N,N'-(methylenedi-p-phenylene)bis(aziridine-1-carboxamide)

Endpoint summary

Description of key information

Skin irritation. Key study. Method according to OECD guideline 439, GLP study. The test item is not a skin irrtant (no category) since the mean percent viability of the treated tissues was 93.1% in a RHE test.

Eye irritation.Key study.Method according to OECD guideline 437, GLP study. The test item is not irritating to the eye (no category) since IVIS score was found to be -1.53.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 December 2017 - 16 December 2017

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Remarks:

SkinEthic RHE® model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: foreskin (number of donors not specified)

Source strain:

not specified

Justification for test system used:

This study makes use of reconstructed human epidermis (RhE) (obtained from human derived non-transformed epidermal keratinocytes) which closely mimics the histological, morphological, biochemical and physiological properties of the upper parts of the human skin, i.e., the epidermis. Use of reconstructed human epidermis (RhE) is also recommended by OECD and other regulatory authorities. SkinEthicTM RHE model has been validated and is part of OECD validated reference methods (VRMs) and is also a recommended model for conducting in vitro skin irritation studies.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic RHE® model
- Tissue batch number(s): 17-RHE-127
- Production date:
- Shipping date:
- Delivery date:
- Date of initiation of testing: 12/12/2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37ºC

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 25 x 1 mL of DPBS
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 300 µL of a MTT solution at 1.0 mg/mL
- Incubation time: 3 hours at 37°C, 5% CO2
- Spectrophotometer: Synergy™ microplate reader (BioTek)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 1.1 (CV = 4.5%) specification OD > 0.7. Historical negative control mean OD range = 1.964-2.776.
- Barrier function: 4.5 h (Specification 4h < ET50< 10h)
- Morphology: 6 Cell layers, absence of significant histological abnormalities, well differentiated epidermis, specification > 4
- Contamination: no

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE: no interference.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the tissue viability after 42 minutes exposure is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the tissue viability after 42 minutes exposure is greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 mg (32mg/cm2)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL
- Concentration (if solution): 5% SDS

Duration of treatment / exposure:

42 minutes

Duration of post-treatment incubation (if applicable):

42 hours ± 60 minutes

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

93.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

(DPBS)

Positive controls validity:

valid

Remarks:

1.2% viability (5% SDS)

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes. A full demonstration of proficiency was performed for the EpiSkin-SM model, plus a reduced validation with the SkinEthic RHE model. Adequate results were obtained for the evaluated chemicals.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, SD of the negative control group was 0.03% (acceptablility criteria, SD ≤ 18%) and OD mean is 2.002 (acceptability criteria, 0.8≤OD≤3).
- Acceptance criteria met for positive control: yes, SD of the positive control group was 0.06% (acceptablility criteria, SD ≤ 18%)
- Acceptance criteria met for variability between replicate measurements: yes. SD of test item was 1.21% (acceptablility criteria, SD ≤ 18%).

Table 1: Data Summary of Percent Viability

Treatment	Tissue Replicate	O.D. at 570 nm	Blank Corrected O.D.	Mean of Corrected O.D.	Mean O.D. of Three Tissues	% Viability/ Tissue	Mean % Viability	S.D. of % Viability	C.V. of % Viability	Corrosivity Class
Negative Control (Dulbecco’s Phosphate Buffered Saline (DPBS))	1	2.067	2.026	2.004	2.002	100	100	0.03	1.50	NA
		2.017	1.976
		2.051	2.010
	2	2.021	1.980	1.988
		2.033	1.992
		2.034	1.993
	3	2.019	1.978	2.014
		2.105	2.064
		2.042	2.001
N,N’-(Methylenedi-P-Phenylene)Bis(Aziridine-1-Carboxamide) (Poly-U-01)	1	1.947	1.906	1.889	1.864	94.4	93.1	1.21	1.30	No Category
		1.910	1.869
		1.932	1.891
	2	1.847	1.806	1.842		92.0
		1.896	1.855
		1.905	1.864
	3	1.900	1.859	1.861		93.0
		1.895	1.854
		1.910	1.869
Positive control (Sodium dodecyl sulphate (5% aq.))	1	0.068	0.027	0.026	0.025	1.3	1.2	0.06	5.00	Category 2
		0.067	0.026
		0.066	0.025
	2	0.067	0.026	0.025		1.2
		0.065	0.024
		0.066	0.025
	3	0.066	0.025	0.025		1.2
		0.065	0.024
		0.066	0.025

Key: O.D. = Optical density, S.D. = Standard deviation, C.V. = Coefficient of variation, NA = Not applicable

Note: For Negative control SD and CV of % viability was calculated using OD at 570 nm and for test item and positive control SD and CV of % viability was calculated using % viability/tissue.

