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 July 2017 - 14 August 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
According to the OECD Test Guideline, evaluation of LLNA results is based on the measurement of 3H-thymidine incorporation into the lymph node cells in the first place, it is state that “other endpoints for assessment of proliferation may be employed provided there is justification and appropriate scientific support”. Lymph node cell count is more direct measurement of cell proliferation than determination of DNA synthetis and therefore considered an appropriate parameter for evaluation of cell proliferation in the assay.
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
Phosphonic acid, calcium salt (2:1)
Cas Number:
13780-04-6
Molecular formula:
Ca·2H3O3P
IUPAC Name:
Phosphonic acid, calcium salt (2:1)
impurity 1
Chemical structure
Reference substance name:
Phosphonic acid
EC Number:
237-066-7
EC Name:
Phosphonic acid
Cas Number:
13598-36-2
Molecular formula:
H3O3P
IUPAC Name:
Phosphorous acid
impurity 2
Reference substance name:
Minor impurities not relevant for classification
IUPAC Name:
Minor impurities not relevant for classification
additive 1
Chemical structure
Reference substance name:
Silicon dioxide
EC Number:
231-545-4
EC Name:
Silicon dioxide
Cas Number:
7631-86-9
Molecular formula:
O2Si
IUPAC Name:
dioxosilane
Test material form:
solid: particulate/powder
Details on test material:
- Density: 2.13 g/cm3- Moisture content: 4.0% (w/w)

In vivo test system

Test animals

Species:
mouse
Strain:
CBA:J
Remarks:
CBA/JRj
Sex:
female
Details on test animals and environmental conditions:
Female CBA/J (CBA/JRj) strain mice (SPF caw) were supplied by Elevage Janvier Labs (F-53941 Le Genest Saint Isle). On receipt the animals were randomly allocated to cages. The animals werenulliparous and non-pregnant. After an acclimatisation period of at least five days under stabling and nutritional conditions identical to those of the test, the animals were selected at random and given anumber unique within the study by indelible ink-marking on the tail and a number written on a cage card.At the start of the main study the animals were 8 or 10 weeks old.The animals were weighed at the beginning and at the end of the study.

Study design: in vivo (LLNA)

