citric acid, esters with dodecan-1-ol

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25.07.2006 - 23.08.2006

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:

2006

Report date:

2006

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Standard Operating Procedure (SOP) T 7 "Hühnerei-Test" of the test facility

Version / remarks:

The test method is based on publications by N. P. Lüpcke, Institute for Pharmacology anfd Toxicology, University of Münster (for example in H. Schöffl, Ersatz- und Ergänzungsmethoden zu Tierversuchen: Möglichkeiten und Grenzen der Reduktion von Tierversuchen, Springer Verlag, 1992).
According to French regulations the HET-CAM test is an accepted alternative method to the Draize test for the control of innocuity of cosmetics and toiletries (Journal Officiel de la RépubliqueFrancaise, 1996, pp. 19137-19138)

Principles of method if other than guideline:

- Principle of test: For the evaluation of the acute irritation potential of a substance a definitive dose is applied onto the chorioallantoic membrane (CAM), a highly vascularized membrane that forms to surround a developing chick embryo. Possible reactions are recorded in dependance on the time.
- Short description of test conditions: Fresh, intact chicken eggs are stored in an incubator between 15 and 22 °C for nine days. After incubation the egg shell around the air space is peeled away and the visible white egg membrane is wetted with physiolocal saline. After approximately 1 minte the saline is poured off and the membrane is carefully removed with forceps. Two prepared eggs under a cold light lamp and the test substance is immediately placed on the CAM. 20 seconds after application the test substance is rinsed off with tap waer. Then the blood vessels are continously observed over a period of 5 minutes. Untreated areas serve as control. After the end of the observation period the next two freshly prepared eggs are exposed to the test substance in the same manner. A total of 6 eggs is used for the assay.
- Parameters analysed / observed: Possible effects are scored in dependance of the time of the reaction.The first appearance of the effect is relevant for scoring and not the lasting of the effect. Reactions observed are Vascular injection, haemorrage and coagulation.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Didodecyl hydrogen citrate

EC Number:

247-146-3

EC Name:

Didodecyl hydrogen citrate

Cas Number:

25637-88-1

Molecular formula:

C30H56O7

IUPAC Name:

didodecyl hydrogen citrate

Constituent 2

Reference substance name:

Tridodecyl 2-hydroxypropane-1,2,3-tricarboxylate

EC Number:

265-671-6

EC Name:

Tridodecyl 2-hydroxypropane-1,2,3-tricarboxylate

Cas Number:

65277-53-4

Molecular formula:

C42H80O7

IUPAC Name:

tridodecyl citrate

Constituent 3

Reference substance name:

Dodecyl dihydrogen 2-hydroxypropane-1,2,3-tricarboxylate

EC Number:

278-950-2

EC Name:

Dodecyl dihydrogen 2-hydroxypropane-1,2,3-tricarboxylate

Cas Number:

78568-45-3

Molecular formula:

C18H32O7

IUPAC Name:

2-[2-(dodecyloxy)-2-oxoethyl]-2-hydroxysuccinic acid

Test material form:

solid

Test animals / tissue source

Species:

other: chicken egg

Strain:

other: SPAFAS

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability: According to French regulations the HET-CAM test is an accepted alternative method to the Draize test for the control of innocuity of cosmetics and toiletries (Journal Officiel de la RépubliqueFrancaise, 1996, pp. 19137-19138).
German authorities agreed to accept the classification of sevwerely eye irritating chemicals by using the HET-CAM test (Spielmann et al., 1996, Results odf a validation study in German on two in vitro alternatives to the Draize eye irritation test, the HET-CAM test and the 3T3 NRU Cytotoxicity test, ATLA 24, pp. 741-858).

Test system

Vehicle:

water

Remarks:

tap water

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 200 µL
- Concentration (if solution): 10 % dispersion in tap water

Duration of treatment / exposure:

20 seconds

Observation period (in vivo):

5 minutes

Number of animals or in vitro replicates:

6

Details on study design:

SELECTION AND PREPARATION OF EGGS

QUALITY CHECK OF THE EGGS: From the 6th incubation day on the eggs were illuminated two-day with a candeling lamp. Eggs, which were not fertilized or not developed are rejected.

