1,1,3-trimethylindan-4-amine

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 - 15 Dec 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

adopted 26 Jun 2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Freie und Hansestadt Hamburg, Behörde für Justiz und Verbraucherschutz

Test material

Test material information

Constituent 1

Reference substance name:

1,1,3-trimethyl-2,3-dihydro-1H-inden-4-amine

EC Number:

838-724-1

Cas Number:

94568-76-0

Molecular formula:

C12H17N

IUPAC Name:

1,1,3-trimethyl-2,3-dihydro-1H-inden-4-amine

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: obtained from slaughterhouse (Otto Vollertsen GmbH, Mittelangeln, Germany)
- Characteristics of donor animals: 6 - 12 months old; no further characteristics given in the report
- Storage, temperature and transport conditions of ocular tissue: On collection, the eyes were completely submerged in Hanks´ Balanced Salt Solution (HBSS). No further transport conditions given in the report.
- Indication of any existing defects or lesions in ocular tissue samples: no; only corneas from eyes free of defects were used
- Indication of any antibiotics used: yes, penicillin at 100 IU/mL and streptomycin at 100 µg/mL

SELECTION AND PREPARATION OF CORNEAS
The corneas were dissected with a 2 to 3 mm rim of sclera and mounted in corneal holders with anterior (epithelium) and posterior (endothelium) chambers.

QUALITY CHECK OF THE ISOLATED CORNEAS
At the end of the equilibration period, the basal opacity was determined (t0). Corneas that had opacity greater than seven opacity units or equivalent for the opacitometer and cornea holders used after an initial one-hour equilibration period had to be discarded. A minimum of three corneas with opacity values close to the median value for all corneas were selected as negative control corneas. The remaining corneas were then distributed into treatment and positive control groups.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 750 µL (also for neg. and pos. controls)

Duration of treatment / exposure:

10 min

Observation period (in vivo):

not applicable

Duration of post- treatment incubation (in vitro):

2 hours at 32°C ± 1°C

Number of animals or in vitro replicates:

triplicates for each treatment and control group

Details on study design:

TREATMENT METHOD: Open-Chamber method: Beginning with the posterior chambers, the chambers were filled to excess with pre-warmed Eagle's Minimum Essential Medium (EMEM), while preventing bubble formation. The corneal holder was equilibrated at 32°C ± 1°C for at least one hour. The glass window from the anterior chamber was removed prior to treatment. The controls or test substance were applied directly to the epithelial surface of the cornea using a pipet. After the exposure period of 10 minutes the exposition solution, namely the test item, the negative and positive controls, were removed from the chamber.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: At least 3. The epithelium was washed with EMEM containing phenol red. Washing was repeated (at least two times) until no test item or discolouration of phenol red was visible. The corneas were rinsed a final time with EMEM without phenol red only to remove any remaining phenol red from the chamber.

POST-EXPOSURE INCUBATION:
The corneas were incubated at 32 ± 1°C for two hours.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was determined by the amount of light transmission through the cornea measured quantitatively via an opacitometer (BASF OP-3.0, Ludwigshafen am Rhein, Germany) resulting in opacity values measured on a continuous scale.
- Corneal permeability: passage of sodium fluorescein dye measured quantitatively with the aid of a microfilter plate reader (Tecan infinite 200Pro, Tecan Deutschland GmbH, Crailsheim, Germany). Measurements at 490 nm were recorded as optical density (OD490).

SCORING SYSTEM:
In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
Test substance with an IVIS > 55 was regarded as serious eye damage and labelled Category 1 according to CLP and GHS-UN, respectively.
Test substance with an IVIS ≤ 3 was regarded as non-irritant and labelled in no category.
Test substance with an IVIS > 3; ≤ 55 no prediction can be made.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Remarks:

mean value of 3 corneas

Run / experiment:

10 min exposure

Value:

1.84

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
-Visible damage on test system: not reported

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, the negative control resulted in opacity (-0.053 ± 0.564) and permeability (0.025 ± 0.016) values that were less than the established upper limits for background opacity and permeability values for bovine corneas treated with the respective negative control (see 'any other information on results incl. tables', table 1).
- Acceptance criteria met for positive control: Yes, the positive control resulted in an IVIS (87.426 ± 18.762) which was within two standard deviations of the current historical mean and well above the cut-off value of 55.

