2-(methylnitroamino)ethyl nitrate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 February 2020

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 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

Test material information

Constituent 1

Reference substance name:

2-(methylnitroamino)ethyl nitrate

EC Number:

241-168-7

EC Name:

2-(methylnitroamino)ethyl nitrate

Cas Number:

17096-47-8

Molecular formula:

C3H7N3O5

IUPAC Name:

methyl(nitro)[2-(nitrooxy)ethyl]amine

Test material form:

solid

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 phlegmatized in 15% water for safety reasons during transportation. Thus, the test item was dried under vacuum in the desiccator and weighed. This procedure was repeated until a constant weight was obtained for every replicate used in the test. Content of water obtained:
Replicate 1: 13.5%
Replicate 2: 15.1%
Replicate 3: 14.0%

Test animals / tissue source

Species:

cattle

Strain:

other: Bos primigenius Taurus

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Müller Fleisch GmbH, Industriestraße 42, 75217 Birkenfeld, Germany.
- Number of animals: Not specified. 9 corneas received for the test.
- Characteristics of donor animals (e.g. age, sex, weight): Between 12 and 60 months old.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): The eyes were transported to the test facility in Hanks’ Balanced Salt Solution with 1% Penicillin-Streptomycin solution (Penicillin 100 U/mL, Streptomycin 100 µg/mL) in a suitable cooled container within 1 hour and 5 minutes.
- Time interval prior to initiating testing: the test was performed on the day of the transport.
- indication of any existing defects or lesions in ocular tissue samples: None of the corneas received showed tissue damage; therefore, all corneas were used.
- Indication of any antibiotics used: Penicillin 100 U/mL, Streptomycin 100 µg/mL.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 749.2 mg (replicate1), 614.9 mg (replicate2) and 668.1 mg (replicate3).
- Concentration (if solution): undiluted

Duration of treatment / exposure:

4 hr

Duration of post- treatment incubation (in vitro):

Not performed.

Number of animals or in vitro replicates:

3 replicates.

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
After the arrival of the corneas, they were examined and only corneas which were free from damages were used. The corneas were excised with a scalpel and cut from the globe with a 2-3 mm ring of sclera around the outside. Each cornea was transferred to a cornea holder in which pre-warmed cMEM (32 ± 1ºC) without phenol red was filled. The holders were then incubated for 1 hour in the incubation chamber at 32 ± 1ºC.
MEM: Minimum Essential Medium
cMEM (complete MEM): MEM supplemented with sodium bicarbonate, L-glutamine and 1% fetal calf serum

QUALITY CHECK OF THE ISOLATED CORNEAS
Corneas which show any tissue damages or an opacity greater than seven opacity units should be discarded.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: yes, Hank’s Balanced Salt Solution (HBSS).

POSITIVE CONTROL USED: yes, 20% imidazole solution in HBSS.

APPLICATION DOSE AND EXPOSURE TIME: 615 to 750 mg (test item tested neat) and 750 μL (negative and positive controls) , 4 h exposure.

TREATMENT METHOD:
The open chamber method was used for the test item.
The closed chamber method was used for the controls.

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least three times or until no visual evidence of test chemical was observed.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacitometer BASF OP 3.0.
- Corneal permeability: passage of sodium fluorescein dye (optical density at 492 nm) measured with microtiter plate photometer (Anthos Reader 2010 Flexi).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

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

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

3.52

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

(0.99)

Positive controls validity:

valid

Remarks:

(111.39)

Results 2

Irritation parameter:

cornea opacity score

Run / experiment:

mean

Value:

3.53

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

(0.89)

Positive controls validity:

valid

Remarks:

(87.20)

Results 3

Irritation parameter:

other: permeability

Run / experiment:

mean

Value:

-0.001

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

(0.0071)

Positive controls validity:

valid

Remarks:

(1.6128)

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 control: yes, HBSS response resulted in opacity and permeability values that are less than established upper limits for negative control opacity (2.81) and permeability values (0.07)
- Acceptance criteria met for positive control: yes, IVIS score of imidazole 20% was 111.39 which falls between two standard deviations of the historical mean i.e. 75.56 – 145.51.

