Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 Sep 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Principles of method if other than guideline:
- Principle of test: With the HET-CAM (Hen's Egg Test – Chorioallantoic Membrane) test (according to INVITTOX Protocol No. 47, https://ntp.niehs.nih.gov/iccvam/docs/protocols/ivocular-hetcam.pdf) an irritative or corrosive potential of the test substance can be determined by the detection of damages in blood vessels under the chorioallantoic membrane (CAM) of fertilized and incubated chicken eggs. The CAM is a vital vascular membrane with a closed blood vessel system. After application of a test item on the membrane the underlying blood vessels could become damaged.
- Short description of test conditions: Eggs were incubated at 37.5 ± 1 °C for eight days. During incubation the eggs were rotated to prevent an attachment of the embryo to one side of the egg. On day eight the eggs were candled (held in front of a strong lamp in order to determine the position of the chicken fetus) and non-viable eggs (eggs containing no fetus) were discarded. The remaining eggs were placed upwards in the incubator and incubated for another day without rotation. On day 9, the shell above the oxygen bubble was removed. The egg skin was removed from the membrane. The test substance was applied to the membrane and the incidence and severity of damage was recorded.
- Parameters analysed / observed: In particular three events are observed: haemorrhage, vessel lysis and coagulation of the blood in the vessels.
GLP compliance:
yes (incl. certificate)
Remarks:
Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals / tissue source

Species:
chicken
Strain:
other: Eggs from Lohmann Selected Leghorn chicken

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 76 to 77 mg
Duration of treatment / exposure:
300 s
Duration of post- treatment incubation (in vitro):
not applicable
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION: Eggs were incubated at 37.5 ± 1 °C for eight days. During incubation the eggs were rotated to prevent an attachment of the embryo to one side of the egg. On day eight the eggs were candled (held in front of a strong lamp in order to determine the position of the chicken fetus) and non-viable eggs (eggs containing no fetus) were discarded. The remaining eggs were placed upwards in the incubator and incubated for another day without rotation. On day 9, the day of the performance of the experiment, the eggs were candled once again in order to determine the position of the egg's oxygen bubble. The position was marked on the eggshell. Along the marking the eggshell was sawed with an electric saw. Afterwards, the shell above the oxygen bubble was removed. The egg skin beneath was covered with saline in order to separate the egg skin from the chorioallantoic membrane beneath the skin. After a short incubation period at 37.5 ± 1 °C the egg skin was removed from the membrane easily using forceps.The test item was applied directly to the membrane in order to cover at least 50% of the membrane.

NUMBER OF REPLICATES: 3 eggs per group (treatment, negative control, positive control (NaOH), positive control (SDS)

NEGATIVE CONTROL USED: 300 µL of a 0.9% (w/v) NaCl solution in deionised water

POSITIVE CONTROL USED: 300 µL of a 1% SDS solution in deionised water; 300 µL of a 0.1 N NaOH solution in deionised water

APPLICATION DOSE AND EXPOSURE TIME: 76 to 77 mg undiluted test item; 300 s exposure period (concurrent observation period)

OBSERVATIONS: The membranes of the eggs were observed for 300 seconds. Lesions of the underlying blood vessels were monitored and noted. In particular three endpoints were observed and the time point at which an effect occurs was recorded. The three endpoints were haemorrhage, coagulation and lysis of the blood vessel.

EVALUATION OF RESULTS: A strong irritating effect shows very fast and strong lesions on the blood vessels underlying the membrane. A weak irritating effect shows very slow and weak reactions. Based on the time recorded for each endpoint an irritancy index (R) can be calculated as follows:

R = ((301-h) × 5)/300 + ((301-l) × 7)/300 + ((301-c) × 9)/300

h = time in seconds at which haemorrhages appear
l = time in seconds at which lysis occurs first
c = time in seconds at which coagulation of protein or blood was noticed first

The mean score for each experimental group was calculated from the individual scores of each egg. The score obtained enabled assessment of the irritancy potential of the test items as follows:

CAM Mean Irritancy Index
0 – 0.9: Not irritant
1 – 4.9: Slight irritant
5 – 8.9: Moderate irritant
9 – 21: Severe irritant

Results and discussion

Results of ex vivo / in vitro study
Irritation parameter:
other: CAM Mean Irritancy Index
Value:
0
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes for NaOH and SDS
- Range of historical values: Irritancy index R [%]: Negative control: 0.00 ± 0.00 (0.9 % NaCl); Positive control: 19.14 ± 0.39 (0.1 N NaOH), 9.94 ± 0.76 (1 % SDS)

Any other information on results incl. tables

Table 1: Results with the test item and the controls

Test Group

Time until Haemorrhage [s]

Time until Lysis [s]

Time until Coagulation [s]

Irritancy Index R

Mean Irritancy Index R

Evaluation

Negative Control

301

301

301

0.00

0.00

not irritant

301

301

301

0.00

301

301

301

0.00

Positive Control 0.1 N NaOH

10

30

15

19.75

19.54

severe irritant

12

39

18

19.42

12

36

19

19.46

Positive Control 1% SDS

11

65

301

10.34

10.40

severe irritant

13

54

301

10.56

12

66

301

10.30

Test Item

301

301

301

0.00

0.00

not irritant

301

301

301

0.00

301

301

301

0.00

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Conclusions:
According to the ECHA Guidance on IR and CSA ´Chapter R.7a Endpoint specific guidance`(Version 4.1 October 2015), the HET-CAM test method may be used to classify a substance as Category 1 for serious eye damage. The test may not be used to determine the non-classification of a substance.