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Diss Factsheets

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:
23 June 2014 to 15 December 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
2,6,10-trimethylundec-9-enal
EC Number:
205-460-8
EC Name:
2,6,10-trimethylundec-9-enal
Cas Number:
141-13-9
Molecular formula:
C14H26O
IUPAC Name:
2,6,10-trimethylundec-9-enal
Test material form:
liquid
Specific details on test material used for the study:
Batch SC00010875
Expiration date 09 April 2015

Test animals / tissue source

Species:
other: Bovine eyes
Strain:
other: Corneas
Details on test animals or tissues and environmental conditions:
The eyes were removed after slaughter, completely immersed in Hanks’ Balanced Salt Solution (containing penicillin at 100 IU/mL and streptomycin at 100 μg/mL) in a suitably sized container and transported on the same day to the testing facility.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 750 µL
- Concentration: Undiluted
Duration of treatment / exposure:
10 minute incubation at 32°C ± 1°C.
Duration of post- treatment incubation (in vitro):
After the 1st incubation (10 minute incubation at 32°C ± 1°C) with exposure (750 µL) , each cornea was washed with media containing phenol red followed by media without phenol red and the corneas assessed for opacity. The corneas were then incubated (horizontally) for 2 hours ± 10 minutes after which, corneal opacity was measured.
Number of animals or in vitro replicates:
Three corneas

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Value:
1.03
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Corneal Opacity :
The mean corrected opacity reading for the test article was 1.0.
The mean corrected opacity reading for the negative control was 0.0.
The mean corrected opacity reading for the positive control was 35.3.

Corneal Permeability:
The mean group corrected optical density for the test article was 0.002.
The mean group corrected optical density for the negative control was 0.000.
The mean group corrected optical density for the positive control was 0.183

The assay was considered valid as the assay acceptance criteria were met.

Any other information on results incl. tables

Table 1: Corneal Opacity

Test substance

Cornea No.

Initial Opacity

Post Incubation Opacity

Change in Opacity

Mean Change in Opacity

Corrected Opacity

Mean Corrected Opacity

Test material

2

3

4

1

N/A

-0.3

1.0

5

3

7

4

2.7

15

3

5

2

0.7

NaCl (negative control)

6

2

3

1

1.33

-0.3

0.0

17

2

4

2

0.7

21

2

3

1

-0.3

DMF (positive control)

16

3

52

49

N/A

47.7

35.3

18

3

22

19

17.7

24

3

45

42

40.7

 

Table 2: Corneal Permeability

Test substance

Cornea No.

Mean Blank OD490

OD490

Corrected OD490

Mean Corrected OD490

Final Corrected OD490

Test material

2

0.022

0.022

N/A

0.003

0.002

5

0.021

0.021

0.002

15

0.019

0.019

0.000

NaCl (negative control)

6

0.000

0.021

0.021

0.019

0.002

0.000

17

0.016

0.016

-0.003

21

0.019

0.019

0.000

DMF (positive control)

16

0.163

0.163

N/A

0.144

0.183

18

0.168

0.168

0.149

24

0.273

0.273

0.254

 

Table 3: IVIS scores

Test substance

IVIS

Test material

1.03

NaCl (negative control)

0.00

DMF (positive control)

38.07

Applicant's summary and conclusion

Interpretation of results:
not classified
Conclusions:
The test article, Adoxal, produced an In Vitro Irritancy Score (IVIS) of 1.03 and does not require classification for eye irritation.
Executive summary:

In the in vitro study (Dreher, 2014), the potential of the test material to cause serious eye irritation was investigated in vitro in a study conducted in accordance with the standardised guidelines OECD 437 and EU Method B.47 under GLP conditions using bovine corneas.

750 µL of undiluted test material was applied to each of three corneas and incubated for 10 minutes at 32 °C ± 1 °C. After incubation, each cornea was washed with media containing phenol red followed by media without phenol red and incubated horizontally for 2 hours ± 10 minutes and then measured for opacity.

Permeability of the corneas was measured by replacing the media in the anterior chamber with 1 mL of 4 mg/mL sodium fluorescein. The posterior chamber was filled with fresh media. The corneas were incubated vertically for 95 minutes at 32 °C ± 1 °C. The posterior chambers were the removed and three 350 µL aliquots of the media were taken for analysis of optical density at 490 nm.

The mean corrected opacity reading for the test material was determined to be 1.0. The mean corrected opacity reading for negative and positive controls was 0.0 and 35.3, respectively. The corneas treated with the positive control were cloudy following treatment.

The mean corrected optical density for the test material was determined to be 0.002. The mean corrected optical densities for negative and positive controls were 0.000 and 0.183, respectively.

Under the conditions of the test, the test material was determined to not require classification for eye irritation. The test material produced an in vitro irritancy score (IVIS) of 1.03 in bovine corneas.