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Eye irritation

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Administrative data

eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31-03-2010 to 18-05-2010
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study, according to the OECD 438 technical guideline

Data source

Reference Type:
study report

Materials and methods

Test guideline
according to guideline
other: ICE Test OECD TG 438
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Flue dust, portland cement
EC Number:
EC Name:
Flue dust, portland cement
Cas Number:
Molecular formula:
It is a UVCB.
Flue dust from Portland cement clinker production
Details on test material:
- Name of test material (as cited in study report): Flue Dust T (REACH)
- Physical state: Solid, beige/grey powder
- Analytical purity:
Na2O 0.14 %
K2O 6.35 %
SO4 as SO3 7.17 %
Cl 3.14 %
CO2 4.98 %
SiO2 15.90 %
Al2O3 4.63 %
TiO2 0.23 %
P2O5 0.14 %
Fe2O3 2.34 %
Mn2O3 0.05 %
MgO 1.62 %
CaO 54.99 %

- Composition of test material, percentage of components (Mineralogical content):
C3S 10 %
C2S 16 %
C3A 4 %
C4AF 6 %
CA 4 %
C4A3SO4 4 %
Spurrite 5 %
Free Lime 20 %
Periclase 2 %
Calcite 10 %
Quartz 4 %
Anhydrite 1 %
Langbeinite 4 %
Arcanite 1 %
Aphtitalite 3 %
Sylvite 6 %

- Purity test date: 2010-01-15
- Lot/batch No.: 12-2009
- Expiration date of the lot/batch: 1.12.2010
- Storage condition of test material: ambient temperature

Test animals / tissue source

other: Isolated Chicken eye
Details on test animals or tissues and environmental conditions:
Test with isolated chicken eyes. Approximately 7 weeks old, male or female chickens (ROSS, spring chickens), body weight range approximately 1.5 - 2.5 kg, were used as eye-donors. Heads of these animals were obtained from poultry slaughterhouse v.d.Bor, Amersfoortseweg 118, Nijkerkerveen, the Netherlands. Heads of the animals were cut off immediately after sedation of the animals by electric shock and incision of the neck for bleeding, and before they have reached the next station on the process line. The heads were placed in small plastic boxes on a bedding of paper tissues moistened with isotonic saline. Next, they were transported to the testing facility. During transportation, the heads were kept at ambient temperature.

Test system

unchanged (no vehicle)
Amount / concentration applied:
30 mg
Duration of treatment / exposure:
Test group 1: 10 seconds
Test group 2: 30 seconds
Observation period (in vivo):
0, 30, 75, 120, 180, 240 minutes
Number of animals or in vitro replicates:
9 test eyes
Details on study design:
Within 2 hours after kill, eyes were carefully dissected and placed in a superfusion apparatus (see Figure 1) using the following procedure: First the eye lids were carefully removed without damaging the cornea and a small drop of Fluorescein sodium BP 2.0% w/v (Minims, Smith & Nephew Ltd., Romford, England) was applied to the corneal surface for a few seconds and subsequently rinsed off with isotonic saline at ambient temperature. Next, the head with the fluorescein treated cornea were examined with a slit lamp microscope (Slit lamp 900 BP, Haag Streit AG, Liebefeld Bern, Switzerland) to ensure that the cornea is not damaged. If undamaged, the eye was further dissected from the head without damaging the eye or cornea. Care was taken to remove the eye ball from the orbit without cutting off the optical nerve too short.

The enucleated eye was placed in a stainless steel clamp with the cornea positioned vertically and transferred to a chamber of the superfusion apparatus (TNO, Zeist, the Netherlands). The clamp holding the eye was positioned in such a way that the entire cornea was supplied with isotonic saline from a bent, stainless steel tube, at a rate of ca 0.10 0.15 mL/min (peristaltic pump, Watson-Marlow 205CA, Rotterdam, the Netherlands). The chambers of the superfusion apparatus as well as the saline were temperature controlled at approximately 32 oC (water pump set at 34 oC; Lauda 103, Germany).

After placing in the superfu¬sion apparatus, the eyes were examined again with the slit lamp microscope to ensure that they are not damaged. Corneal thickness was measured using the Depth Measuring Attachment No. I for the Haag Streit slit lamp microscope. Corneal thickness was expressed in instrument units. An accurate measurement was taken at the corneal apex of each eye.

Eyes with a corneal thickness deviating more than 10% of the average corneal thickness of the eyes, eyes that show opacity (score higher than 0.5), or are unaccep¬tably stained with fluores¬cein (score higher than 0.5) indicating the cornea to be permeable, or eyes that show any other signs of damage, were rejected as test eyes and replaced.

