Heparin, sodium salt

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

Experimental start date : 7 November 2012 - Experimental completion date: 12 November 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP, guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Heparin, sodium salt

Cas Number:

9041-08-1

IUPAC Name:

Heparin, sodium salt

Details on test material:

Test article identification: Heparin sodium (P10043)
Test article description: Powder
Batch 12PL5716
Storage conditions bulk material: room temperature
Retest or expiry date: 11 june 2005

Test system

Details on study design:

Principle of the test:
The Rhe-based test method measures the initiating events occuring in skin irritation, beginning with penetration of the stratum corneum and damage to the underlying layers of keratinocytes.
It moreover directly covers the initial step of the inflammatory cascade/mechanism of action (cell damage and tissue damage resulting in localized trauma) that occurs during irritation in vivo.

Test description:
The experimental design is based on the assessment of the potential skin irritation induced by the test article applied on three epidermis with comparison with negative and positive controls. The test article, the positive and negative controls are applied topically to each epidermis, depending on the physical form of the test article:
- solid powder: 11+/- 1.5 mg (to be crushed to a fine powder if needed using a mortar and a pestle)
The duration of exposure is approximately 15 minutes. The post-treatment incubation is approximately 42 hours at 37+/-2°C, 5+/-1% CO2. The viability is assessed after approximately a 3-hour incubation with MTT solution in a 12-well plate (0.3 mg/ml; 2ml per well).

Reading of optical density - viability determination
Cell viability in RhE model is measured by enzymatic conversion of the yellow vital dye MTT into blue formazan salt that is quantitatively measured after extraction from tissues. The precipitated formazan is extracted with acidified isopropanol (0.5 ml) and quantified spectrophotometrically at 570 nm +/- 30 nm (200 µl/well). The Mean Optical Density (MOD) is the mean calculated from measurement on 6 wells, with 2 wells per epidermis.

Data evaluation
the relative survival (mean viability in %) of each treated epidermis is determined by comparing the corrected MOD of the 3 wells treated with the test article or positive control to the corrected MOD of the 3 negative control wells.

Experiment acceptance criteria
The experiment is considered valid when the following acceptance criteria are fulfilled:
- mean OD value from the 3 tissues treated with NC should be >= 0.6 and =<1.5;
- mean PC viability expressed as % of NC should be <=40%;
- SD of % viability from NC and PC should be <= 18%
A test article is considered to induce an acceptable response in the skin irritation test when the criteria listed below are fulfilled:
- test article induces the same effect on each epidermis: all individual values for viability should be either <= 50% or all individual values should be >50%
- SD of viability from test article-treated epidermis should be <=18%
Results which only partially satisfy one of the above criteria are dealt on a case by case basis, taking into account the reproducibility and the biological relevance.
When the experiment does not meet the acceptance criteria, additional experiment may be performed.


Positive control: 5 % aqueous sodium dodecyl sulphate (SDS)
Negative control: phosphate buffered saline (D-PBS)

Results and discussion

Any other information on results incl. tables

Mean results for optical density, viability (in%) and standard deviation

	Mean OD	Mean viability (%)	Standard deviation OD Viability (%)
Negative control	0.801	100.0	0.09	10.72
Positive control	0.172	21.4	0.05	6.53
Heparin sodium	0.798	99.6	0.10	13.06

With Heparin sodium, the mean viability was higher than 50% which indicated a non-irritant potential.

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

In conclusion, under these experimental conditions, the test article Heparin sodium was considered as non-irritant to the skin.