2-Thiophenecarboxylic acid, 5-amino-4-cyano-3-methyl-, methyl ester

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: test method is under evaluation

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Materials and methods

Principles of method if other than guideline:

Undissolved diazokomponenten Rot B was applied topically to reconstituted human ocular epithelia (HCE :Human Cornea Epithelial) tissue for 60 min followed by a 16 hour post -treatment incubation period. The potential ocular irritation of the test substance is evaluated by measuring the HCE cell viability using the MTT (Methylthiazoletetrazolium) assay. The HCE model is currently involved in the eye irritation validation conducted by the COLIPA following ECVAM guidelines

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

methyl 5-amino-4-cyano-3-methylthiophene-2-carboxylate

EC Number:

700-422-1

Cas Number:

61320-65-8

Molecular formula:

C8 H8 N2 O2 S

IUPAC Name:

methyl 5-amino-4-cyano-3-methylthiophene-2-carboxylate

Details on test material:

content 99.1 %
batch No. BOS 2589

Test animals / tissue source

Species:

other: in vitro toest

Strain:

other: in vitro test

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: in vitro - assay: --negative control: phosphat buffered saline --positive control 1H-1,2,4 -Triazole-3 -thiol

Amount / concentration applied:

30 mg (RT;three inserts per period of incubation time)

Duration of treatment / exposure:

60 min incubation time

Observation period (in vivo):

After the exposure period (RT; three inserts per period of incubation time) the inserts were washed with PBS. After a post-exposure incubation of 16 hours MTT reduction assay was performed

Number of animals or in vitro replicates:

not applicable

Details on study design:

Undissolved diazokomponenten Rot B was applied topically to reconstituted human ocular epithelia (HCE :Human Cornea Epithelial) tissue for 60 min followed by a 16 hour post -treatment incubation period. The potential ocular irritation of the test substance is evaluated by measuring the HCE cell viability using the MTT (Methylthiazoletetrazolium) assay.
--negative control: phosphat buffered saline (PBS, 30µl)
--positive control 1H-1,2,4 -Triazole-3 -thiol (30 mg, plus 30µlPBS to moisten and ensure good contact with the skin, in three replicates)

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

other: absorption at 570 nm

Basis:

other: negative control

Time point:

other: 60 min + 16 hours

Score:

1.03

Max. score:

1.16

Reversibility:

not specified

Remarks on result:

other: Optical Density (OD) 1.03 = 100 % viability;

Results 2

Irritation parameter:

other: absorption at 570 nm

Basis:

other: test substance

Time point:

other: 60 min + 16 hours

Score:

1.02

Max. score:

1.03

Reversibility:

not specified

Remarks on result:

other: Optical Density (OD) 1..03 = 100 % viability; OD 1.902 = 99.14 % viability

Results 3

Irritation parameter:

other: absorption at 570 nm

Basis:

other: positive control

Time point:

other: 60 min + 16 hours

Score:

0.23

Max. score:

1.03

Reversibility:

not specified

Remarks on result:

other: Optical Density (OD) 1.03 = 100 % viability; OD: 0.23 = 22.63 % viability

Irritant / corrosive response data:

Compound Cell viability [%] Evaluation
Diazokomponente Rot B 99.14 non-irritant
Positive control 22.63 irritant
Negative control 100.00 non-irritant

Any other information on results incl. tables

Sample No	Test item	OD mean*	StdDev	% Viability
1-3	Negative control PBS	1.03	0.04	100.00
4-6	Positive control 1H-1,2,4-Triazole-3-thiol	0.23	0.11	22.63
7-9	Diazokomponente Rot B	1.02	0.02	99.14

* 6 values

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information

Executive summary:

This in vitro study for assessing ocular irritation of compounds using a human epithelial corneal cell model was performed in 2011. The model used is standardized and commercially available (SkinEthicTM Human Corneal Epithelial Model (HCE); SkinEthic, France).

Undisolved Diazokomponente Rot B was applied topically to the HCE tissue, i.e. 30 mg per insert (three replicates). After an exposure period of 60 minutes, followed by a 16 hours post-treatment incubation period, the cell viability was 99.14% as measured by a MTT conversion assay.

The results of the concurrent negative (NC, PBS) and positive control

(PC, 1H-1,2,4-Triazole-3-thiol) demonstrated the viability (NC) and sensitivity (PC) of the test model.

The results show that Diazokomponente Rot B is predicted as non-irritant under the conditions of this test method (Bayer 2011).