Vehicle:
other: Pluronic L92 (1%) (CAS 9003-11-6)
Concentration:
0 - 10 - 25 - 50 %
No. of animals per dose:
4
Details on study design:
PRE-SCREEN TESTS:As no information was available regarding irritant potential or systemic toxicity of the test item in the mouse, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 μL of the test item diluted at 50% in Pluronic® L92 at 1% (maximun concentration administrable) to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed daily from Day 1. Any signs of toxicity or excessive local irritation noted during this period were recorded. Ear thickness was recorded on Day 1, Day 3 and on Day 6. The bodyweight of the mouse was recorded on Day 1 (prior to dosing) and Day 6.MAIN STUDYANIMAL ASSIGNMENT AND TREATMENTCriteria used to consider a positive response:Ear thickness measurements and recording of local reactions were performed in order to assess any possible irritant effect of the test item, as possible irritancy may be involved in false positive lymphoproliferative responses.On day 1 and on day 3 (before application) as well as on day 6 (after sacrifice) of each experiment, the thickness of the right ear of each animal of the vehicle control and treated groups was measured by a micrometer.Furthermore, on day 6, punch biopsies of 8 mm in diameter of the apical area of both ears were prepared and weighed in order to assess the irritation potential of the test item and the two lymph nodes per mouse were weighed.Any irritation reaction (erythema and oedema) was recorded in parallel. Any other observation (dryness, presence of residual test item...) was noted.The irritation level of the test item is determined according to the following table:% increase in ear thickness between day 1 and day 3 and/or between day 1 and day 6Interpretation< 10 %Non irritant10 – 25 %Slightly irritant*> 25 %IrritantThe test item should also be considered as an irritant if the score of erythema is higher or equal to 3.On day 6 (end of the test), the animals were euthanized with sodium pentobarbital (Dolethal®). The draining auricular lymph nodes from the four mice were excised.A single cell suspension of the lymph node cells of 4 mice of each group was prepared by gentle mechanical tissue disaggregation through a 200-mesh cell strainers in 4 mL of PBS (Ca2+ / Mg2+ - free) containing 0.5% BSA into a well of a multi-well 6.10 μL of this cell suspension was diluted in 10 mL of physiological saline solution (NaCl 0.9%). The lymphocyte cells were counted using a cell counter (Beckman Coulter Z2).For the run, the lower size selected was 5 μm and the upper size selected was 15 μm (the average size of a lymphocyte is 8 μm).TREATMENT PREPARATION AND ADMINISTRATION:Groups of four mice were treated with the test item diluted at the concentrations of 50%, 25% and 10% in Pluronic® L92 at 1%. The mice were treated by daily application of 25 μL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2 and 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.A further group of four mice received the test vehicle alone in the same manner.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
An EC1.4 value of 7.41% was observed for the test item α-Hexylcinnamaldehyde, resulting in a classification as a sensitiser in Category 1, Sub-category 1B, in accordance with the Regulation EC No. 1272/2008 on classification, labelling and packaging of substances and mixtures.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
0.94
Test group / Remarks:
10%
Key result
Parameter:
SI
Value:
1.16
Test group / Remarks:
25%
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
50%
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATADETAILS ON STIMULATION INDEX CALCULATIONThe proliferation response of lymph node cells was expressed as the number of lymphocytes per lymph node and as the ratio of lymphocytes into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).Results are expressed as the Stimulation Index (SI).When using the pooled approach, the SI is calculated according to the following formula: SI = cell count of treated group / cell count of control groupThe test item will be regarded as a sensitiser if at least one concentration of the test item results is greater than 1.4 compared to control values.EC CALCULATION:The EC1.4 value (theoretical concentration resulting in a SI value of 1.4) was determined by linear interpolation of points on the dose-response curve, immediately above and below the 1.4-fold threshold. The equation used for calculation of EC1.4 was:EC1.4 = c + [(1.4 – d) / (b – d)] x (a – c)Legend: a=thelowestconcentrationgivingstimulationindex>1.4b = the actual stimulation index caused by ac = the highest concentration failing to produce a stimulation index of 1.4 d = the actual stimulation index caused by cAccording the validated protocol based on the use of cell counting (by cell counter) to measure cell proliferation, the EC1.4 replace the EC3 to subcategorise the skin sensitizer.According to the O.E.C.D. Test Guideline, evaluation of LLNA results is based on the measurement of 3H-thymidine incorporation into the lymph node cells in the first place, it is state that “other endpoints for assessment of proliferation may be employed provided there is justification and appropriate scientific support”. Lymph node cell count is more direct measurement of cell proliferation than determination of DNA synthetis and therefore considered an appropriate parameter for evaluation of cell proliferation in the assay.For cell count indices such cut-off values are much lower: 1.4 times increase of stimulation index. This is understandable by the facts that cell count indices have:- lower individual variance compared to 3H-thymidine incorporation- lower maximum stimulation indices compared to radioactive labellingCLINICAL OBSERVATIONS:No mortality and no signs of systemic toxicity were noted in the test and control animals during the test.BODY WEIGHTS:Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

Any other information on results incl. tables

No mortality and no sign of systemic toxicity were noted in the treated and control animals during the test.

No cutaneous reaction was observed.

No increase in ear thickness and ear weight was recorded at the concentrations of 10%, 25% and 50%. Therefore, the test item has to be considered as not excessively irritant at these concentrations.

Groups

Test item

Ear thickness increase D6/D1 (%)

Biopsy ear weight Increase (%)

Excessive irritation

1

Pluronic 1%

0.0

n.a

No

2

10%

1.3

-0.1%

No

3

25%

3.9

1.6%

No

4

50%

-3.6

-2.3%

No

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In view of these results, under these experimental conditions, the test item CALCIUM DIHYDROGENPHOSPHITE does not have to be classified as a sensitizer, in accordance with the criteria for classification, packaging and labelling of dangerous substances and mixtures of the Regulation No. 1272/2008.No signal word or hazard statement is required.