Three test eyes, one negative control eye and three positive control eyes were selected for testing. Three additional test eyes were treated for 30 seconds in order to mimic possible prolonged exposure (up to one hour) which may occur in the in vivo rabbit eye irritation test. Each eye will provide its own baseline values for corneal swelling, corneal opacity and fluorescein retention. For that purpose, after an equilibrati¬on period of 45 60 minutes, the corneal thickness of the eyes was measured again to determine the zero reference value for corneal swelling calculati¬ons.
At time t = 0 (i.e. immedia¬tely after the zero reference measure¬ment), the following procedure was applied for each test eye: The clamp holding the test eye was placed on paper tissues outside the chamber with the cornea facing upwards. Next, the eyes (corneas) were treated with the study substances according to the following scheme:

Group Treatment Exposure conditions Number of eyes
Volume Duration Rinsing
Negative control Saline 30 μL 10 seconds 20 ml saline 1
Positive control NaOH 30 mg 10 seconds 20 ml saline 3
Test group 1 Flue Dust T (REACH) 30 mg 10 seconds 20 ml saline 3
Test group 2 Flue Dust T (REACH) 30 mg 30 seconds Saline drip 3

After rinsing, each eye in the holder was returned to its chamber.
The eyes were examined at ca 0, 30, 75, 120, 180 and 240 minutes after treatment, using the criteria and scoring system given in Annex 2. Fluorescein retention was only scored at ca 30 minutes after treatment. All examinations were carried out with the slit lamp microscope.
After the final examination the test substance treated eyes and the negative and positive control eyes were preserved in a neutral aqueous phosphate buffered 4% solution of formaldehyde. The corneas were embedded in paraffin wax, sectioned at 5 µM and stained with PAS (Periodic Acid-Schiff). The microscopic slides were filed in the archives and kept available for histopathological examination if considered relevant. In this case, histopathological examination was not considered relevant, because severe corneal effects were observed.

Results and discussion

In vivo

Irritation parameter:
other: Irritation index
Time point:
other: 240 min
> 140

Any other information on results incl. tables

An overall summary of the results based on maximum mean values for corneal swelling, corneal opacity, and fluorescein retention, the irritation categories assigned to the parameters and the general irritancy classification of the negative control, the test sample (two exposures) and the positive control is presented in Table 1.

In addition, an Irritation Index was given for the samples tested to allow for numerical ranking and comparison. The index was based on the addition of the maximum mean scores obtained for the parameters according to the following formula:


Irritation Index = Maximum Mean Corneal Swelling + Maximum Mean Opacity (times 20) + Mean Fluorescein Score (times 20).


A factor of 20 was included to give equal weight to the scores obtained for opacity and fluorescein retention in the index compared to the maximum swelling possible (circa 60%). The maximum Irritation Index possible is circa 200.


The mean values for corneal swelling, corneal opacity and fluores­cein retention recorded at the various observation time points forFlue Dust T (REACH);10-second exposure and 30-second exposure,and the positive control are presented in Tables 2, 3 and 4, respectively.

Individual values for corneal swelling, corneal opacity and fluorescein retention forFlue Dust T (REACH);10-second exposure and 30-second exposure,the positive control and the negative control and are presented in Appendices 1, 2, 3 and 4, respectively.


The negative control eye did not show any significant corneal effect and demonstrated that the general conditions during the test were adequate.

The positive control eyes showed very severe corneal effects and demonstrated the suitability and sensitivity of the ICE to detect severe eye irritants.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye)
Migrated information Criteria used for interpretation of results: EU
Applying the EC/GHS-classification criteria of the ICE, Flue Dust T (REACH) is classified as Category 1 (irreversible effects on the eye/serious damage to the eye).
Executive summary:

1.      Flue Dust T (REACH) was evaluated neat in the Isolated ChickenEye (ICE) test. Chicken eyes were obtained from slaughter animals used for human consumption. The isolated chicken eyes were exposed to a single application of the test sample for 10 seconds using an application amount of 30 mg.Three additional test eyes were treated for 30 seconds in order to mimic possible prolonged exposure (up to one hour) which may occur in thein vivorabbit eye irritation test with solids. Three main parameters were measured to disclose possible adverse eye effects: corneal thickness (expressed as corneal swelling), corneal opacity and fluorescein retention of damaged epithelial cells.


2.      The negative control eye showedno corneal effects.


3.      The positive controlcaused very severe corneal effects. The calculated Irritation Index was higher than 140.


4.      Flue Dust T (REACH) with a 10-second applicationcaused a mixed picture of corneal effects, i.e. overall moderate swelling, moderate opacity and severe fluorescein retention, and focal areas with severe corneal opacity. The calculated Irritation Index was 148.


5.      Flue Dust T (REACH) with a 30-second applicationcaused very severe opacity and severe fluorescein retention. Corneal thickness could not be measured, because of the presence of very severe opacity. The calculated Irritation Index was higher than 140.


6.      Applying the EC/GHS-classification criteria of the ICE, Flue Dust T (REACH) is classified as Category 1 (irreversible effects on the eye/serious damage to the eye).