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Toxicological information

Exposure related observations in humans: other data

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Administrative data

Endpoint:
exposure-related observations in humans: other data
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: non GLP

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2001
Report Date:
2011

Materials and methods

Type of study / information:
Test that correlates in vitro data with in vivo data for assessing skin irritation potential was made.
Endpoint addressed:
skin irritation / corrosion
Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
The test substance was chosen as model irritants and a spontaneously immortalized human keratinocyte line, HaCaT, was used as an in vitro model to predict the cutaneous irritation. The end-point used to assess toxicity was uptake of the vital dye neutral red 24 h after dosing. The cytotoxicity data from theses assays were compared with the irritant responses obtained after 24 h application of the test substance (100 μL of 20 mmol/L aqueous solution ) to the volar forearm of human volunteers.
GLP compliance:
not specified

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
No data given.

Method

Ethical approval:
confirmed and informed consent free of coercion received
Details on study design:
Cytotoxicity
Cytotoxicity was determined by NR absorption. This assay is based on the uptake of this slightly cationic dye by lysosomes of living cells. 24 h after adding the test substance, the medium as replaced by NR-containing medium (50 μg/mL). After 3 h incubation, the NR-containing medium was removed, cell cultures were carefully washed with phosphate-buffered saline and NR was extracted with 1% acetic acid/50% ethanol. Absorption at 540 nm was measured in an enzyme-linked immunosorbent assay reader and values were expressed as percentage of controls.

Cell culture
The spontaneously immortalized human keratinocyte line HACAT6 was provided by NEFusenig. The keratinocytes were cultured in Dulbecco’s modified Eagle’s medium supplemented with 5% FCS and 1% L-glutamine. The 56th to 78th passages were seeded in subconfluent density (300000 to 500000 cells/mL ) into 96 well plates, and incubated (37 ℃: 95% O2, 5% CO2). After 24 h, cell culture medium was replaced by fresh medium containing the test compounds in various concentrations.

Volunteer studies
Test substance was evaluated at identical concentrations (20 mmol/L). One hundred microlitres were applied to the volar forearm of adult human volunteers according to a randomized and double-blind protocol. The test substance was applied in occlusive polypropylene chambers and left in place for 24 h. After the chambers were removed from the skin the application sites were rinsed with tap water and patted dry with a soft paper towel. The responses were evaluated by objective, noninvasive techniques 4 h after removal the test substance following at least 20 min acclimatization at constant ambient conditions (20-23 ℃, 55-70% relative humidity).
Transepidermal water loss is a measure of the integrity of stratum corneum barrier function. It was measured with a tewameter. The technique has been described in detail elsewhere. Briefly, it is based on water vapour gradient determination between tow hygrosensors placed at different distances perpendicular to the skin. Actual values were corrected to a skin standard temperature of 30 ℃. Erythema was quantified with a tri-stimulus chromameter. In the Lab mode, parameter a represents the coluor spectrum form total green to pure red. It correlates closely with erythema values. All values are given as means of three measurements.
Exposure assessment:
measured
Details on exposure:
TYPE OF EXPOSURE: skin irritation

TYPE OF EXPOSURE MEASUREMENT: Personal sampling

EXPOSURE LEVELS: 100 μL of 20 mmol/L aqueous solution

EXPOSURE PERIOD: 24 h

POSTEXPOSURE PERIOD: 4 h

DESCRIPTION / DELINEATION OF EXPOSURE GROUPS / CATEGORIES: adult human volunteers

Results and discussion

Results:
Test substance had cytotoxic effects as demonstrated by decreased NR uptake, which showed a clear dose-response relationship. Test substance concentration resulting in 50% inhibition of NR uptake (IC50) was 20 mmol/L. A good overall correlation between in vitro cytotoxicity and in vivo irritation potential in humans was found. The epidermal barrier, which greatly limits the percutaneous penetration of xenobiotics in vivo, does not exist in cell culture models.

Any other information on results incl. tables

No in vivo results on the test substance were presented. It was stated that the reaction of non-mentoned compounds (including the test substance) did not induce an increase of erythema compared to control values.

Applicant's summary and conclusion

Conclusions:
Test substance concentration resulting in 50% inhibition of NR uptake (IC50) was 20 mmol/L.
Executive summary:

The test substance was chosen as model irritants and a spontaneously immortalized human keratinocyte line, HaCaT, was used as an in vitro model to predict the cutaneous irritation. The end-point used to assess toxicity was uptake of the vital dye neutral red 24 h after dosing. The cytotoxicity data from theses assays were compared with the irritant responses obtained after 24 h application of the test substance (100 μL of 20 mmol/L aqueous solution ) to the volar forearm of human volunteers.

Test substance concentration resulting in 50% inhibition of NR uptake (IC50) was 20 mmol/L. No data on in vivo results were presented, but the test substance was stated to be non-irritant.