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Diss Factsheets

Administrative data

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 
 
MIXED ASH  Mean  1.346  1.405 
  SEM  0.039  0.017 
 
  NS  NS 
Positive control  Mean  NA  0.000 
  SEM  NA  0.001 
 
  (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%)  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 
  SEM 
 
Positive control  Mean  14 
  SEM 
 
MIXED ASH  Mean  12 
  SEM 
 

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.