2-(methylnitroamino)ethyl nitrate

Endpoint summary

Description of key information

Skin irritation/corrosion: Data waiving (study scientifically not necessary): According to column 2 of REACH Annex VII, the study does not need to be conducted because an acute toxicity study by the dermal route is available for the test substance and does not indicate skin irritation up to 2 000 mg/kg body weight (limit dose test).

Serious eye damage/eye irritation: Key study. Method according to OECD guideline 437, GLP study. 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.

Serious eye damage/eye irritation: Key study. Method according to OECD guideline 492, GLP study. Under the conditions of the test, no prediction can be made as the test substance is considered either eye irritant or inducing serious eye damage in the EpiOcular™ Eye Irritation Test.

Serious eye damage/eye irritation: Final conclusion on classification of the test substance: Based on the result obtained in the BCOP Test (OECD 437) it can be excluded that the test item should be classified as “Category 1”. Based on the result obtained in the EpiOcular™ Test (OECD 492) it can be excluded that the test item should be classified as “No category”, but no prediction can be made between Category 1 and Category 2. Thus, Category 2 (eye irrtation) should be the correct classification for the test substance.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because an acute toxicity study by the dermal route does not indicate skin irritation up to the relevant limit dose level (2 000 mg/kg body weight)

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
According to column 2 of REACH Annex VII, the study does not need to be conducted because an acute toxicity study by the dermal route is available for the test substance and does not indicate skin irritation up to 2 000 mg/kg body weight (limit dose test).

Reason / purpose for cross-reference:

data waiving: supporting information

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

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

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)

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%

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.

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).

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)

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)

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.

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)

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.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 May 2020 - 07 May 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Deviations:

no

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 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: 14.6%
Replicate 2: 13.6%
Replicate 3: 10.5%
Replicate 4: 8.7%

Species:

human

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability: The EpiOcular™ model and the corresponding test method have been validated for irritation testing in an international validation study and its use is recommended by the relevant OECD guideline (OECD No. 492), and the method is applicable to mixtures, solids, liquids, semi-solids and waxes. Therefore, it was considered to be suitable for this study.

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: The test was performed using EpiOcular™ Reconstructed Human Corneum Epithelium (RhCE) from MatTek Corporation. The system consists of normal, human-derived corneal keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells. These cells are not transformed or transfected with genes to induce an extended life span. The EpiOcularTM tissues are cultured in specially prepared cell culture inserts with a porous membrane through which nutrients can pass to the cells. The tissue surface is 0.6 cm2. The certificate of Analysis of the test system is included in the report. The tissue viability and barrier function tests are within the acceptable ranges and indicate the appropiate formation of the mucosal barrier and a viable basal cell layer. Tissues were used within 48 h of their delivery.
The cells used to produce EpiOcular (TM) tissue are screened for potential biological contaminants:
- HIV-1 virus - oligonucleotide-directed amplification: Not detected.
- Hepatitis B virus - oligonucleotide-directed amplification: Not detected.
- Hepatitis C virus - oligonucleotide-directed amplification: Not detected.
- Bacteria, yeast and other fungi - long term antibiotic, antimycotic free culture: Not detected.

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): 51.5 and 54.6 mg for each replicate.

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

18 hours +15 min

Number of animals or in vitro replicates:

2 tissues per test substance and for each control were used.

Details on study design:

- Details of the test procedure used: Two tissues (0.6 cm2) were pre-wetted with 20 µL DPBS buffer, incubated for 30 min, then dosed topically with 50 mg test item and exposed for 6 hours at 37ºC, 5% CO2, ≥ 95% RH. After exposure, the tissues were rinsed with DPBS, transferred to fresh assay medium, and incubated for 25 min. Then, they were transferred to fresh medium again and incubated for 18 hours and 15 min at 37ºC, 5% CO2, ≥ 95% RH.
The MTT assay for cell viability evaluation was performed on the tissues, by incubating them for 3 hours with MTT solution at 37ºC, 5% CO2, ≥ 95% RH. The precipitated formazan crystals were then extracted using isopropanol and quantified spectrophotometrically.
- RhCE tissue construct used, including batch number: RhCE EpiOcular™, Source: MatTek Corporation (Mylnské Nivy 73, 82105 Bratislava, Slovakia.), Tissue batch number(s): 30657, delivery date: 05. May 2020. The EpiOcular tissues were used within 48 hours of their delivery.
- Doses of test chemical and control substances used: 50 mg test item, 50 µL in controls.
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: exposure 6 h at 37ºC, 5% CO2; post-exposure immersion 25 min at room temperature; post-exposure incubation 18 h at 37ºC, 5% CO2.
- Number of tissue replicates used per test chemical and controls: 2 tissues per test substance and for each control were used.
- Wavelength and band pass used for quantifying MTT formazan, and linearity range of measuring device: Wavelength: 570 nm.
- Description of the method used to quantify MTT formazan: Optical density by microtiter plate photometer.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: According to OECD 492, the percentage tissue viability cut-off value distinguishing classified from non-classified test chemicals is 60%. Therefore, the test item is identified as not requiring classification if the mean percent tissue viability after exposure and post-exposure incubation is > 60%. Otherwise, the test item is identified as potentially requiring classification and labelling.
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: see table below.
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: The validity of the RhCE EpiOcular™ test at laboratory facility was demonstrated in a proficiency study. For this purpose 15 proficiency chemicals (indicated by the OECD 492 guideline) were tested. All of the 15 proficiency chemicals were correctly categorized.
- Positive and negative control means and acceptance ranges based on historical data: see table below.
- Acceptable variability between tissue replicates for positive and negative controls: yes, 4.7% (negative control) and 1.9% (positive control). The values for negative and positive controls were below 20% (OECD Guideline 492).
- Acceptable variability between tissue replicates for the test chemical: yes, 1.6% below 20% (OECD Guideline 492).

