Talc (Mg3H2(SiO3)4)

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation / corrosion, other

Remarks:

validated "in vitro" test method

Type of information:

other: Published data

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Reliability score 2 on the basis of the weight of evidence found during review of public documents existing relating to toxicological data

Justification for type of information:

Pure talc has the formula Mg3Si4O10(OH)2 and a chemical composition of 31.88% by weight (wt) magnesium oxide (MgO), therefore the health effects of Magnesium chloride also should be taken into account

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Skin sensitization, false positives and false negatives: experience with guinea pig assays

Author:

Basketter DA, Kimber I.

Year:

2010

Bibliographic source:

J Appl Toxicol. 2010 Jul;30(5):381-6

Reference 2

Reference Type:

publication

Title:

The ECVAM international validation study on in vitro tests for acute skin irritation: report on the validity of the EPISKIN and EpiDerm assays and on the Skin Integrity Function Test

Author:

Spielmann H, Hoffmann S, Liebsch M, Botham P, Fentem JH, Eskes C, Roguet R, Cotovio J, Cole T, Worth A, Heylings J, Jones P, Robles C, Kandárová H, Gamer A, Remmele M, Curren R, Raabe H, Cockshott A, Gerner I, Zuang V.

Year:

2007

Bibliographic source:

Altern Lab Anim. 2007 Dec;35(6):559-601.

Reference 3

Reference Type:

publication

Title:

In vitro skin irritation testing: Improving the sensitivity of the EpiDerm skin irritation test protocol.

Author:

Kandárová H, Hayden P, Klausner M, Kubilus J, Kearney P, Sheasgreen J.

Year:

2009

Bibliographic source:

Altern Lab Anim. 2009 Dec;37(6):671-89.

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: EU method B46 (irritation)

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

other: ECVAM international validation study on in vitro tests for acute skin irritation (Altern Lab Anim. 2007 Dec; 35 (6):559-601)

Deviations:

no

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline for the testing of chemicals; Draft proposal for a new guideline, in vitro skin irritation: Reconstructed Human Epidermis (RhE) Test method,Version 4, 11 December 2009.

Deviations:

no

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

Magnesium chloride

EC Number:

232-094-6

EC Name:

Magnesium chloride

Cas Number:

7786-30-3

IUPAC Name:

magnesium dichloride

Test material form:

solid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: epidermal keratinocytes

Cell source:

other: adult human-derived epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis.

Details on animal used as source of test system:

The test was carried out with the reconstituted three-dimensional human skin model EPISKIN-SM™ (Skinethic). This skin model consists of normal (non-cancerous), adult human-derived epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK are cultured on chemically modified, collagen-coated cell culture inserts. A highly differentiated and stratified epidermis model is obtained after 13-day culture period ans comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Test animals

Species:

human

Strain:

other: epidermal keratinocytes

Details on test animals or test system and environmental conditions:

The test was carried out with the reconstituted three-dimensional human skin model EPISKIN-SM™ (Skinethic). This skin model consists of normal (non-cancerous), adult human-derived epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK are cultured on chemically modified, collagen-coated cell culture inserts. A highly differentiated and stratified epidermis model is obtained after 13-day culture period ans comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Test system

Vehicle:

water

Controls:

other: not applicable

Amount / concentration applied:

TEST MATERIAL AND VEHICLE
- Amount(s) applied (volume or weight with unit): Firstly, 5µl distilled water (A. dest.) were applied by a pipette to the epidermal surface in order to improve further contact between the test item and the epidermis. The water was gently spread with the pipette. Afterwards approximately 10 mg (26.3 mg/cm^2) of powder were applied to the epidermis surface.

Duration of treatment / exposure:

15 +/- 0.5 min.

Observation period:

42 h postincubation period and immediate determination of cytotoxic effects via MTT reduction assay

Number of animals:

not applicable

Details on study design:

CONTROLS:
Controls were set up in parallel to the test item cultures in order to confirm the validity of the test.
Negative control: Sterile water (Delta Select, Lot No. 12903C)
Positive control: 5% sodium dodecyl sulfate (SDS; AppliChem, Art.-No.A1112,0500, CAS No.: 151-21-3, Lot No. 8T009926) in A. dest.

DOSE GROUPS:
1. Negative control: 10 µL sterile water
2. Positive control: 10 µL 5 % SDS solution
3. Test item: 10 mg +/- 5 µL A. dest.
The test was performed on a total of 3 tissues per dose group.

TEST SYSTEM:
The test was carried out with the reconstituted three-dimensional human skin model EPISKIN-SMTM (Skinethic). This skin model consists of normal (non-cancerous), adult human-derived epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK are cultured on chemically modified, collagen-coated cell culture inserts. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
MTT SOLUTION:
- MTT stock solution: 3 mg/mL MTT in PBS
- MTT medium: MTT stock solution will be diluted 1 + 9 with assay medium (final concentration 0.3 mg/mL). The solution was kept at -20 °C for not longer than 1 year and was not thawn more than 10 times.

PRE-EXPERIMENT:
To check the MTT-reducing capability of the test item 10 mg of the test item were mixed with 2 mL MTT medium and incubated for 1 h at room temperature. If the mixture turns blue/purple, the test item is presumed to have reduced MTT.

