Reaction mass of ethoxylated N-oleyl-1,3-propanediamine, hydrofluorides of and ethoxylated N-palmityl-1,3-propanediamine, hydrofluorides of and ethoxylated N-stearyl-1,3-propanediamine, hydrofluorides of and olaflur

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2009-09-28 to 2009-11-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: ICCVAM Test Method Evaluation Report: Appendix G: ICCVAM recommended HET-CAM Method Protocol (Nov. 2006)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Test animals / tissue source

Species:

other: not applicable (HET-CAM test in vitro)

Strain:

other: not applicable (HET-CAM test in vitro)

Details on test animals or tissues and environmental conditions:

Freshly laid Lohmann Leghorn chicken eggs were obtained from the LSL Rhein Main Geflügelvermehrungsbetrieb (Dieburg, Germany) nine days before the start of the test (arrived on 21. September 2009). Then the eggs were incubated at 38.3 +- 0.2 °C and 58% +- 2% humidity in a still air incubator for eight days. During incubation, the eggs were rotated to prevent an attachment of the embryo to one side of the egg.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: not applicable (HET-CAM test in vitro)

Amount / concentration applied:

As the test item consists of a wax-like substance, a small amount was weighed into each of six small beakers. The weight of each filled beaker was recorded. For each application, the necessary volume was taken from one of the beakers. The substance was then carefully slid onto the surface of the membrane.

Duration of treatment / exposure:

301 sec

Observation period (in vivo):

not applicable

Number of animals or in vitro replicates:

6 eggs (test substance), 3 eggs (negative and positive controls each)

Details on study design:

Preparation of Test System
24 hours before the start of the experiment (eight days after obtaining the eggs), the chicken eggs were candled and non-viable eggs were discarded. The rest of the eggs were placed upward in the incubator until the next day.

Experimental Parameters
Date of treatment 30. September 2009
Incubation time 301 seconds
Positive controls Na-dodecylsulphate, 1% NaOH, 0.1 mol/L

Method Description
On the day of the test, the eggs were removed from the incubator for use in the assay.
At first, the eggs were candled and the air bubble was marked. The egg was opened on the air bubble with a rotating dentist’s saw blade. The inner membrane was moistened with 0.9 % NaCl, then the egg was left to stand into the incubator for 30 minutes. After this period, the solution was decanted and the inner membrane was carefully removed with a forceps.
Then the test item was given directly onto the CAM surface.
As the test item consists of a wax-like substance, a small amount was weighed into each of six small beakers. The weight of each filled beaker was recorded. For each application, the necessary volume was taken from one of the beakers. The substance was then carefully slid onto the surface of the membrane.
The test item was given on the membrane in such a manner that 50% of the surface of the membrane was covered with test item.
The reactions on the CAM were observed over a period of 301 seconds. Note: The observation period was chosen as 301 seconds (instead of 300 seconds as recommended by the guideline) due to mathematical reasons in case no reactions on the CAM are observed at all.

The following endpoints are described in the guideline:
- haemorrhage (bleeding from the vessels)
- vascular lysis (blood vessel disintegration)
- coagulation (intra- and extra-vascular protein denaturation)
The time for the appearance of each of the noted endpoints was monitored and recorded in seconds.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

mean value

Value:

17.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: For details please refer to "Any other information on results incl. tables"

Other effects / acceptance of results:

In the negative control, no signs of irritation were observed.
Both positive controls showed severe irritation.
The test item Olaflur showed severe irritation.

Any other information on results incl. tables

Table 1: Irritation Score

Test Group	Irritation Score	Mean Irritation Score
Negative Control	0.00	0.00
Negative Control	0.00
Negative Control	0.00
Positive Control 0.1-N NaOH	19.97	19.87
Positive Control 0.1-N NaOH	19.73
Positive Control 0.1-N NaOH	19.91
Positive Control 1% SDS	10.64	10.55
Positive Control 1% SDS	10.54
Positive Control 1% SDS	10.46
Olaflur	16.61	17.20
Olaflur	17.46
Olaflur	17.31
Olaflur	17.28
Olaflur	17.35
Olaflur	17.22

Applicant's summary and conclusion

Interpretation of results:

other: severe irritation

Remarks:

Criteria used for interpretation of results: other: based on ICCVAM recommendation

Conclusions:

The test item Olaflur showed severe irritation in HET-CAM test.

Executive summary:

For this HET-CAM test, hen’s eggs with an age of nine days were used. The test item Olaflur was brought onto the surface of the CAM of a hen’s egg which had been incubated at 38.3°C and 58% humidity for nine days. Observation time was 5 minutes at room temperature.

Physiological sodium chloride solution was used as negative control. The negative control showed no irritating effect on the blood vessels under the membrane.

Solutions of SDS (1%) and NaOH (0.1N) were used as positive controls. The positive controls induced a severe irritation on the blood vessels.

The test item Olaflur showed severe effects on the blood vessels of the CAM. The calculated mean irritation score is 17.20.

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item Olaflur possesses severe irritation potential.