HISTORICAL CONTROL DATA
See ''any other information on results incl. tables', table 4'

Any other information on results incl. tables

Table 1: Opacity values and corrected opacity values of the BCOP assay.

Parameter	Opacity [Opacity Units]	Corrected Opacity
		After subtraction of the background opacity (neg. ctrl. = 0.053)	Mean of group	Standard deviation
Negative control (0.9% NaCl)	0.597	-	-0.053	0.564
	-0.358
	-0.399
Test substance	2.112	2.165	1.275	1.039
	0.080	0.133
	1.475	1.528
Positive control (1% NaOH)	62.151	62.204	46.321	13.916
	40.438	40.491
	36.216	36.269

 

Table 2: Permeability OD values (490 nm) of the BCOP assay.

Parameter	Permeability [OD]	Mean of Triplicates	Corrected Permeability [OD]
			After subtraction of the background permeability (neg. ctrl. = 0.025)	Per Cornea		Per Group
				Mean	SD	Mean	SD
Negative control (0.9% NaCl)	0.020	0.020	-	0.020	0.001	0.025	0.016
	0.020		-
	0.019		-
	0.044	0.043	-	0.043	0.001
	0.043		-
	0.042		-
	0.012	0.013	-	0.013	0.001
	0.013		-
	0.013		-
Test substance	0.036	0.036	0.011	0.011	0.000	0.038	0.029
	0.036		0.011
	0.036		0.011
	0.058	0.058	0.033	0.033	0.000
	0.058		0.033
	0.058		0.033
	0.094	0.094	0.069	0.069	0.001
	0.094		0.069
	0.095		0.070
Positive control (1% NaOH)	2.875	2.897	2.850	2.872	0.026	2.740	0.579
	2.925		2.900
	2.890		2.865
	3.280	3.267	3.255	3.242	0.013
	3.265		3.240
	3.255		3.230
	2.145	2.132	2.120	2.107	0.013
	2.120		2.095
	2.130		2.105

 

Table 3: In-Vitro Irritancy Score (IVIS) values of the BCOP assay.

Parameter	Per Cornea	Per Group
		Mean	SD
Negative control (0.9% NaCl)	0.897	0.327	0.552
	0.287
	-0.204
Test substance	2.330	1.840	1.056
	0.628
	2.563
Positive control (1% NaOH)	105.284	87.426	18.762
	89.121
	67.874

 

 

Table 4: Historical Control Data of the BCOP assay with liquid test items (2017 to 2020 (n=18)).

	Parameter	Mean	SD	Lower limit of acceptance (mean – 2x SD)	Upper limit of acceptance (mean + 2x SD)
NaCl 0.9 %	Opacity	-0.083	0.844	-1.771	1.604
	Permeability	0.009	0.008	-0.007	0.025
	IVIS	0.053	0.830	-1.608	1.713
NaOH 1%	Opacity	58.768	14.710	29.349	88.188
	Permeability	2.329	0.896	0.537	4.121
	IVIS	93.702	18.606	56.489	130.914

 

 

Applicant's summary and conclusion

Interpretation of results:

other: Eye Irrit. 2 (H319) according to Regulation (EC) No 1272/2008

Conclusions:

This study was part of an integrated testing strategy. A bottom-up approach starting with the Epiocular™ test (according to OECD 492) was conducted. A positive in vitro irritation response was revealed for the test substance, which is not conclusive with respect to classification of the test substance as eye irritant (Eye Irritant Cat. 2) or serious eye damage (Eye Damage Cat. 1). Therefore, the BCOP was chosen as follow-up test (according to OECD 437) and, as already described, the test substance turned out not to be irritating. It is therefore concluded that the test substance 1,1,3-trimethylindan-4-amine should be classified as Eye Irrit. Cat. 2 according to Regulation (EC) No 1272/2008.