Any other information on results incl. tables

Table1.1. Illuminance Values (Unit: LUX)

Parameter	Negative Control			Test Item			Positive Control
	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.
(I) Measured values before exposure	1042	1050	1053	1016	1023	1027	1004	1004	1016
(I) Measured values after exposure	1020	1027	1033	914	932	935	342	324	337

Table 1.2. Opacity Values Negative Control

Parameter	Negative Control
	1. Rep.	2. Rep.	3. Rep.
Opacity before exposure	2.84	2.52	2.40
Opacity after exposure	3.75	3.46	3.21
Opacity Difference	0.91	0.94	0.81
Mean Opacity Difference	0.89

Table 1.3. Opacity Values Test Item and Positive Control

Parameter	Test Item			Positive Control
	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.
Opacity before exposure	3.92	3.62	3.46	4.44	4.44	3.92
Opacity after exposure	8.76	7.83	7.68	89.33	96.48	91.24
Opacity Difference	4.84	4.20	4.22	84.89	92.05	87.32
Opacity Difference corrected	3.95	3.32	3.33	84.01	91.16	86.43
Mean Opacity Difference corrected	3.53			87.20

Table 2.1. Optical density at 492 nm of Blank

Parameter	cMEM without phenol red
1. Measurement	0.038
2. Measurement	0.039
3. Measurement	0.037
Mean	0.038

Table 2.2. Optical density at 492 nm of Negative Control, Test Item and Positive Control

Parameter	Negative Control			Test Item			Positive Control
	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.	1.Rep.	2.Rep.	3.Rep.
1. Measurement	0.050	0.045	0.041	0.047	0.045	0.039	1.705	1.787	1.469
2. Measurement	0.050	0.044	0.039	0.047	0.047	0.040	1.710	1.813	1.456
3. Measurement	0.050	0.045	0.042	0.044	0.051	0.040	1.705	1.814	1.462
1. Measurement – blank	0.0120	0.0070	0.0030	0.0090	0.0070	0.0010	1.6670	1.7490	1.4310
2. Measurement – blank	0.0120	0.0060	0.0010	0.0090	0.0090	0.0020	1.6720	1.7750	1.4180
3. Measurement – blank	0.0120	0.0070	0.0040	0.0060	0.0130	0.0020	1.6670	1.7760	1.4240
Mean of each replicate	0.0120	0.0067	0.0027	0.0080	0.0097	0.0017	1.6687	1.7667	1.4243
Mean of the 3 replicates	0.0071			--			--
Corrected	--	--	--	0.0009	0.0026	-0.0054	1.6616	1.7596	1.4172
Corrected mean of the 3 replicates	--			-0.0007			1.6128

Table 3. IVIS

Test Group	IVIS	Mean IVIS	Relative Standard Deviation IVIS
Negative Control HBSS	1.09	0.99	12.81%
	1.04
	0.85
Test Item Methyl-NENA	3.96	3.52	10.93%
	3.35
	3.25
Positive Control 20% imidazole solution	108.93	111.39	4.82%
	117.55
	107.69

*IVIS = Mean Opacity value + (15 × Mean Permeability value)

Applicant's summary and conclusion

Interpretation of results:

other: No prediction can be made (CLP Regulation EC no. 1272/2008)

Conclusions:

The test item cannot be predicted as not classified for eye irritation/serious eye damage or as causing serious eye damage with the BCOP test method.

Executive summary:

An in vitro Bovine Corneal Opacity and Permeability (BCOP) 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. Three sets consisting of three corneas each were tested: the first set was the negative control and was treated using the closed chamber method with 750 μL Hank’s Balanced Salt Solution (HBSS); the second set was the positive control and was treated using the closed chamber method with 750 μL of 20% imidazole solution and the third set was treated using the open chamber method with 615 to 750 mg of test item without dilution. The corneas were exposed for 4 hours at 32ºC. After exposure, opacity of the corneas and fluorescein permeability were measured. Negative control showed No category (IVIS score 0.99) and resulted in opacity and permeability values that are less than the established upper limits for the background values. The positive control induced serious eye damage on the cornea (IVIS score 111.39) and was within two standard deviations of the current historical mean. The test item exhibited an IVIS score of 3.52, thus cannot be predicted as not classified for eye irritation/serious eye damage or as causing serious eye damage with the BCOP test method.