Interpretation of results:

other: No category (CLP Regulation EC no. 1272/2008)

Conclusions:

The test substance was found to not be a skin irritant in a reconstructed human epidermis test.

Executive summary:

An in vitro skin irritation test of the test item was performed in a reconstructed human SkinEthic RHE® model, according to OECD TG 439 (GLP study). Three epidermis units were treated with 16 mg test item for 42 minutes at room temperature. Exposure of the test item was terminated by rinsing with 25 x 1 mL of DPBS. The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues with MTT, extracting the precipitated formazan crystals using isopropanol and incubating the tissues for overnight in a refrigerator protected from light, and finally measuring the concentration of formazan by determining the OD at 570 nm. Under the test conditions, the mean percent viability of the treated tissues was 93.1%, versus 1.2% of the positive control (5% Sodium Dodecyl Sulfate). Therefore, the test item must be considered as not classified for skin irritation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 December 2017 - 30 December 2017

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 i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Deonar Abattoir slaughter house, Mumbai, Maharashtra.
- Characteristics of donor animals (e.g. age, sex, weight): 1-5 years of age.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): transported under cold condition in Hank's Balanced Salt Solution containing antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL).
- Time interval prior to initiating testing: eyes were used within 24 h from the slaughtering.
- indication of any existing defects or lesions in ocular tissue samples: Eyes were examined prior to use. Corneas from eyes free of visible defects were used.
- Indication of any antibiotics used: yes, penicillin at 100 IU/mL and streptomycin at 100 μg/mL.

Vehicle:

other: corn oil

Controls:

yes, concurrent vehicle

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 μL
- Concentration (if solution): 20% (w/v).

VEHICLE
- Amount(s) applied (volume or weight with unit): 750 μL
- Concentration (if solution): not applicable
- Lot/batch no. (if required): MKBZ9899V (Sigma Aldrich)

Duration of treatment / exposure:

4 h ± 5 min

Number of animals or in vitro replicates:

3 replicates.

Details on study design:

SELECTION AND PREPARATION OF CORNEAS: All eyes were examined prior to use, corneas free from defects were dissected to a 2-3 mm rim and transferred to a container with Hank's Balanced Salt Solution. Corneas from eyes free of visible defects and with an opacity value inferior than 7 were used. The selected corneas were mounted on the corneal holders with the endothelial side against the O-ring of the posterior half of the holder, the anterior half of the holder was then placed on top of the cornea and fixed with screws. Both chambers were filled with pre-warmed phenol red free Eagle's Minimum Essential Medium (EMEM) and equilibrated at 32 ± 1ºC for at least 1h. Following the equilibration period, the medium was removed from both chambers and baseline opacity readings were taken for each cornea.

QUALITY CHECK OF THE ISOLATED CORNEAS: Corneas from eyes free of visible defects and with an opacity value inferior than 7 were used

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: yes. Normal saline (Sodium Chloride Injection I.P.), source: Realcade Life Sciences Pvt. Ltd., batch no: R2010266.

SOLVENT CONTROL USED: yes. Corn oil, source: Sigma Aldrich, batch no: MKBZ9899V.

POSITIVE CONTROL USED: yes. Imidazole (20% w/v solution), source: Sigma Aldrich, batch no: SLBP2962V.

APPLICATION DOSE AND EXPOSURE TIME: 750 μL of 20% (w/v) test item solution in corn oil, 4h exposure.

TREATMENT METHOD: closed chamber.

POST-INCUBATION PERIOD: no.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: the corneal epithelium was washed until no visual evidence of test item was observed using EMEM (containing phenol red) and, once the medium was free of test item, a final rinse with phenol red-free EMEM was performed.

- POST-EXPOSURE INCUBATION: no.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: corneal opacity was measured with an opacitometer BASF-OP3.0 (Duratec, Germany)
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: as indicated in the TG (see table below).

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

-1.53

Vehicle controls validity:

valid

Remarks:

1.63

Negative controls validity:

valid

Remarks:

1.32

Positive controls validity:

valid

Remarks:

118.42

Irritation parameter:

cornea opacity score

Run / experiment:

mean

Value:

-1.45

Vehicle controls validity:

valid

Remarks:

1.41

Negative controls validity:

valid

Remarks:

1.19

Positive controls validity:

valid

Remarks:

88.24

Irritation parameter:

other: permeability

Run / experiment:

mean

Value:

-0.005

Vehicle controls validity:

valid

Remarks:

0.014

Negative controls validity:

valid

Remarks:

0.009

Positive controls validity:

valid

Remarks:

2.012

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no.

