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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
Study period:
4 May 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: 1a. GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD guideline no. 438: “Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants”, adopted on 7 September 2009.
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Date of receipt : 27 April 2011
Test substance name : N-methylaniline_EC 202-870-9
Chemical name : N-methylaniline
Purity : 93,56%
Lot number : 20110120
CAS. Reg. No. : 100-61-8
Storage conditions : ambient
Appearance : colourless to clear yellow liquid
Expiry date : 27 April 2012

Test animals / tissue source

Species:
other: In vitro test with chicken eyes.
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Approximately 7 weeks old, male or female chickens (ROSS, spring chickens), body weight range approximately 1.5 2.5 kg, were used as eye donors. Heads of these animals were obtained from a poultry slaughterhouse in the Netherlands. Heads of the animals were cut off immediately after sedation of the animals by electric shock and incision of the neck for bleeding. The heads were placed in small plastic boxes on a bedding of paper tissues moistened with isotonic saline. Next, they were transported to the testing facility. During transportation, the heads were kept at ambient temperature.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
other: Negative control - 1 eye
Amount / concentration applied:
30 μL undiluted.
Duration of treatment / exposure:
10 seconds
Observation period (in vivo):
Eyes were examined at approximately 0, 30, 75, 120, 180 and 240 minutes after treatment
Number of animals or in vitro replicates:
Negative control - 1 eye
Positive control - 3 eyes
Test group - 3 eyes
Details on study design:
Within 2 hours after kill, eyes were carefully dissected and the eye lids were carefully removed without damaging the cornea. Next, the head with the fluorescein treated cornea was examined to ensure that the cornea is not damaged. If undamaged, the eye was further dissected from the head without damaging the eye or cornea. The enucleated eye was placed in a stainless steel clamp with the cornea positioned vertically and transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with isotonic saline from a bent, stainless steel tube, at a target rate of 0.10 - 0.15 mL/min (temperature controlled at approximately 32˚C). Corneal thickness was measured using the Depth Measuring Attachment No. I for the Haag Streit slit lamp microscope. An accurate measurement was taken at the corneal apex of each eye. Eyes with a corneal thickness deviating more than 10% of the average corneal thickness of the eyes, eyes that show opacity (score higher than 0.5), or are unacceptably stained with fluorescein (score higher than 0.5) indicating the cornea to be permeable, or eyes that show any other signs of damage, was rejected as test eyes and replaced. Each eye provided its own baseline values for corneal swelling, corneal opacity and fluorescein retention. For that purpose, after an equilibration period of 45 - 60 minutes, the corneal thickness of the eyes was measured again to determine the zero reference value for corneal swelling calculations.

Results and discussion

In vivo

Resultsopen allclose all
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
other: 240 min
Score:
65
Max. score:
65
Reversibility:
not specified
Remarks on result:
other: Irritation Index = maximum mean corneal swelling + maximum mean opacity (x 20) + mean fluorescein score (x 20)
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
Swelling %
Time point:
other: 240 min
Score:
9
Max. score:
9
Reversibility:
not specified
Remarks on result:
other: Positive control: 27%
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
opacity
Time point:
other: 240 min
Score:
1.8
Max. score:
1.8
Reversibility:
not specified
Remarks on result:
other: Positive control: score of 3 (Severe loosening of epithelium)
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
Fluorescein retention
Time point:
other: 30 min
Score:
1
Max. score:
1
Reversibility:
not specified
Remarks on result:
other: Positive control: 3.0

Any other information on results incl. tables

Mean valuesand irritancy categories: N-methylaniline:

 

Time intervals

[min]

 

 

Swelling %

 

Opacity

 

Fluorescein

30

2

1.0

1.01

75

4

1.0

 

120

7

1.5

 

180

8

1.5

 

240

9

1.8

 

 

 

Parameter

 

 

Maximum score

 

Irritancy category

Swelling %

9

II

Opacity

1.8

III

Fluorescein retention

1.0

II

 

Applicant's summary and conclusion

Interpretation of results:
Category 2A (irritating to eyes)
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
N-methylaniline caused slight corneal swelling, slight to moderate opacity, and slight fluorescein retention. The calculated Irritation Index was 65 (max possible score is 200).
Executive summary:

1.    A sample of N-methylaniline_EC 202-870-9was evaluated neat for eye irritation potential in the Isolated Chicken Eye (ICE) test. In addition, the test included one negative control (saline) eye and three positive control eyes (BAC 5%). Chicken eyes were obtained from slaughter animals used for human consumption. The isolated chicken eyes were exposed to a single application of 30 µL of the test sample for 10 seconds followed by a 20 ml saline rinse. Three main parameters were measured to disclose possible adverse eye effects: corneal thickness (expressed as corneal swelling), corneal opacity and fluorescein retention of damaged epithelial cells.

 

2.    N-methylaniline_EC 202-870-9caused slight corneal swelling, slight to moderate opacity, and slight fluorescein retention.The calculated Irritation Index was 65 (max possible score is 200).

 

3.    The negative control (saline) caused no corneal effects.

 

4.    The positive control BAC 5% caused moderate corneal swelling, severe opacity and severe fluorescein retention. The calculated Irritation Index was 147.

 

5.    According to theGHS and the EU-CLP classification schemes of the ICE,N-methylaniline_EC 202-870-9is considered to be Category 2B: “Mild irritant/causes eye irritation” (GHS classification) and Category 2: “Irritating to eyes” (EU-CLPclassification).