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Toxicological information

Eye irritation

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Administrative data

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guidelineopen allclose all
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

Test material

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 / tissue source

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%

Test system

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

Results and discussion

In vivo

Results
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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.