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

Endpoint summary

Description of key information

The potential of N1,N3-diallylpropane-1,3-diamine dihydrochloride to induce skin irritation/corrosion was evaluated in two suitable in vitro test methods (OECD 439 and OECD 431). Based on the results, N1,N3-diallylpropane-1,3- diamine dihydrochloride (100% purity) must be considered as irritant to skin and classification as Skin Irrit. 2, H315 is warranted in accordance with CLP regulation 1272/2008.

In addition, effects on eye irritation were investigated in two in vitro tests according to OECD 437 and OECD 492. Following the combined assessment of the two studies, classification for Eye Irrit. 2, H319 in accordance with CLP Regulation 1272/2008 is warranted.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2020-03-09 to 2020-04-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

adopted 18th June 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- 25 mg of the test item were applied directly atop the EpiDerm^TM tissue using an application spoon avoiding compression of the test item. To ensure good contact with the skin the test item was moistened with 25 μL H2O. The volume of H2O was increased, and the test item was spread to match size of the tissue.

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The EpiDerm^TM Skin Model is a well-established organotypic, three-dimensional model of the human epidermis and is used for in vitro experiments since many years. It is known for its similarity to human skin.

Vehicle:

water

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm(TM), EPI-200 (MatTek)
- Tissue batch number(s): Lot No. 30851 A

EpiDerm Kit:
The EpiDerm tissues were provided as kits (MatTek), consisting of the following components relevant for this study:
1x sealed plate containing e.g. 24 reconstructed epidermis units (area: 0.63 cm²); each reconstructed epidermis is attached to a cell culture insert and maintained on nutritive agar for transport (Lot: 30851 A)
2x 24-well plates
4x 6-well plates
1x bottle of assay medium (DMEM-based medium; Lot: 030520MJC)
1x bottle of DPBS Rinse Solution (Lot: 112719ISE)
25 pieces Nylon Mesh circles (8 mm diameter, 200 µm pore)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: After 3 min or 60 min of application, the inserts were removed from the 6-well plate with forceps. Using a wash bottle, the tissue was gently rinsed about 20 times with PBS (phosphate buffered saline) to remove any residual test item.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT stock solution (5 mg/mL) was diluted 1 + 4 with DMEM-based medium (final concentration 1 mg/mL)
- Incubation time: 3 h
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2

PREDICTION MODEL / DECISION CRITERIA: For more details see Table 1 in section "Any other information on materials and methods incl. tables"
See Table 1 in box "Any other information on materials and methods incl. tables".

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 25 mg + 50 μL H2O (the volume was increased to spread the test item on the surface of the tissue)

VEHICLE : water
- Amount(s) applied: 50 µl

NEGATIVE CONTROL : distilled water
- Amount(s) applied: 50 μL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL 8 N KOH
- Concentration (if solution): 8 N

Duration of treatment / exposure:

3 min and 60 min

Duration of post-treatment incubation (if applicable):

3 h

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min exposure period, mean of replicates

Value:

107.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: non-corrosive

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 min exposure period, mean of replicates

Value:

68.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: non-corrosive

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: The mixture of 25 mg test item per 1 mL MTT medium showed no reduction of MTT as compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT is determined to be 0%.
- Colour interference with MTT: The mixture of 25 mg test item per 300 μL Aqua dest. and 300 µL isopropanol showed no colouring as compared to the solvent. Therefore NSC is determined to be 0%.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative & positive control: The controls confirmed the validity of the study. The mean OD570nm of the two negative control tissues was ≥ 0.8 and ≤ 2.8 for each exposure period (1.687, 1.823). The mean relative tissue viability (% negative control) of the positive control was < 15% (3.4%) after 60 min treatment. The coefficient of variation (CV) (in the range of 20 – 100% viability) of replicate tissues of all dose groups was ≤ 30% (0.3% - 6.4%).

For details and raw data results please refer to Tables 2 and 3 in section "Any other information on results incl. tables".

