Reaction mass of 2-ethyl-4-methyl-1H-imidazole-1-propiononitrile and 2-ethyl-5-methyl-1H-imidazole-1-propiononitrile

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 -14 Dec 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

adopted 28 Jul 2015

Deviations:

yes

Remarks:

- the test substance showed reduction of MTT and colouring as compared to the vehicle control. - the non-specific reduction of MTT was not determined - no killed tissue controls were performed

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

adopted 9 Oct 2017

Deviations:

yes

Remarks:

- the test substance showed reduction of MTT and colouring as compared to the vehicle control. - the non-specific reduction of MTT was not determined - no killed tissue controls were performed

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Deutschland

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 2-ethyl-4-methyl-1H-imidazole-1-propiononitrile and 2-ethyl-5-methyl-1H-imidazole-1-propiononitrile

EC Number:

947-727-8

Cas Number:

568591-00-4

Molecular formula:

C9H13N3

IUPAC Name:

Reaction mass of 2-ethyl-4-methyl-1H-imidazole-1-propiononitrile and 2-ethyl-5-methyl-1H-imidazole-1-propiononitrile

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

- in vitro test method: Eye Irritation Test (EIT) with reconstructed human cornea-like epithelium (RhCE) tissue, EpiOcular™ (MatTek)
- Lot number: 27017
- Keratinocyte strain: 4F1188
- no biological contaminants (virus, bacteria, yeast or fungi) detected
- Acceptance criteria for tissue viability met: 1.604 ± 0.08 (Acceptance criteria: OD (540-570 nm) [1.1 - 3.0])
- Acceptance criteria for Barrier function met: 21.7 min (Acceptance criteria: ET-50 [12.2 - 37.5 min])

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 50 µL undiluted

NEGATIVE CONTROL
- Amount(s) applied: 50 µL
- Lot/batch no.: Sigma, RNBF7110

POSITIVE CONTROL
- Amount(s) applied: 50 µL
- Lot/batch no.: Merck, S6943111

Duration of treatment / exposure:

30 ± 2 min

Duration of post- treatment incubation (in vitro):

12 ± 2 min (post-soak plate)
120 ± 15 min (post-treatment plate)

Number of animals or in vitro replicates:

duplicates for each treatment and control group; from each tissue, 2 absorbance measurements after MTT incubation were performed

Details on study design:

- RhCE tissue construct used, including batch number: Epiocular™, Test Kit name: OCL-200-EIT (MatTek), Lot number: 27017

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: exposure: 30 ± 2 min at 37 ± 1°C, post exposure: 12 ± 2 min at room temperature (post-soak plate), 120 ± 15 min (post-treatment plate) at 37 ± 1°C

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: The ability of the test substance to directly reduce MTT and to change the color of the MTT medium was assessed in a pre-experiment.

Mixture of the test substance with MTT medium showed non-specific reduction of MTT compared to the solvent. The mixture turned blue/purple. Since the mean relative tissue viability of the test substance treated tissues (TM) was below the 60% threshold value no killed tissue controls were performed, since the test substance has to be classified as irritant in any case.
Mixture of test substance with distilled water or isopropanol showed colouring as compared to the solvent. Since the mean relative tissue viability of the test substance treated tissues (TM) was below the 60% threshold value no coloured tissue controls were performed, since the test substance has to be classified as irritant in any case.

- Number of tissue replicates used per test chemical and controls: 2

- Wavelength: 570 nm ± 30 nm

- Spectrophotometer: plate spectrophotometer

- Description of evaluation criteria used: The test substance is considered to be not irritating to the eye if the tissue viability is > 60%. The test substance is considered to be irritating to eye if the tissue viability is ≤ 60% (no distinction between category 1 or 2 possible).

The test meets acceptance criteria if:
- mean absolute OD570 of the negative control is > 0.8 and < 2.5,
- mean relative tissue viability of the positive control is < 50%,
- mean relative tissue viability difference between replicate tissues is < 20%.

Results and discussion

In vitro

Results

Irritation parameter:

other: % tissue viability mean value of 2 tissues

Value:

6.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Mixture of the test substance with MTT medium showed non-specific reduction of MTT compared to the solvent. The mixture turned blue/purple. Since the mean relative tissue viability of the test substance treated tissues (TM) was below the 60% threshold value no killed tissue controls were performed, since the test substance has to be classified as irritant in any case.

- Mixture of test substance with distilled water or isopropanol showed colouring as compared to the solvent. Since the mean relative tissue viability of the test substance treated tissues (TM) was below the 60% threshold value no coloured tissue controls were performed, since the test substance has to be classified as irritant in any case.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, the OD of the negative control was 1.720, 1.808, 1.959 and 1.952, and thus within the acceptibility range (OD > 0.8 and < 2.5).
- Acceptance criteria met for positive control: Yes, the mean positive control reduced the cell viability at 2.0% and fulfilled the acceptance criteria (< 50%).
- Acceptance criteria for variabilties: Differences of two tissue cell viabilities in the negative control substance, the positive control substance and the test substance groups were 10.3%, 0.3% and 0.4%, respectively, and thus < 20%.

Any other information on results incl. tables

Table 2: Summary of Results

Name	Negative Control		Positive Control		Test Substance
Tissue	1	2	1	2	1	2
OD570 values (blank-corrected)	1.720225	1.959125	0.039525	0.035025	0.109525	0.118925
	1.808325	1.952425	0.040225	0.032025	0.109225	0.115125
mean of the duplicates	1.764	1.956	0.040	0.034	0.109	0.117
mean OD	1.860^*		0.037		0.113
mean SD OD	0.12		0.00		0.00
tissue viability [%]	94.9	105.1	2.1	1.8	5.9	6.3
relative tissue viability difference [%]^***	10.3		0.3		0.4
mean tissue viability [%]	100.0		2.0^**		6.1

*         Corrected mean OD570 of the negative control corresponds to 100% absolute tissue viability

**        mean relative tissue viability of the positive control is < 50%

***       relative tissue viability difference of replicate tissues is < 20%.

SD = standard deviation

OD = optical density

Applicant's summary and conclusion

Interpretation of results:

other: irritating potential according to OECD 492

Conclusions:

Under the conditions of the RhCE test method the test substance showed irritant effects, but no prediction on the potential for serious eye damage can be made and additional testing should be conducted for classification and labeling purposes.