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Toxicological information

Eye irritation

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Administrative data

eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
12 March 2008
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
The test has been performed based on international recommendations. Although the HET-CAM test method is not yet validated, the EU national regulatory authorities accept positive outcomes (severe eye irritants) of this test method for classification and labelling severe eye irritants. Where a negative result is obtained, and in vivo test is subsequently required.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
no guideline available
Principles of method if other than guideline:
The study procedures described in this report were based on the following documents and follows international recommendations (ref. 1-5)

- The Ocular Toxicity Working Group (OTWG) of the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the National Interagency Centre for the Evaluation of Alternative Toxicological Methods (NICEATM), Background Review Document (BRD): current status of in vitro test methods for identifying ocular corrosives and severe irritants: The Hen’s Egg Test - Chorioallantoic Membrane (HET-CAM) test method, March 2006.
- INVITTOX protocol 47. The HET-CAM test - Method of Spielmann and Liebsch, January 1992.

ref 1: Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals, Section 4, Health Effects, No. 405, "Acute Eye Irritation / Corrosion", Paris Cedex, 2002.
ref 2: UN 2003. Globally Harmonized System of Classification and Labelling of Chemicals (GHS). United Nations Publications: New York & Geneva.
ref 3: EC 2004. Commission directive 2004/73/EC adapting to technical progress for the 29th time Council Directive 67/548/EEC on the approximation of laws, regulations and administrative provisions related to classification, packaging and labelling of dangerous substances. Official Journal of the European Union L152:1-316. April 2004.
ref 4: EC 2004. Manual of Decisions for Implementation of the 6th and 7th amendments to Directive 67/548 on Dangerous Substances (Directives 79/831/EEC and 92/32/EEC). July 2004.
ref 5: EPA 1998. Health Effects Test Guideline, OPPTS 870.2400 Acute Eye Irritation. EPA 712-C-98-195. Washington, DC: U.S. Environmental Protection Agency.

The HET-CAM assay measures three important components which are predictive for conjunctival injury of the eye, heamorrhage, vessel lysis and coagulation.

GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Lithium hexafluorophosphate(1-)
EC Number:
EC Name:
Lithium hexafluorophosphate(1-)
Cas Number:
Molecular formula:
lithium(1+) hexafluoro-λ⁵-phosphanuide
Details on test material:
- Name of test material (as cited in study report): LiPF6
- Substance type: inorganic
- Physical state: white powder
- Analytical purity: > 99.9%
- Lot/batch No.: P07102202
- Expiration date of the lot/batch: 31 December 2008
- Stability under storage conditions: stable
- Storage condition of test material: At room temperature protected from light under nitrogen

Test animals / tissue source

other: Fresh, fertilised brown leghorn chicken eggs
not specified
Details on test animals or tissues and environmental conditions:
Eggs were obtained from a non-GLP chicken-breeding center (Het Anker Ochten, the Netherlands).

Test system

unchanged (no vehicle)
Amount / concentration applied:
Three eggs per treatment group were treated for 300 seconds with 0.3 ml of the negative or positive control solutions. The appearance of haemorrhage, vessel lysis and coagulation on the CAM was monitored and recorded over this 300-second period.

Three eggs were treated for 20 seconds with 300 mg LiPF6. After the treatment period the eggs were carefully rinsed with approximately 5 ml of tepid water (Milli-RO water). The appearance of haemorrhage, vessel lysis and coagulation on the CAM was monitored and recorded over the following 280-second period.
Duration of treatment / exposure:
20 seconds
Observation period (in vivo):
280 seconds
Number of animals or in vitro replicates:
3 eggs per treatment group
Details on study design:
CAM preparation:
The number of eggs used in the study was 3 per treatment group. The air cell of the eggs were marked and cut off with a rotating saw blade. The uncovered inner membrane was moistened with physiological saline and subsequently the egg was kept at room temperature for 1-10 minutes. After this period the physiological saline was removed and the inner egg membrane carefully eliminated using forceps to expose the CAM.
Negative control:
A negative control (0.9% (w/v) NaCl (Merck, Darmstadt, Germany) in Milli-RO water (Millipore Corp., Bedford, MA., USA) (physiological saline) was included to detect non-specific changes in the test system and to provide a baseline for the assay endpoints.
Positive control:
The positive controls was 1% (w/v) Sodium dodecyl sulphate solution prepared in physiological saline.

Results from the three test method endpoints were evaluated separately for each egg.

The irritancy score (IS) was calculated using the formula:
IS = [((301-Haemorrhage time)/300)x5] + [((301-Lysis time)/300)x7] + [((301-Coagulation time)/300)x9]

IS = irritancy score for total effect
Haemorrhage time = observed start (in seconds) of haemorrhage reactions on CAM
Lysis time = observed start (in seconds) of vessel lysis on CAM
Coagulation time = observed start (in seconds) of coagulation formation on CAM

Data evaluation:
The mean score for each treatment was calculated from the final scores obtained for each egg in that treatment and evaluated according to the following irritancy classification scheme of Luepke (1985) and Kalweit et al. (1987).

HET-CAM Irritancy Score (IS) Range Irritation Category
0 – 0.9 Not irritant
1 – 4.9 Slight Irritant
5 – 8.9 Moderate Irritant
9 – 21 Severe Irritant

Acceptability of the assay:
The assay will be acceptable if:
- The positive controls give an in vitro irritancy score that is in range or close to the historical in vitro irritancy score mentioned in the BRD document: 1% (w/v) Sodium dodecyl sulphate: 8-14: NOTOX: mean 14.8 ± 0.4 (range: 14.1 – 15.3).
- The negative control gives an in vitro irritancy score of less than 1.

Results and discussion

In vitro

Irritation parameter:
in vitro irritation score
Remarks on result:
positive indication of irritation
mean of 3 eggs
Other effects / acceptance of results:
The coagulation was observed directly after rinsing the test substance of from the chorioallantoic membrane. The haemorrhages occurred on the treated parts of the membrane and vessel lysis on the non-treated parts of the membrane.
The mean irritancy score of the negative control was 0.
The mean irritancy score of the 1% Sodium dodecyl sulphate was 15.2.

Applicant's summary and conclusion

Interpretation of results:
other: severe irritant
Criteria used for interpretation of results: other: Luepke (1985) and Kalweit et al. (1987)
The mean in vitro irritancy score obtained with the negative control was less than 1 indicating that the negative control did not induce irritancy on the chorioallantoic membrane. The mean in vitro irritancy score of the positive control (1% (w/v) Sodium dodecyl sulphate) was within the historical control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.
The mean in vitro irritancy score obtained during a 280 seconds observation period after 20 seconds treatment with LiPF6 was 16.0.

Finally, it is concluded that this test is valid and that LiPF6 is a severe irritant in the Hen’s Egg Test – Chorioallantoic Membrane Test under the experimental conditions described in this report.