Table 2: Results of 3 min Experiment

Name	Negative Control		Positive Control		Test Item
Replicate Tissue	1	2	1	2	1	2
Absolute OD570	1.673	1.716	0.120	0.109	1.908	1.755
	1.654	1.683	0.120	0.108	1.872	1.714
	1.725	1.674	0.120	0.110	1.882	1.719
Mean Absolute OD570	1.687***		0.114		1.808
OD570- Blank Corrected	1.623	1.666	0.070	0.059	1.858	1.705
	1.604	1.633	0.070	0.058	1.822	1.664
	1.675	1.624	0.070	0.060	1.832	1.669
Mean OD570of 3 Aliquots (Blank Corrected)	1.634	1.641	0.070	0.059	1.838	1.679
SD OD570 of 3 Aliquots	0.037	0.022	0.000	0.001	0.019	0.022
Total Mean OD570of 2 Replicate Tissues (Blank Corrected)	1.637*		0.064		1.758
SD OD570 of 2 Replicate Tissues	0.005		0.008		0.112
Mean Relative Tissue Viability [%]	100.0		3.9		107.4
Coefficient Of Variation [%]^**	0.3		12.1		6.4

*corrected mean OD570of the negative control corresponds to 100% absolute tissue viability.

**coefficient of variation (CV) (in the range of 20 – 100% viability) between two tissues treated identically is ≤ 30%.

***The mean absolute OD570of the negative control is ≥ 0.8 and ≤ 2.8

Table 3: Results of 60 min Experiment

Name	Negative Control		Positive Control		Test Item
Replicate Tissue	1	2	1	2	1	2
Absolute OD570	1.886	1.821	0.103	0.122	1.305	1.276
	1.765	1.868	0.103	0.116	1.279	1.194
	1.772	1.829	0.100	0.118	1.281	1.210
Mean Absolute OD570	1.823****		0.110		1.257
OD570- Blank Corrected	1.836	1.771	0.053	0.072	1.255	1.226
	1.715	1.818	0.053	0.066	1.229	1.144
	1.722	1.779	0.050	0.068	1.231	1.160
Mean OD570of 3 Aliquots (Blank Corrected)	1.757	1.789	0.052	0.069	1.238	1.177
SD OD570 of 3 Aliquots	0.068	0.025	0.001	0.003	0.015	0.043
Total Mean OD570of 2 Replicate Tissues (Blank Corrected)	1.773*		0.060		1.207
SD OD570 of 2 Replicate Tissues	0.023		0.012		0.044
Mean Relative Tissue Viability [%]	100.0		3.4**		68.1
Coefficient Of Variation [%]^***	1.3		19.7		3.6

*corrected mean OD570of the negative control corresponds to 100% absolute tissue viability.

**mean relative tissue viability of the 60 min positive control is ≤ 15%,

***coefficient of variation (CV) (in the range of 20 – 100% viability) between two tissues treated identically is ≤ 30%.

****The mean absolute OD570of the negative control is ≥ 0.8 and ≤ 2.8

Interpretation of results:

other: non-corrosive

Conclusions:

In this study under the given conditions the test item showed no corrosive effects. The mean relative tissue viability (% negative control) was above 15% after 60 min treatment and above 50% after 3 min treatment. Therefore, the test item is classified as “non-corrosive“.

Executive summary:

In a primary skin corrosion study conducted according to OECD guideline 431, two EpiDerm tissues per exposure time were exposed to 25 mg of N1,N3 -diallylpropane-1,3 -diamine dihydrochloride (100% purity) for 60 min and 3 min. Cytotoxicity was measured in comparison to the concurrent negative controls via the MTT reduction assay and irritation was scored by the method of mean relative tissue viability. The test item showed no corrosive effects. The mean relative tissue viability (% negative control) was ≥15% (68.1%) after 60 min treatment and ≥50% (107.4%) after 3 min treatment. The controls confirmed the validity of the study and all acceptance criteria were fulfilled.

Based on the results from this study, the test item can be classified as non-corrosive. To specify the classification in accordance to CLP regulation 1272/2008 the results from an OECD 439 study must be additionally consulted.

The study is acceptable and satisfies the guideline requirements for an in vitro skin corrosion study (OECD 431).

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2020-04-20 to 2020-05-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

adopted 18th June 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Firstly, 25 μL of sterile DPBS were applied to the epidermal surface in order to improve the contact between the powder and the epidermis. Afterwards, 25 mg (39 mg/cm²) of the test item were applied directly atop the EpiDerm(^TM) tissue using an application spoon avoiding compression of the test item. The test item was spread to match size of the tissue by using a bulb-headed Pasteur pipette.

