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EC number: 231-034-6 | CAS number: 7417-99-4
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Eye irritation
Administrative data
- 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
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 018
- Report date:
- 2018
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)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
Test material
- Reference substance name:
- N,N'-(methylenedi-p-phenylene)bis(aziridine-1-carboxamide)
- EC Number:
- 231-034-6
- EC Name:
- N,N'-(methylenedi-p-phenylene)bis(aziridine-1-carboxamide)
- Cas Number:
- 7417-99-4
- Molecular formula:
- C19H20N4O2
- IUPAC Name:
- N-[4-({4-[(aziridine-1-carbonyl)amino]phenyl}methyl)phenyl]aziridine-1-carboxamide
- Test material form:
- solid
1
Test animals / tissue source
- 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.
Test system
- 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).
Results and discussion
In vitro
Resultsopen allclose all
- 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)
Any other information on results incl. tables
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)
Applicant's summary and conclusion
- 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).
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