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- A test is considered acceptable if: the positive control gives an IVIS that falls within two standard deviations of the current historical mean and negative/solvent controls gives values of opacity and permeability lower than upper limits for background values.
- Acceptance criteria met for negative/solvent controls: yes (negative: opacity upper limit=2.52, permeability upper limit=0.099; solvent: permeability upper limit=0.065)
- Acceptance criteria met for positive control: yes (historical value range: 63.28 - 257.23, SD: 56.79; mean = 159.82, 2*SD = 113.57)

Table 1. In vitro Irritation Score.

 

Group : Normal Saline, 0.75 mL
Cornea Holder N°	Io  (LUX)	I (Initial) (LUX)	Initial Opacity Value	I (Post Treatment) (LUX)	Post Treatment Opacity Value	Corr. Opacity Value	OD490 Value	Corr. OD490 Value	IVIS
2	1116	1027	3.87	1010	4.60	0.73	0.06	0.010	0.88
9	1101	1115	-0.08	1094	0.68	0.76	0.053	0.003	0.81
12	1136	1051	3.64	1003	5.71	2.07	0.063	0.013	2.27
Mean						1.19	-	0.009	1.32
SD						0.77	-	0.005	0.82

Group : Imidazole at 20% (w/v) in normal saline, 0.75 mL
Cornea Holder N°	Io  (LUX)		I (Initial) (LUX)		Initial Opacity Value	I (Post Treatment) (LUX)		Post Treatment Opacity Value	Corr. Opacity Value	Final Opacity Value		OD490 Value	Corr. OD490Value	Final OD490Value		IVIS
3	1106		970		6.01	383		75.63	69.62	68.43		2.124	2.074	2.065		99.41
6	1162		1141		1.16	337		97.96	96.80	95.61		1.858	1.808	1.799		122.60
10	1140		1134		0.63	320		102.51	101.88	100.69		2.23	2.18	2.171		133.26
Mean										88.24		-	2.021	2.012		118.42
SD										17.35		-	0.192	0.192		17.31
Group : Corn Oil, 0.75 mL
Cornea Holder N°		Io  (LUX)		I (Initial) (LUX)		Initial Opacity Value	I (Post Treatment) (LUX)		Post Treatment Opacity Value		Corr. Opacity Value		OD490 Value		Corr. OD490 Value		IVIS
5		1132		1063		3.01	1002		5.59		2.58		0.068		0.018		2.85
8		1159		1120		1.81	1085		3.14		1.33		0.061		0.011		1.50
11		1127		1115		0.85	1106		1.18		0.33		0.064		0.014		0.54
Mean											1.41		-		0.014		1.63
SD											1.13		-		0.004		1.16

Group : N,N’-(methylenedi-p-phenylene)bis(aziridine-1-carboxamide) (POLY-U-01) (suspension) at 20% (w/v) in Corn Oil, 0.75 mL
Cornea Holder N°	Io (LUX)	I (Initial) (LUX)	Initial Opacity Value	I (Post Treatment) (LUX)	Post Treatment Opacity Value	Corr. Opacity Value	Final Opacity Value	OD490 Value	Corr. OD490 Value	Final OD490 Value	IVIS
1	1129	1088	1.92	1108	1.18	-0.74	-2.15	0.059	0.009	-0.005	-2.23
7	1162	1078	3.53	1080	3.45	-0.08	-1.49	0.058	0.008	-0.006	-1.58
13	1182	1113	2.89	1095	3.59	0.70	-0.71	0.06	0.01	-0.004	-0.77
Mean							-1.45	-	0.009	-0.005	-1.53
SD							0.72	-	0.001	0.001	0.73

Keys: IVIS = In Vitro Irritation Score, Io =Baseline Reading (With medium but without cornea), I = LUX Reading with Medium and Cornea, OD₄₉₀= Optical Density at 490 Wave Length, - = Not Applicable, Corr. = Corrected. Blank OD₄₉₀value = 0.050.

- InitialOpacity Value = [((Io⁄I)-b)/a)], Where I = Initial LUX, Io = Baseline Reading, Note: a (0.0251) and b (0.9894) are constant.