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™-Standard Model (EPI-200-SIT, MatTek)
- Tissue batch number(s): Lot No.: 30858

EpiDerm Kit:
The EpiDerm™ tissues were provided as kits (e.g. EPI-200-SIT, MatTek), consisting of the following components relevant for this study:
1x sealed 24-well plate containing e.g. 24 reconstructed epidermis units (area: 0.63 cm²); each reconstructed epidermis is attached to a cell culture insert and maintained on nutritive agar for transport (Lot No.: 25864)
2x 24-well plates
8x 6-well plates
1x bottle of assay medium (DMEM-based medium, Lot No.: 041620MJD)
1x bottle of DPBS Rinse Solution (Lot No.:012120MJA)
1x 1 vial 5% SDS Solution (TC-SDS-5%)
25 pieces Nylon Mesh circles (8 mm diameter, 200 μm pore)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature for 25 min, 35 min at 37 ± 1 °C
- Temperature of post-treatment incubation (if applicable): 37 ± 1 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After dosing of all tissues, all plates were incubated for 25 ± 1 min under the sterile flow and for the remaining time of 35 ± 1 min transferred to the incubator.Then, the tissues were washed by filling and emptying the inserts 15 times with DPBS using a constant stream in about 1.5 cm distance from the tissue surface. Subsequently, the inserts were completely submerged three times in 150 mL DPBS and shaken to remove rests of the test item. Finally, the inserts were rinsed once from the inside and the outside with sterile DPBS.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT stock solution (5 mg/mL) was diluted 1 + 4 with DMEM-based medium (final concentration 1 mg/mL)
- Incubation time: 3 h ± 5 min
- Spectrophotometer: yes
- Wavelength: 570 nm
- Filter bandwidth: ± 30 nm

NUMBER OF REPLICATE TISSUES: three

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if viability after 1 hour exposure is less than 50% relative to the negative control.
- The test substance is considered to be non-irritant to skin if the viability after 1 hour exposure is greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 25 mg (39 mg/cm²) + 25 µL DPBS

NEGATIVE CONTROL : Dulbecco’s phosphate buffered saline (DPBS)
- Amount(s) applied (volume or weight): 30 µl

POSITIVE CONTROL : Sodium dodecyl sulfate (SDS)
- Amount(s) applied (volume or weight): 30 µl
- Concentration (if solution): 5%

Duration of treatment / exposure:

60 min (25 ± 1 min under the sterile flow and for the remaining time of 35 ± 1 min transferred to the incubator)

Duration of post-treatment incubation (if applicable):

42 h post-incubation

Number of replicates:

three per treatment group

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean of triplicates

Value:

2.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, OD570 nm ≥ 0.8 and ≤ 2.8 (1.945)
- Acceptance criteria met for positive control: yes, mean relative tissue viability of the three positive control tissues is ≤ 20% (3.7%)
- Acceptance criteria met for variability between replicate measurements: yes, standard deviation (SD) of relative tissue viability obtained from each three concurrently tested tissues is ≤ 18%. (0.1-7.1%)

For detailed results see Table 1 in box "Any other information on results incl tables".

Table 1: Result of the Test Item N1,N3-diallylpropane-1,3-diamine dihydrochloride

Name	Negative Control			Positive Control			Test Item
Replicate Tissue	1	2	3	1	2	3	1	2	3
Absolute OD570	1.961	1.837	2.108	0.119	0.119	0.115	0.105	0.104	0.106
	1.913	1.79	2.06	0.116	0.122	0.122	0.105	0.101	0.103
Mean Absolute OD570	1.945****			0.119			0.104
OD570 (Blank Corrected)	1.913	1.789	2.06	0.071	0.07	0.067	0.057	0.056	0.058
	1.865	1.742	2.012	0.067	0.074	0.074	0.057	0.053	0.055
Mean OD570 of the Duplicates (Blank Corrected)	1.889	1.766	2.036	0.069	0.072	0.07	0.057	0.055	0.056
Total Mean OD570 of the 3 Replicate Tissues (Blank Corrected)	1.897*			0.071			0.056
SD of Mean OD570 of the 3 Replicate Tissues (Blank Corrected)	0.135			0.002			0.001
Relative Tissue Viability [%]	99.6	93.1	107.3	3.6	3.8	3.7	3	2.9	3
Mean Relative Tissue Viability [%]	100			3.7**			2.9
SD of Relative Tissue Viability [%]***	7.1			0.1			0.1
CV [% Viabilities]	7.1			2.2			2.1
* Blank-corrected mean OD570 of the negative control corresponds to 100% absolute tissue viability.
** Mean relative tissue viability of the three positive control tissues is ≤ 20%.
*** Standard deviation (SD) obtained from the three concurrently tested tissues is ≤ 18%.
**** The mean absolute OD570 of the negative control is ≥ 0.8 and ≤ 2.8.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

