Di(µ-2,2´,2´´-nitrilotris(ethanol)-diperchlorato)dinatrium

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 7th, 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

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)

Principles of method if other than guideline:

SOP 118 00 830, edition 1, dated 10 April 2008.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

480-340-8

EC Name:

-

Cas Number:

156157-97-0

Molecular formula:

C12H30Cl2N2Na2O14

IUPAC Name:

Di(µ-2,2´,2´´-nitrilotris(ethanol)-diperchlorato)dinatrium

Test animals / tissue source

Species:

other: Lohmann Leghorn chicken's eggs

Details on test animals or tissues and environmental conditions:

- Description of the cell system used: chlorionallantoic membrane (CAM) of 9 day incubated chicken eggs; freshly laid Lohmann Leghorn chicken eggs were obtained from the LSL Rhein Main Geflugelvermehrungsbetried (Dieburg, Germany) and incubated for eight days.
- Incubation of eggs: 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:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amounts applied: 300 μl of the test item were measured using a centrifuge vial. The amounts were weighed before application to test system: 91.4; 106.0; 91.2; 105.7; 107.3; 91.9 mg.

Duration of treatment / exposure:

incubation time: 300 seconds.

Number of animals or in vitro replicates:

six replicates for test item, three replicates for each positive control, three replicates for negative control.

Details on study design:

CAM PREPARATION: 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. 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 forceps. The inner membrane was moistened with 0.9 % NaCl, then the egg was left to stand into the incubatror for 30 minutes. After this period, the solution was decanted and the inner membrane was carefully removed with a forceps.

TREATMENT OF CAM WITH TEST SUBSTANCE: after the preparation of CAM, the test item was given directly onto the CAM surface; 300 μl (approx. 100 mg) were measured using a centrifuge vial. Prior to the application of the test system the amounts were weighed. The test item was applied on the membrane in such a manner that 50 % of the surface of the membrane was covered with test item.

OBSERVATIONS: the reactions on the CAM were observed over a period of 300 seconds. These reactions include: haemorrhage (bleeding from the vessels), vascular lysis (blood vessel disintegration) and coagulation (intra- and extra-vascular protein denaturation). The time for the appearance of each of the noted observations was monitored and recorded in seconds.

DECISION CRITERIA
- The test substance is considered to be irritant to eye according to the irritation score (IS). Between 0 - 1.9 the substance is not irritant, between 2- 4.9 the substance is slightly irritant, between 5 - 8.9 the substance is moderately irritant and between 9 - 21 the substance is severely irritant.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

17.69

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: no signs of irritation were observed. IS for negative control was within the historical control data (0.00).
- Acceptance criteria met for positive control: both positive controls showed severe irritation. IS for both positive controls were within the historical control data 10 -19.

Any other information on results incl. tables

The observations noted with the corresponding calculated irritation scores are presented in the following table.

Table: experimental observations and irritation score.

Test group	Time until Haemorrhage (s)	Time until Lysis (s)	Time until Coagulation (s)	Irritation score	Mean Irritation score
	seconds	seconds	seconds
Negative control	301	301	301	0.00	0.00
Negative control	301	301	301	0.00
Negative control	301	301	301	0.00
Positive control 0.1 NaOH	15	29	40	18.94	19.03
Positive control 0.1 NaOH	11	28	39	19.06
Positive control 0.1 NaOH	16	27	36	19.09
Positive control 1% SDS	14	87	301	9.78	10.15
Positive control 1% SDS	14	55	301	10.52
Positive control 1% SDS	12	72	301	10.16
Test item	10	116	65	16.25	17.69
Test item	8	98	29	17.78
Test item	9	74	39	18.02
Test item	9	100	35	17.54
Test item	10	68	34	18.30
Test item	9	76	30	18.25

Applicant's summary and conclusion

Interpretation of results:

other: classified in Category 1 for serious eye damage according to the CLP Regulation (EC) No.1272/2008.

Conclusions:

The substance is considered as corrosive to eye.

Executive summary:

The irritation potential of the substance was assessed in the in-vitro study by detection of damages in blood vessels on the chorionalantoic membrane (CAM) of incubated chicken eggs (nine days old). The reactions on the CAM were observed over a period of 300 seconds and the time of the appearance of haemorrhage, lysis and coagulation were recorded in order to estimate the irritation score. Negative control and two positive controls run in parallel.

Both positive controls induced severe irritation on the blood vessels whereas the negative control showed no irritation. Both positive and negative control data lay within the accepted historical data. A mean IS (of six replicates) of 17.69 was calculated for the test substance.

The substance is considered to be as corrosive to eye.