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

Endpoint summary

Description of key information

Eye irritating and skin corrosive properties of Ash were studied with two in vitro guideline studies in a GLP laboratory. Although Ash has a high pH (>11.5), it was found to be non-corrosive for skin, which indicates high buffer capacity. Instead, it was observed to be highly irritating to eye. Eye irritation properties of Ash were also determined in a GLP compliant laboratory in vivo with a rabbit as a test organism. Two hours after treatment up to the end of the study (time72 hours), severe chemosis with lacrimation, redness of the conjunctivae and corneal lesions on greater than three-quarters up to whole area of the cornea were present. Based on high pH value, Ash is presumed to irritate the respiratory tract as well.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

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.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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

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

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'.

Irritation parameter:

other: Cell viability as %

Basis:

mean

Time point:

other: 3 min

Score:

ca. 97

Max. score:

100

Reversibility:

no data

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.

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.

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation, other

Remarks:

other: Chorioallantoic membrane test method (HET-CAM)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study was conducted on 31 May 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study.

Qualifier:

according to guideline

Guideline:

other: the general requirements of French Official Text, Annex IV, dated on December 26th, 1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Invitox protocol No. 47 dated on January 1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: NIH publication No. 06-4515 (March 2006)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

4 December 2006

Species:

other: fertile chicken eggs

Strain:

other: Isa Brown

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Couvoir d'Ile de France, ZI Equillemont, 3 rue Helene Boucher, 28700 Auneau
- Age at study initiation: 10 days after fertilization and incubation
- Weight at study initiation: Between 53.3 and 64.4 g on the first day of the study
- Housing: Eggs were delivered few days after fertilization. Observations were performed at the time of delivery of the eggs. Eggs were directly placed into a specified incubator at 37.8°C ± 1°C until the start of the experiment (D10). The incubator was provided with manual rotation. Eggs were rotated twice a day.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 37.8°C ± 1°C
- Humidity (%): 43 - 62%

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

TEST MATERIAL, MIXED ASH
- Amount(s) applied (volume or weight with unit): 0.3 ml of grounded test material
- Concentration (if solution): undiluted

POSITIVE CONTROL ITEM
A 0.1N sodium hydroxide solution (SIGMA-Aldrich, Batch 017K6098, Expiry date: Feb 2014) was used as positive control item.

NEGATIVE CONTROL ITEM
A 0.9% sodium chloride solution (Cooper, Batch 19BM05GA, Expiry date: Dec 2013) was used as a negative control item.

Duration of treatment / exposure:

20 seconds

Observation period (in vivo):

5 minutes

Number of animals or in vitro replicates:

12 eggs: 4 eggs for each treatment group. See Table 1.

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): With 0.9% sodium hydrochloride solution
- Time after start of exposure: 20 s

SCORING SYSTEM: Scores were calculated according to HET-CAM scoring system Table 2

TOOL USED TO ASSESS SCORE: Halogen candling light, Super Flash

Irritation parameter:

other: Haemorrhage and coagulation

Basis:

mean

Time point:

other: 30 - 120 s

Score:

ca. 12

Max. score:

21

Reversibility:

not specified

Remarks on result:

other: See Tables 4-6.

Irritant / corrosive response data:

See Tables 4-6.

Irritation score and irritation score mean

In the negative control, 4/4 eggs showed no hyperemia, no hemorrhage and no coagulation at the end of the 5-minute observed period. In the positive control, 4/4 eggs showed an hemorrhage within the first 30 seconds and coagulation before the end of the first 2 minutes of observation. For instance, the negative and positive controls induced a response that falls within the classification of non-irritating (ISmean=0) and severely irritating (ISmean=14) respectively. For this reason, the test is considered valid.

All eggs treated with test item MIXED ASH showed haemorrhage and coagulation between 30 and 120 seconds of observation period. Mean irritation score calculated after treatment with test item MIXED ASH was 12. The results are summarised in the Table 4.

Table 4 Mean irritation score and irritation category

Treatment	Cotation (IS Mean)	Irritation category
Negative control (NaCl 0.9%)	0	Non irritant
Positive control (NaOH 0.1N)	14	Severe irritation
MIXED ASH	12	Severe irritation

Individual and mean results are presented in Table 5 and 6.

Table 5 Irritation Score Mean (mean values)

Treatment		IS
Negative control	Mean	0
	SEM	0
	N	4
Positive control	Mean	14
	SEM	0
	N	4
MIXED ASH	Mean	12
	SEM	0
	N	4

No statistical analysis. IS: Irritation score calculated for each eggs as follows: IS = score obtained for hyperemia + score obtained for haemorrhage + score obtained for coagulation

Table 6 Appearance time of hyperemia, hemorrhage and coagulation. Results are expressed in second (s)

Treatment	Egg number	Time of hyperemia (s)	Time of hemorrhage (s)	Time of coagulation (s)
Negative control	20100001	Not appeared	Not appeared	Not appeared
	20100002	Not appeared	Not appeared	Not appeared
	20100003	Not appeared	Not appeared	Not appeared
	20100004	Not appeared	Not appeared	Not appeared
Positive control	20100005	Not appeared	14	43
	20100006	Not appeared	15	36
	20100007	Not appeared	15	37
	20100008	Not appeared	12	34
MIXED ASH	20100009	Not appeared	35	80
	20100010	Not appeared	46	65
	20100011	Not appeared	60	66
	20100012	Not appeared	42	75

Interpretation of results:

highly irritating

Remarks:

severe irritant Criteria used for interpretation of results: EU

Conclusions:

Ash was found to be severe irritant.

Executive summary:

Eye irritation properties of Ash were determined in a GLP compliant laboratory with an in vitro Hen's Egg Test Chorioallantoic Membrane (HET-CAM) test. The test method used is based on the general requirements of French Official Text, Annex IV, dated on December 26th, 1996, Invitox protocol No. 47 dated on January 1992 and NIH publication No. 06-4515 (March 2006). After 10-day incubation of fertilised eggs, 0.3 ml of pulverised Ash was placed onto the CAM for 20 second. The CAMs were observed for a period of 5 min by using candling light. The observed reactions were haemorrhage and coagulation of the CAM. Thus, Ash is severe irritant for the eye.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Additional information

Justification for selection of skin irritation / corrosion endpoint:
GLP compliant guideline study.

Justification for selection of eye irritation endpoint:
GLP compliant guideline study (HET-CAM) showing severe irritant properties.

Effects on skin irritation/corrosion: irritating

Effects on eye irritation: corrosive

Effects on respiratory irritation: irritating

Justification for classification or non-classification

Classification is required under Directive 67/548/EEC and under Regulation EC 1272/2008 for skin, eye and respiratory tract irritation. Skin irritation: In vitro study for skin corrosion, OECD 431: No effect; pH > 11.5.

Eye irritation: severity and duration scores above threshold for classification.

Respiratory irritation: pH > 11.5.