- Post TreatmentOpacity Value = [((Io⁄I)-b)/a)], Where, I = Post Treatment LUX, Io = Baseline Reading. Note: a (0.0251) and b (0.9894) are constant.

- Corr. Opacity Value = Post Treatment Opacity Value - Initial Opacity Value

- Final Opacity Value = Corr. Opacity Value – Mean Corr. Opacity Value of Control (Group I)

- Corr. OD₄₉₀Value = OD₄₉₀Value – Blank OD₄₉₀Value, Where Blank Value for this trial was 0.050

- Final OD₄₉₀Value = Corr. OD₄₉₀Value –Mean Corr.OD₄₉₀Valueof Control (Group I)

- IVIS (Control) = Corr. Opacity Value + (15 x Corrected OD₄₉₀)

- IVIS (Treatment) = Final Opacity Value + (15 x Final OD₄₉₀)

Interpretation of results:

other: Not classified (CLP Regulation EC no. 1272/2008)

Conclusions:

The mean IVIS score for the test item was found to be -1.53 in the BCOP test. Therefore, the test item is not irritating to the eye (no category).

Executive summary:

An in vitro (ex vivo) Bovine Corneal Opacity and Permeability study was conducted in order to determine the potential severe eye damaging effects of the test item according to the OECD guideline 437 under GLP conditions. Four sets consisting of three corneas each were tested: the first set was the negative control, and was treated with 750 μL normal saline; the second set was the positive control, and was treated with 750 μL of 20% (w/v) imidazole in normal saline; the third set was the solvent/vehicle control and was treated with 750 μL corn oil and the fourth set was treated with 750 μL of 20% (w/v) of test item suspension in corn oil for 4h at 32ºC. After exposure, opacity of the corneas was measured. Then, to determine permeability, 1 mL fluorescein solution (5 mg/mL) was applied on the anterior surface of the corneas, while fresh EMEM (phenol red-free) was added to the posterior chamber and, after 90 min incubation at 32ºC, the OD (490 nm) of the medium in the posterior chamber was measured. Acceptance criteria were met for positive, negative a solvent controls. The mean corneal opacity for the test item treated corneas was -1.45, the mean permeability was -0.005 and the mean IVIS score was found to be -1.53. Therefore, the test item is not irritating to the eye (no category).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation. Key study. An in vitro skin irritation test of the test item was performed in a reconstructed human SkinEthic RHE®model, according to OECD TG 439 (GLP study). Three epidermis units were treated with 16 mg test item for 42 minutes at room temperature. Exposure of the test item was terminated by rinsing with 25 x 1 mL of DPBS. The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues with MTT, extracting the precipitated formazan crystals using isopropanol and incubating the tissues for overnight in a refrigerator protected from light, and finally measuring the concentration of formazan by determining the OD at 570 nm. Under the test conditions, the mean percent viability of the treated tissues was 93.1%, versus 1.2% of the positive control (5% Sodium Dodecyl Sulfate). Therefore, the test item must be considered as not classified for skin irritation.

Eye irritation. Key study. An in vitro (ex vivo) Bovine Corneal Opacity and Permeability study was conducted in order to determine the potential severe eye damaging effects of the test item according to the OECD guideline 437 under GLP conditions. Four sets consisting of three corneas each were tested: the first set was the negative control, and was treated with 750 μL normal saline; the second set was the positive control, and was treated with 750 μL of 20% (w/v) imidazole in normal saline; the third set was the solvent/vehicle control and was treated with 750 μL corn oil and the fourth set was treated with 750 μL of 20% (w/v) of test item suspension in corn oil for 4h at 32ºC. After exposure, opacity of the corneas was measured. Then, to determine permeability, 1 mL fluorescein solution (5 mg/mL) was applied on the anterior surface of the corneas, while fresh EMEM (phenol red-free) was added to the posterior chamber and, after 90 min incubation at 32ºC, the OD (490 nm) of the medium in the posterior chamber was measured. Acceptance criteria were met for positive, negative a solvent controls. The mean corneal opacity for the test item treated corneas was -1.45, the mean permeability was -0.005 and the mean IVIS score was found to be -1.53. Therefore, the test item is not irritating to the eye (no category).

Justification for classification or non-classification

Based on the available information, the test item is not classified for skin irritation/corrosion in accordance with CLP Regulation (EU) No. 1272/2008.

Based on the available information, the test item is not classified for serious eye damage/eye irritation in accordance with CLP Regulation (EU) No. 1272/2008.