The product from the burning of a combination of carbonaceous materials.

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation / corrosion, other

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study was conducted between 2 June and 3 June 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study.

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:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD Guideline No. 431 (April 13, 2004)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: the NIH Publication No. 04-4510 dated on May 2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

No. B 18-023-01

Test material

Test material information

Constituent 1

Reference substance name:

The product from the burning of a combination of carbonaceous materials.

EC Number:

931-597-4

Molecular formula:

UVCB substance, not available. View remarks field.

IUPAC Name:

The product from the burning of a combination of carbonaceous materials.

Details on test material:

- Name of test material: Mixed ash
- Physical state: powder
- Composition range of test material (% (w/w)): Aluminium (Al) 13.07, Calcium (Ca) 45.52 , Iron (Fe) 4.15 , Magnesium (Mg) 3.49, Phosphorus (P) 0.79 , Potassium (K) 2.56, Silicon (Si) 28.07, Sodium (Na) 1.08, Sulphur (S) 1.27.
- The critical minor components examined (mg/kg d.w.): Arsenic (As) 18, Barium (Ba) 670 Cadmium (Cd) 3.6, Copper (Cu) 410, Lead (Pb) 180 and Antimony (Sb) 22.
- Purity test date: the substance is UVCB substance
- Lot/batch No.: MIXED ASH 1-01032010
- Expiration date of the lot/batch: March 2011
- Stability under test conditions: Stable
- Storage condition of test material: Room temperature
- Other: Mixed Ash is named in dossier as Ash. Mixed in the substance name has meant that there have been several (mixed) fuels when producing ash.

Test animals

Species:

other: a three-dimensional human skin model, comprising at least a reconstructed epidermis with a functional stratum corneum

Details on test animals or test system and environmental conditions:

TEST ANIMALS Reconstructed Human Epidermis RHE/S/17
- Source: SkinEthic Laboratories - 45 rue Saint Philippe - Le Palmeira - 06000 Nice - France
- Age at study initiation: generally 17 days at the start of the experiment
- Pre-incubation time: 1 hour

ENVIRONMENTAL CONDITIONS
Maintenance medium

Test system

Type of coverage:

not specified

Preparation of test site:

not specified

Vehicle:

other: The culture medium, a non-corrosive vehicle

Controls:

other: reconstructed epidermis

Amount / concentration applied:

TEST MATERIAL, MIXED ASH
- Amount(s) applied (volume or weight with unit): 19.8 mg, grounded powder

Positive control item
- Potassium hydroxide solution at 8 mol/l (SIGMA-Aldrich, Batch 028K07551, Expiry date: 25 Jun 2014) was used as positive control item.
- Amount(s) applied: 39.7 μL

Negative control item
- Sodium chloride solution at 0.9% (Cooper, batch 19BM05GA, Expiry date: Dec 2013) was used as negative control item.
- Amount(s) applied: 39.7 μL

REAGENTS
- Reconstructed Human Epidermis (SkinEthic Laboratories, Batch 10 022A 0507, Expiry date: 07 Jun 2010)
- Maintenance Medium (SkinEthic Laboratories, Batch 1005 0115645, Expiry date: 07 Jun 2010)
- MTT [3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide] (SIGMA, batch 027K5308, expiry date: 08 Jun 2012)
- Phosphate buffer saline solution (Gibco, Batch 645392 and 756589, Expriry date: Jun 2011 and Apr 2012, respectively)

Duration of treatment / exposure:

1 hour

Observation period:

3 minutes and 1 hour

Number of animals:

15 units of reconstructed epidermis

Details on study design:

See text in "Any other information on materials and methods" part 'Main study'.

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

other: Cell viability as %

Basis:

mean

Time point:

other: 3 min

Score:

ca. 97

Max. score:

100

Reversibility:

no data

Results 2

Irritation parameter:

other: Cell viability as %

Basis:

mean

Time point:

other: 1 hour

Score:

ca. 100

Max. score:

100

Reversibility:

no data

Irritant / corrosive response data:

See below.

Any other information on results incl. tables

Preliminary study

Since the MTT solution did not turn blue/purple when in contact with the test item for 1 hour (step 1), no interference between MTT and test item was concluded. For this reason, the second step of the preliminary study was not performed.

Main study

Mean results are presented in Table 3. Individual results are presented in Table 4.

Table 3 Effect on optical density (mean values)

Treatment		T=3min	T=1H
Negative control	Mean	1.387	1.340
	SEM	0.021	0.073
	N	9	9
MIXED ASH	Mean	1.346	1.405
	SEM	0.039	0.017
	N	9	9
	P	NS	NS
Positive control	Mean	NA	0.000
	SEM	NA	0.001
	N	0	9
	P	(n<5)	**
	Treshold	NA	0.144

NS:P>0.05, **:P60.01, when compared with control group

Analysis of variance with Dunnet's test if P <=0.05

(n<5): not not included in statistical analysis because of insufficient number

Threshold: smallest difference being statistically significant (P <= 0.05) estimated from Dunnet's test

Table 4 Optical density and cell viability

Treatment	Optical density T=3min	Viability (%) T=3min	Optical density T=1H	Viability (%) T=1H
Negative control	1.387	100	1.340	100
MIXED ASH	1.346	97	1.405	100
Positive control	NA	NA	0.000	0

After 1 hour of treatment, the positive control item showed a cell viability percentage of 0.2%. As expected and according to the OECD Guideline No.431, the positive control item was classified as corrosive. This result validated the ongoing sensitivity of the method used.

After 3 minutes and 1 hour of treatment with the undiluted test item MIXED ASH, the percentage of cell viability was 97.1% and 100% respectively as summarised in Table 6:

Table 6 Prediction of corrosivity

Treatment	Cell viability (%) T=3min	Cell viability (%) T=1H	Classification
Negative control (NaCl 0.9%)	100	100.0	Non-corrosive
Test item MIXED ASH (undiluted)	97	100.0	Non-corrosive
Positive control (KOH 8 mol/L)	/	0.2	Corrosive

CONCLUSION

Under the experimental conditions adopted, test item MIXED ASH was classified as noncorrosive on the SkinEthic human reconstructed epidermis.

Applicant's summary and conclusion

Interpretation of results:

other: non-corrosive

Remarks:

Criteria used for interpretation of results: OECD GHS

Conclusions:

Ash was found to be non-corrosive.

Executive summary:

Skin corrosivity of Ash was studied with an in vitro method Human Skin Model Test (OECD 341). In the test, skin epidermis was exposed to grounded Ash powder. Indications of corrosivity were observed after 3 minutes and 1 hours after the exposure. After the exposure time, tissues were rinsed and further incubated with MTT medium. After this incubation step, tissues were washed with a buffer solution and optical density of formazan extract was determined spectrophotometrically. No evidence of corrosivity was observed.