Irritation parameter:

other: cell viability (%)

Run / experiment:

mean

Value:

23.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

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

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes (mean OD = 1.9; demanded > 0.8 and < 2.8)
- Acceptance criteria met for positive control: yes (mean cell viability = 30.4%; demanded < 50% of negative control)

Table 1. Absorbance Values Blank Isopropanol (OD at 570 nm)

Replicate	1	2	3	4	5	6	7	8	Mean
Absorbance	0.032	0.033	0.033	0.035	0.033	0.034	0.034	0.035	0.034

Table 2. Absorbance Values Negative Control, Positive Control and Test Item (OD at 570 nm)

Designation	Measurement	Negative Control	Positive Control	Test Item
Tissue 1	1	2.003	0.619	0.495
	2	1.864	0.613	0.490
Tissue 2	1	1.861	0.583	0.461
	2	1.831	0.579	0.464

Table 3. Mean Absorbance Negative Control, Positive Control and Test Item

Designation	Negative Control	Positive Control	Test Item
Mean – blank (Tissue 1)	1.900	0.582	0.459
Mean – blank (Tissue 2)	1.812	0.547	0.429

Table 4. % Viability Positive Control and Test Item

Designation	Positive Control	Test Item
% Viability (Tissue 1)	31.4%	24.7%
% Viability (Tissue 2)	29.5%	23.1%
% Viability Mean	30.4%	23.9%

Table 5. Validity

Criterion	Demanded	Found
Mean OD of negative control	>0.8 and< 2.8	1.9
% mean relative viability of positive control	< 50% of negative control	30.4%
Variation within replicates	< 20%	4.7% (negative control) 1.9% (positive control) 1.6% (test item)

Interpretation of results:

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

Conclusions:

Under the conditions of the test, no prediction can be made as the test substance is considered either eye irritant or inducing serious eye damage in the EpiOcular™ Eye Irritation Test.

Executive summary:

An in vitro EIT study according to OECD 492 was conducted under GLP conditions to assess the irritation potential of the test item. Preliminary tests were performed to detect the ability of the test item to directly reduce MTT as well as its colouring potential. Based on the preliminary tests, two tissues (0.6 cm2) were pre-wetted with 20 µL DPBS buffer, incubated for 30 min, then dosed topically with 50 mg test item and exposed for 6 hours at 37ºC, 5% CO2, ≥ 95% RH. After exposure, the tissues were rinsed with DPBS, transferred to fresh assay medium, and incubated for 25 min. Then, they were transferred to fresh medium again and incubated for 18 hours and 15 min at 37ºC, 5% CO2, ≥ 95% RH. The MTT assay for cell viability evaluation was performed on the tissues, by incubating them for 3 hours with MTT solution at 37ºC, 5% CO2, ≥ 95% RH. The precipitated formazan crystals were then extracted using isopropanol and quantified spectrophotometrically (OD at 570 nm) in order to determine cell viability. Concurrent positive and negative controls were run in parallel. All validity criteria were met. The relative mean viability of the EpiOcular™ tissues treated with the test item was 23.9%. This value is below the threshold for eye irritation potential (≤ 60%). Test items that induce values below the threshold are considered either eye irritant or inducing serious eye damage. Thus, no prediction can be made.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Skin irritation/corrosion: Based on the available data, the substance is not classified for skin irritation according to CLP Regulation no. 1272/2008.

Serious eye damage/eye irritation: Based on the available data, the substance is classified in category 2 (eye irritation) according to CLP Regulation no. 1272/2008.