EXPERIMENTAL PROCEDURE:
Upon receipt of the Episkin-SMTM, the tissues were transferred into 12-well plates containing 2 mL prewarmed maintenance medium per well. The 12-well plates were incubated in a humidified incubator at 37 +/- 1 °C, 5.0 % CO2 for at least 24 h.
After this pre-incubation the tissues were treated with each dose group in triplicate, starting with the negative control. Start time was recorded with dosing of the first tissue. Then the tissues were incubated at room temperature for 15 +/- 0.5 min. Afterwards, the tissues were washed with PBS to remove any residual test item. Excess PBS was removed by blotting bottom with blotting paper. The inserts were placed in a prepared 12-well plate containing 2 mL prewarmed fresh maintenance medium and post-incubated at 37 +/- 1 °C, 5.0 % CO2, humidified to 95 % for 42 +/- 1h.
After this incubation period the inserts were transferred in a prepared 12-well plate containing 2 mL prewarmed MTT medium and further incubated for approximately 3 h +/- 5 min. at 37 +/- 1 °C, 5.0% CO2, humidified to 95%.
After the 3 h MTT incubation period the tissues were placed on blotting paper to dry the tissues. Afterwards a total biopsy of the epidermis by using the special biopsy punch was performed and the epidermis was separated from the collagen mytrix with the aid of forceps. Both parts (epidermis and collagen matrix) were transferred into suitable tubes and 500 µL of acidic isopropanol (0.04 N HCl in isopropanol) was added. Extraction was carried out protected from light over the weekend at 2 - 8 °C.At the end of the formazan extraction period the tubes were mixed by vortexing until solution colour became homogeneous.
If any visible cell/tissue fragments are in suspension, the tubes were centrifuged at 500 rpm to eliminate the fragments and avoid further possible interference with the absorbance readings.
Per each tissue 2 X 200 µL aliquots of the extract were transferred into a 96-well plate and OD was measured at 550 nm without reference wavelength in a plate spectrophotometer.

DATA ANALYSIS:
Irritant potential of the test item was predicted from the relative mean tissue viabilities compared to the negative control tissues concurrently treated with aqua dest. The test item is considered to be irritating to skin (R38), if the tissue viability after 15 min of exposure and 42 h of post-incubation is less or equal to 50%.

TEST ACCEPTANCE CRITERIA:
The test meets acceptance criteria if:
- mean OD550nm of the three negative control tissues is ≥ 0.6
- mean relative tissue viability of the three positive control tissues is ≤ 30 %
- the standard deviation (SD) obtained from the three concurrently tested tissues is ≤ 18%
No further information on the study design was stated.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: relative tissue viability (%)

Value:

112.9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

The mean relative tissue viability (% negative control) was ≥ 50% (112.9%) after 15 min treatment and 42 h post incubation.

In vivo

Results

Irritation parameter:

other: relative tissue viability (%)

Basis:

mean

Time point:

other: after 15 min incubation

Score:

112.9

Max. score:

128.8

Reversibility:

no data

Remarks on result:

other: OD550 nm

Irritant / corrosive response data:

The test item showed no irritant effects. The mean relative tissue viability (% negative control) was ≥ 50% (112.9%) after 15 min treatment and 42 h post incubation.

Any other information on results incl. tables

PRE-EXPERIMENT:

The mixture did not turn blue/purple, therefore the test item itself did not reduce MTT. The OD550 was 0.079 (blank: 0.084).

EXPERIMENT:

Tissue Replicates	Negative control   1             2           3			Positive control     1            2            3			Test item     1             2            3			Blank    1             2           3
Absolute OD550-values	0.736	0.744	0.817	0.196	0.207	0.214	0.920	0.824	0.819	0.043	0.043	0.044
	0.759	0.764	0.856	0.207	0.208	0.227	0.980	0.864	0.839	0.043	0.044	0.044
net OD550-values	0.693	0.701	0.774	0.153	0.164	0.171	0.877	0.781	0.776
	0.716	0.721	0.813	0.164	0.165	0.184	0.937	0.821	0.796
Net mean of the duplicats	0.704	0.711	0.793	0.158	0.164	0.177	0.907	0.801	0.786
Net mean of 3 replicate tissues	0.736*			0.166			0.831
Tissue viablility [%]	100.0	100.9	112.6	22.4	23.3	25.1	128.8	113.7	111.6
SD tissue viability [%] ***	7.0			1.4			9.4
Mean relative tissue viability	100.0			22.6			112.9

* mean OD550nm of the three negative control tissues is ≥0.6

** mean relative tissue viability of the three positive control tissues is ≤30 %

*** the standard deviation (SD) obtained from the three concurrently tested tissues is ≤ 18%

 

Applicant's summary and conclusion

Interpretation of results:

other: not irritating

Conclusions:

In this study under the given conditions the test item showed no irritant effects. The test item is classified as "non irritant (NI)".
The test item should not be classified and labeled as skin irritant according to Directive 67/548/EEC.
The test item should not be classified and labeled as skin irritant according to regulation (EC) No.: 1272/2008.

Executive summary:

The potential of the test item to induce skin irritation was analysed by using the three-dimensional human skin model EPISKIN-SMä(Skinethic) comprising a reconstructed epidermis with a functional stratum corneum.

In the present study, Magnesium Chloride Hexahydrate was applied topically to the EPISKIN-SMätissue for 15 min followed by a 42 h postincubation period and immediate determination of cytotoxic effects via MTT reduction assay.

Irritant potential of the test item was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with sterile water.

The test item showed no irritant effects. The mean relative tissue viability (% negative control) was =>50 % (112.9 %) after 15 min treatment and 42 h post incubation. The controls confirmed the validity of the study. The mean OD550 of the three negative control tissues was =>0.6. The mean relative tissue viability (% negative control) of the positive control was =< 30 % (22.6 %). The standard deviation of replicate tissues of all dose groups was <= 30 % (1.4 % - 9.4 %).