N1,N3-diallylpropane-1,3-diamine dihydrochloride

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2020-04-21 to 2020-05-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

adopted 09th October 2017

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(prop-2-en-1-yl)({3-[(prop-2-en-1-yl)amino]propyl})amine dihydrochloride

EC Number:

812-745-6

Cas Number:

205041-15-2

Molecular formula:

C9 H20 Cl2 N2

IUPAC Name:

(prop-2-en-1-yl)({3-[(prop-2-en-1-yl)amino]propyl})amine dihydrochloride

Test material form:

solid

Details on test material:

- CAS: 205041-15-2
- Batch: LWDDP00072
- Colour: white
- Purity (GC): 100%
- Expiry date: 2020-08-11
- Storage conditions: room temperature
- Stability: stable under recommended storage conditions

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was tested as supplied.

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
Corneas of calves aged les than 8 months were collected in the slaughterhouse of Sobeval Boulazac 24759 - France, in a short time after slaughtering of the animals and transported in a Hanks´s buffered saline solution with antibiotic to the laboratory.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 750 ± 75 mg

Duration of treatment / exposure:

4 hours at 32 +/- 1 °C

Duration of post- treatment incubation (in vitro):

The permeability at 490 nm was measured upon 90 minutes of incubation with fluorescein after exposure to the test item by using a spectrophotometer.

Number of animals or in vitro replicates:

Three corneas each for the test item, negative control and positive control.

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
- At reception, calve eyes were carefully examined under lighting and these showing a visble defect were eliminated. For each selected eye, an incision with a scalpel was practiced at the level of the scleral ring by means of scissors. Approximately 2 to 3 mm of scleral ring were left to facilitate the further handlings. Corneas were immersed in Hanks' medium at room temperature. For mounting, the corneas were deposited, endothelial side upwards on the posterior part of cornea holders. Then the anterior part was firmly clamped in place with 3 screws. The anteroir (epithelial side) and posterior (endothelial side) compartments were then filled with pre-warmed EMEM without phenol and ensuring that no bubbles were formed. The corneas were incubated for one hour at 32 +/- 1 °C.

INITIAL OPACITY and CORNEA SELECTION
- After pre-incubation, compartments were emptied and fresh EMEM was added. The opacity at t=0 (OPT0) was then determined. Corneas showing a value of opacity greater than seven opacity units were discarded.

NUMBER OF REPLICATES
- 3 corneas for test item, 3 corneas for negative control and 3 corneas for the positive control

NEGATIVE CONTROL USED
- 0.9% NaCl, Cooper batch 13NLP271

POSITIVE CONTROL USED
- Imidazole, diluted at 20% with 0.9% NaCl, Sigma batch: SLBTZ469

APPLICATION DOSE AND EXPOSURE TIME :
- 750 ± 75 mg for 4 hours

TREATMENT METHOD: open chamber

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the end of the exposure period, the test item and controls were removed from the anterior compartment and the epithelium washed at least three times (or until no visual evidence of test item can be observed) with EMEM containing phenol red. After the last effective rinsing, corneas were given a final rinse with EMEM without phenol red, what allowed to make sure that the inside of support was cleared of any phenol red before the next measure of opacity.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacity was determined by the amount of light transmission through the cornea. Corneal opacity was measured quantitatively with the aid of an opacitometer (OP-KIT), resulting in opacity values measured on a continuous scale. The opacity of each cornea was measured at 2 times, just before treatment with the test item (OPT0) and immediately after the end of the exposure period (OPT2)
- Corneal permeability: Passage of sodium fluorescein dye measured with the aid of a spectrophotometer (software VISION lite(TM) version 2.2) (OD490) . The assessment of this parameter was performed after the 2nd opacity measurement.
- Other: Corneas were observed and visible modifications of the cornea (oedema, colouring) were noted.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS) (mean opacity value + (15 x mean permeability OD490 value))

DECISION CRITERIA: The decision criteria as indicated in the TG were used (see Table 1 in box "Any other information on materials and methods incl. tables).

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

mean of triplicates

Value:

10.7

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no prediction can be made

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: epithelium detachment

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the test was considered acceptable if the negative control gave opacity values OP < 12 and the optical density OD < 0.140. They were respectively: OP = 0.7 and OD = 0.004.
- Acceptance criteria met for positive control: yes, the test was considered acceptable if the positive control gives an IVIS that ranged within two standard deviations of the current historical mean (80.3 < IVIS < 179.3) (Result: 133.2 ± 12.0).

Any other information on results incl. tables

Table 2: Results of the main experiment

4 h treatment
Support Nos	Treatment	Opt0	Opt2	Opt2-Opt0	adjusted opacity	OD	OD adjusted	Score (Opa+15xODa)
26	Negative control NaCl 0.9%,	2	3	1		0	0	0
7		4	2	-2		0.006	0.006	0.1
18		0	3	3		0.007	0.007	0.1
Observations: -			Mean		0.7	0.004	0.004	0.1
			SD		2.5	0.004	0.004	0.1
Support Nos	Treatment	Opt0	Opt2	Opt2-Opt0	adjusted opacity	OD	OD adjusted	Score (Opa+15xODa)
4	Positive control Imidazol	0	90	90	89.3	2.418	2.414	125.5
27		0	115	115	114.3	2.186	2.182	147.1
15		0	90	90	89.3	2.51	2.506	126.9
Observations:Oedema and high epithelium detachment			Mean		97.7	2.367		133.2
			SD		14.4	0.167		12
Support Nos	Treatment	Opt0	Opt2	Opt2-Opt0	adjusted opacity	OD	OD adjusted	Score (Opa+15xODa)
22	N1,N3-diallylpropane-1,3-diamine dihydrochloride	0	11	11	10.3	0.066	0.062	11.3
13		0	14	14	13.3	0.158	0.154	15.6
12		2	6	4	3.3	0.125	0.121	5.1
Observations:Epithelium detachment			Mean		9	0.112		10.7
			SD		5.1	0.047		5.3

Applicant's summary and conclusion

Interpretation of results:

other: No prediction can be made

Conclusions:

In conclusion, based on the mean in vitro irritation score of 10.7 obtained in the bovine corneal opacity and permeability assay (BCOP, OECD 437), no prediction can be made regarding the classification of the test substance.

Executive summary:

The eye irritation potential of N1,N3-diallypropane-1,3-diamine dihydrochloride (100% purity) was investigated in the bovine corneal opacity and permeability assay (BCOP, OECD 437). A mean in vitro irritation score of 10.7 was determined. The positive and negative controls induced the appropriate responses, indicating the validity of the assay. Based on the results obtained, no prediction can be made regarding the classification of the substance.

The study is acceptable and satisfies the guideline requirements for an in vitro study for eye damage (OECD 437).