In this in vitro skin irritation study (OECD 439), the test item is considered to be irritant to the skin. In combination with the results from an OECD 431 test, it can be concluded, that classification as skin irritant (Category 2) is warranted.

Executive summary:

In a primary dermal irritation study conducted according to OECD guideline 439, the EpiDerm™-Model (EPI-200-SIT) was topically exposed to N1,N3-diallypropane-1,3-diamine dihydrochloride (100% purity) for 60 min and 42 h post incubation period. Irritant potential of the test item was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with DPBS. The mean relative tissue viability (% negative control) was ≤ 50% (2.9%). The controls confirmed the validity of the study and all acceptance criteria were fulfilled. Based on this result N1,N3-diallypropane-1,3-diamine dihydrochloride is considered to be irritating to the skin. In combination with the results from a second in vitro test (OECD 431), it can be concluded that classification as Skin Irrit. 2, H315 is warranted.

The study is acceptable and satisfies the guideline requirements for an in vitro skin irritation study (OECD 439).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

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

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)

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.

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.

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).

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).

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

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).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In a primary skin corrosion study conducted according to OECD guideline 431, two EpiDerm tissues per exposure time were exposed to 25 mg of N1,N3-diallylpropane-1,3-diamine dihydrochloride (100% purity) for 60 min and 3 min. Cytotoxicity was measured in comparison to the concurrent negative controls via the MTT reduction assay and irritation was scored by the method of mean relative tissue viability. The test item showed no corrosive effects. The mean relative tissue viability (% negative control) was ≥15% (68.1%) after 60 min treatment and ≥50% (107.4%) after 3 min treatment. The controls confirmed the validity of the study and all acceptance criteria were fulfilled.

Based on the results from this study, the test item can be classified as non-corrosive. To specify the classification in accordance to CLP regulation 1272/2008 the results from an OECD 439 study must be additionally consulted.

In the study conducted according to OECD guideline 439, the EpiDerm™-Model (EPI-200-SIT) was topically exposed to N1,N3-diallypropane-1,3-diamine dihydrochloride (100% purity) for 60 min and 42 h post incubation period. Irritant potential of the test item was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with DPBS. The mean relative tissue viability (% negative control) was ≤ 50% (2.9%). The controls confirmed the validity of the study and all acceptance criteria were fulfilled. Based on this result N1,N3-diallypropane-1,3-diamine dihydrochloride is considered to be irritating to the skin. In combination with the results from a second in vitro test (OECD 431), it can be concluded that classification as Skin Irrit. 2, H315 is warranted.

The eye irritation potential of N1,N3-diallylpropane-1,3-diamine dihydrochloride (100% purity) was investigated in two in vitro eye irritation studies. In the bovine corneal opacity and permeability assay (BCOP, OECD TG 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, the conditions for classification for serious eye damage were not fulfilled. But, also the conditions to justify a non-classification were not fulfilled and no prediction on classification can be made by this test alone.

In the second in vitro eye irritation test, conducted according to OECD TG 492, the test item showed irritant effects (mean tissue viability 2.6% compared to negative control). Based on the results, the conditions for non-classification were not fulfilled. No prediction of the classification can be made since from this study alone it cannot be differentiated between UN GHS “Category 1” or “Category 2”, if the relative tissue viability is less or equal to 60%.

Based on the available data and assessing them in a weight-of-evidence approach, classification for serious eye irritation (Eye Irrit. 2, H319) is warranted in accordance with the CLP Regulation 1272/2008.

Justification for classification or non-classification

Based on the available data and assessing them in a weight-of-evidence approach, classification for serious eye and skin irritation (Eye Irrit. 2, H319; Skin Irrit. 2, H315) is warranted in accordance with the CLP Regulation 1272/2008.