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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Acceptable, non validated in vitro test, GLP compliant

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: ICCVAM-Recommended Test Method Protocol: Hen's Egg Test - Chlorioallantoic Membrane (HET-CAM) Test method, published 2010
Deviations:
yes
Remarks:
minor
Principles of method if other than guideline:
The basis of the method is to assess the eye irritancy potential of substances by means of the chorioallantoic membrane of hens' eggs (HET_CAM).
The study was carried out according to : ICCVAM-Recommended Test Method Protocol: Hen’s Egg Test – Chorioallantoic Membrane (HET-CAM) Test Method, published 2010.
This method is increasingly used for the detection of eye mucosa membrane irritants. Eye irritation caused by external contact with chemical substances is characterized by corneal damage and/or conjunctival injury and/or iris defects.
The CAM of fertile eggs incubated for 9 days is a vital vascular membrane with a blood vessel complex. Effects which might produce irritancy in the conjunctiva are assessed by exposing the CAM to liquid or solid test items.



GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
purity: 99.3% (GC Method MS409A.M-Results)

Test animals / tissue source

Species:
other: Chicken eggs
Strain:
other: Fertile white Leghorn chicken eggs
Details on test animals or tissues and environmental conditions:
Weight: 48-51g (according to Study Plan the weight should be 50-60 g)
Age: 7 days

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes
Amount / concentration applied:
300 µL/egg (used undiluted)
Duration of treatment / exposure:
20 seconds
Observation period (in vivo):
300 seconds
Number of animals or in vitro replicates:
3 eggs treated with the test item
Negative control item 3 eggs treated with physiological saline
Positive control items 3 eggs treated with NaOH (0.1 N); 3 eggs treated with SDS (1%)

The solutions were prepared on the day of testing.
Details on study design:
The weight of the eggs ranged from 48 to 60 g and not from 50 to 60 g as stated in the Study Plan.
This minor deviation did not affect the validity and integrity of the scientific results obtained during this study.

Results and discussion

In vivo

Resultsopen allclose all
Irritation parameter:
overall irritation score
Basis:
mean
Score:
10.1
Remarks on result:
other: 1,4-diethylbenzene
Irritation parameter:
overall irritation score
Basis:
mean
Score:
0
Remarks on result:
other: Negative control 0.9% Na Cl solution
Irritation parameter:
overall irritation score
Basis:
mean
Score:
18.6
Remarks on result:
other: Positive control: 0.1N NaOH
Irritation parameter:
overall irritation score
Basis:
mean
Score:
10.2
Remarks on result:
other: Positive control: 1% aqueous solution sodium dodecil sulphate
Irritant / corrosive response data:
20 seconds after application the CAM was carefully rinsed with 0.9% NaCl solution. The reactions on the CAM were observed over a period of 300 seconds. The time for the appearance of each of the noted endpoints was monitored and recorded, in seconds.
The following endpoints were observed:
- haemorrhage (bleeding from the vessels)
- vascular lysis (blood vessel disintegration)
- coagulation (intra- and extra-vascular protein denaturation)

Whereby,
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.

The evaluation consists in to giving scores of each to the three HET-CAM points at the three points 0.5, 2 and 5 minutes.

The numerical time-dependent scores for lysis, haemorrage and coagulation are summed to give a single numercial value indicating the potential of the test substance on a scale with a maximum value of 21. The mean value of three eggs facilitates an assessment by a classification scheme analogous to the Draize categories.

Any other information on results incl. tables

Table 1. Scoring scheme for irritation testing

 Effect  score (0.5min)  score (2min)  score (5min)
 lysis  5  3  1
 haemorrage  7  5  3
 coagulation  9  7  5

The observed effects were grades for each egg by an irritation score (IS) according to a formula

Table 2: IS Classificatio Scheme

 IS score range  Irritation Assessment
 0 -0.9  Practically none
 1 -4.9  Slight
 5 -8.9  Moderate
 9 -21  Strong

Applicant's summary and conclusion

Interpretation of results:
highly irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the present test conditions 1,4-diethylbenzene was a strong irritant in an experiment employing the chorioallantoic membrane of hens' eggs (HET-CAM) as model.
Executive summary:

The purpose of this study was to determine the eye irritancy potential of 1,4-Diethylbenzene by means of the chorioallantoic membrane of hens' eggs (HET-CAM). Eye irritation caused by external contact with chemical substances is characterized by corneal damage and/or conjunctival injury and/or iris defects.

The of fertile eggs incubated for 9 days is a vital vascular membrane with a blood vessel complex.

Three eggs each were treated with 300 µL1,4-Diethylbenzene/egg and with the control items. 0.9% NaCl solution was used as the negative control item. 0.1 NSodium hydroxide (NaOH)and 1% aqueous sodium dodecyl sulphate (SDS) were used as the positive control items. The administration volume for the control items was 300 µL per egg.

After administration of the test item blood vessels including the capillary system and the albumen were examined and scored for irritant effects (haemorrhage, coagulation and lysis) during 5 minutes.

The eggs treated with1,4-Diethylbenzene revealed an effect with an irritation score (IS) of 10.0. The test item was considered to be a strong irritant.

The positive control items 0.1or 1% SDS caused the expected effect with irritation scores (IS) of 18.6 or 10.0, respectively and, hence, were well within the historical data-range. No effects were observed in the negative control 0.9% NaCl solution. Hence,the HET-CAM assay is considered to be valid.