NUMBER OF REPLICATES: 6

NEGATIVE CONTROL USED: tap water

POSITIVE CONTROL USED: Texapon ASV 70 (sodium magnesium lauryl-myristyl-6-ethoxy-sulfate, Henkel KGaA, Düsseldorf, Germany, lot-no.: CS 50090003), 5 % active substance in tap water

APPLICATION DOSE AND EXPOSURE TIME: 200 µL of a 10 % test substance dispersion in tap water

REMOVAL OF TEST SUBSTANCE: rinsing with tap water
- Number of washing steps after exposure period: 1

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

other: Vascular injection, haemmorrage, coagulation

Run / experiment:

1

Value:

3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

other: Vascular injection, haemmorrage, coagulation

Run / experiment:

2

Value:

3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

other: Vascular injection, haemmorrage, coagulation

Run / experiment:

3

Value:

3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 4

Irritation parameter:

other: Vascular injection, haemmorrage, coagulation

Run / experiment:

4

Value:

5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 5

Irritation parameter:

other: Vascular injection, haemmorrage, coagulation

Run / experiment:

5

Value:

5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 6

Irritation parameter:

other: Vascular injection, haemmorrage, coagulation

Run / experiment:

6

Value:

3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Table 1: Summary of the observed effects after application of the test substance as 10 % (w/w) dispersion in tap water onto the CAM

Egg number	Vascular injection			Haemmorrage			Coagulation			Sum
	Appearance in minutes			Appearance in minutes			Appearance in minutes
	0.5	2	5	0.5	2	5	0.5	2	5
1	0	0	0	0	0	3	0	0	0	3
2	0	3	-	0	0	0	0	0	0	3
3	0	3	-	0	0	0	0	0	0	3
4	0	0	0	0	5	-	0	0	0	5
5	5	-	-	0	0	0	0	0	0	5
6	0	0	0	0	0	0	0	0	0	3

Mean:        3.7

Table 2: Summary of the observed effects after application of the negative / vehicle control tap water onto the CAM

Egg number	Vascular injection			Haemmorrage			Coagulation			Sum
	Appearance in minutes			Appearance in minutes			Appearance in minutes
	0.5	2	5	0.5	2	5	0.5	2	5
1	0	0	0	0	0	0	0	0	0	0
2	0	0	0	0	0	0	0	0	0	0
3	0	0	0	0	0	0	0	0	0	0
4	0	0	0	0	0	0	0	0	0	0
5	0	0	0	0	0	0	0	0	0	0
6	0	0	0	0	0	0	0	0	0	0

Mean:        0

Table 3: Summary of the observed effects after application of the reference substance Texapon ASV 70 (5 % active substance preparation in tap water) onto the CAM

Egg number	Vascular injection			Haemmorrage			Coagulation			Sum
	Appearance in minutes			Appearance in minutes			Appearance in minutes
	0.5	2	5	0.5	2	5	0.5	2	5
1	5	-	-	0	5	-	0	0	0	10
2	5	-	-	0	5	-	0	0	0	10
3	5	-	-	0	5	-	0	0	0	10
4	5	-	-	0	5	-	0	0	0	10
5	5	-	-	0	5	-	0	0	0	10
6	5	-	-	0	5	-	0	0	0	10

Mean:        10

Applicant's summary and conclusion

Interpretation of results:

other: slightly irritating

Conclusions:

The test substance was graded as slightly irritating according to the evalaution scheme used in the test.

Executive summary:

The eye irritation potential of the substance was tested in an in vitro HET-CAM test. A 10 % dispersion of the substance caused slight irritation according to the evalaution scheme used in the test. For an anticipation of the irritation potential in vivo, the results obtained with the positive control Texacon ASV 70 can betaken into consideration. A 5 % active substance preparation of Texapon ASV 70 is known to be only slightly irritating in vivo. From this it can be concluded that the test substance as 10 % (w/w) dispersion in tap water will be non irritating to slightly irritating in vivo if the result obtained with the reference substance is taken into consideration.