Strontium nitrate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-06-02 to 2010-06-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study reliable without restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals, Draft Proposal for a New Guideline: In vitro skin irritation: Reconstructed Human Epidermis (RhE) Test method

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Commission regulation (EC) No. 440/2008 B.46

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Strontium nitrate

EC Number:

233-131-9

EC Name:

Strontium nitrate

Cas Number:

10042-76-9

Molecular formula:

Sr(NO3)2

IUPAC Name:

strontium nitrate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Strontium nitrate
- Molecular formula: Sr(NO3)2
- Molecular weight: 211.63
- Physical state: White crystals
- Storage condition of test material: At room temperature in the dark
- Stability under storage conditions: Stable
- pH (1% in water, indicative range): 6.4 - 7.0 /determined at NOTOX)
- Stability in water: Not indicated
- Solubility in water: Yes
No further information on the test material was stated.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: Adult human-derived epidermal keratinocytes

Cell source:

other: three-dimensional human epidermis model

Justification for test system used:

The EPISKIN model has been validated for irritation testing in an international validation study and its use is recommended by the relevant OECD guideline for irritation testing and therefore, it was considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Details on test system:

TEST SYSTEM
EPISKIN Standard Model TM (EPISKIN-SM TM, 0.38 cm2, Lot no.: 10-EKIN-023; Source: SkinEthic Laboratories, Nice, France).
This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

TEST FOR REDUCTION OF MTT BY THE TEST SUBSTANCE
Strontium nitrate was checked for possible direct MTT reduction before the study was started. To assess the ability of the test substance to reduce MTT, approximately 10 mg of test substance was added to a 12 well plate filled with 2 ml MTT solution (0.3 mg/ml in PBS). The mixture was incubated for approximately 3 hours at 37 °C. A negative control, sterile Milli-Q water was tested concurrently.
Because no colour change was observed it was concluded that barium chloride dihydrate did not interact with MTT.

APPLICATION/TREATMENT OF THE TEST SUBSTANCE
The test was performed on a total of 3 tissues per test substance together with a negative control (Phosphate buffered saline (PBS, Invitrogen Corporation, Breda, the Netherlands) and a positive control (5% (aq) Sodium dodecyl sulphate (SDS, Sigma Aldrich, Zwijndrecht, The Netherlands, CAS Number 151-21-3). At least 10 mg solid (with a small glass weight boat) with 5 µl Milli-Q water (Millipore Corp., Bedford, Mass., U.S.A.) was added into 12-well plates on top of the skin tissues. Three tissues were treated with 10 µl PBS (negative control) and 3 tissues with 10 µl 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test substance. After rinsing the cell culture inserts were each dried carefully. The skin tissues were kept in new 12-well plates on 2 ml pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37 °C.

CELL VIABILITY MEASUREMENT
After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 ml MTT-medium (0.3 mg/ml). The tissues were incubated for 3 h at 37 °C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in prelabeled microtubes and extracted with 500 µl isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for 70 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the Multiskan Spectrum (Thermo Labsystems).
Cell viablity was calcuated for each tissue as a percentage of the mean of the negative control tissues. Skin irritation potential of the test substance was classified according to remaining cell viability following exposure of the test substance.
No further information on the study design was stated.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): At least 10 mg of the solid test substance was applied directly on top of the skin tissue. Strontium nitrate was spread to match the size of the tissue.

VEHICLE
- Amount(s) applied (volume or weight with unit): Skin tissue was moistened with 5µl of Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact to the tissue.
No further information on the amount/concentration applied was stated.

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3 tissues

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

118

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Any other information on results incl. tables

TEST FOR REDUCTION OF MTT BY THE TEST SUBSTANCE

Because no colour change was observed it was concluded that strontium nitrate did not interact with MTT.

Results after treatment with test substance:

Table 1. Individual OD measurements at 570 nm

	A (OD570)	B (OD570)	C (OD570)
Negative control OD570measurement 1 OD570measurement 2	0.630 0.564	0.618 0.595	0.619 0.598
Strontium nitrate OD570measurement 1 OD570measurement 2	0.748 0.725	0.703 0.657	0.725 0.714
Positive control OD570measurement 1 OD570measurement 2	0.064 0.062	0.049 0.046	0.054 0.056

OD = Optical density

Triplicate exposures are indicated by A, B and C.

The mean absorption at 570 nm measured after treatment with strontium nitrate and controls are presented in Table 2.

Table 2.  Mean absorption in thein vitroskin irritation test with strontium nitrate

	A (OD570)	B (OD570)	C (OD570)	Mean (OD570)	SD
Negative control	0.597	0.607	0.609	0.604	0.006
Strontium nitrate	0.737	0.680	0.720	0.712	0.029
Positive control	0.063	0.048	0.055	0.055	0.008

 

OD = optical density

SD = Standard deviation

Triplicate exposures are indicated by A, B and C.

In this table the values are corrected for background absorption. Isopropanol was used to measure the background absorption.

 

Table 3 shows the mean tissue viability obtained after 15 minutes treatment with strontium nitrate compared to the negative control tissue.

Table 3. Mean tissue viability in thein vitroskin irritation test with strontium nitrate 

	Mean tissue viability (percentage of control)
Negative control	100
strontium nitrate	118
Positive control	9

The positive control had a mean cell viability after 15 minutes exposure of 9 %.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

It is concluded that this test is valid and that strontium nitrate is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.