N,N-bis(2-ethylhexyl)formamide

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 Mar 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

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)

Version / remarks:

adopted in 2020

Deviations:

yes

Remarks:

demonstration of technical proficiency of the testing facility was not included in the study report but provided separately

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

N,N-bis(2-ethylhexyl)formamide

EC Number:

860-695-9

Cas Number:

88247-41-0

Molecular formula:

C17H35NO

IUPAC Name:

N,N-bis(2-ethylhexyl)formamide

Test material form:

liquid

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Hokkaido Livestock Corporation, Douo Plant, Hayakita Factory
- Characteristics of donor animals: The eyeballs of healthy cattle aged from 12 months to less than 60 months were used.
- Storage, temperature and transport conditions of ocular tissue: The eyeballs collected were immersed in HBSS (Hank’s Buffered Salt Solution) and transported at less than 20 °C until use in test operation. During storage and transportation, the temperature ranged from 10 – 13 °C.
- Time interval prior to initiating testing: The corneas were isolated on the same day after delivery of the eyes and directly used in the BCOP test on the same day.
- Indication of any existing defects or lesions in ocular tissue samples: The eyeballs were macroscopically observed and examined for defects including corneal opacity, scratches and neovascularisation.
- Preparation of corneas: The corneas were dissected with a 2 – 3 mm rim of the sclera remaining and were immersed in HBSS. A specifically designed corneal holder was used to mount each cornea.
- Quality check of the isolated corneas: Following equilibration, the opacity value of each cornea was measured using the opacitometer (BASF-OP3.0).

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL (also negative and positive controls)
- Amount applied: 750 µL

Duration of treatment / exposure:

10 min at 32 ± 1 °C

Duration of post- treatment incubation (in vitro):

2 h at 32 ± 1 °C

Number of animals or in vitro replicates:

Three corneas were used for each treatment group.

Details on study design:

TREATMENT METHOD: Open chamber method
Each isolated cornea was mounted in a specially designed cornea holder, which consists of anterior and posterior compartments, which interfaced with the epithelial and the endothelial sides of the cornea, respectively. Both compartments of the holder were filled with phenol red-free minimum essential medium. Thereafter, the corneas were immersed in the medium and equilibrated for 1 h 7 min at 32 ± 1 °C (actual temperature: 32.0 – 32.2 °C). Following the equilibration, the medium of both chambers was exchanged and the basal opacity was determined. The cornea was then macroscopically observed again for defects including opacity, scratches and neovascularisation. In the measurement and observation, no abnormalities were observed in any cornea. For treatment, the medium in the anterior chamber of the corneal holder was removed and 750 µL of the test item, negative or positive control were added on the surface of the cornea. The cornea holder was kept in a horizontal position and incubated at 32 ± 1 °C for 10 min (actual temperature: 32.1 – 32.2 °C).

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After exposure, the test item or the control items were removed from the anterior chamber of the corneal holder and the epithelial layer was washed with phenol red-containing minimum essential medium at least 3 times until no visual evidence of the test article was observed. At the end of washing with phenol red-containing medium, the epithelial layer was finally washed with phenol red-free medium.

- POST-EXPOSURE INCUBATION: 2 h at 32 ± 1 °C in phenol red-free medium (actual temperature: 31.9 – 32.2 °C).

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Following the 2 h post-incubation after exposure, each cornea was macroscopically observed for tissue peeling, residual test article and non-uniform opacity patterns. No abnormalities were noted in each group. Thereafter, the anterior and posterior chambers were refilled with fresh phenol red-free medium and the opacity value of each cornea was measured using the opacitometer (BASF-OP3.0).
- Corneal permeability: After measurement of the corneal opacity, the medium was removed from the anterior chamber of the cornea holder and 1 mL of sodium fluorescein was injected into the anterior chamber. In a horizontal position, the cornea holder was incubated for 90 min at 32.0 - 32.1 °C. The passage of sodium fluorescein dye was measured at 490 nm and recorded as optical density (OD490) with an ultraviolet visible spectrophotometer (UV-2600, Shimadzu Corporation).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
Test substance with an IVIS > 55 was regarded as Category 1.
Test substance with an IVIS ≤ 3 was regarded as No Category.
Test substance with an IVIS > 3 ≤ 55: no stand-alone prediction can be made.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Remarks:

mean value of 3 corneas

Run / experiment:

10 min exposure

Value:

5.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Demonstration of technical proficiency of the testing laboratory was not included in the study report but provided as a separate document. 8 of the 13 proficiency chemicals indicated by the OECD guideline 437 were tested in the BCOP test. All proficiency chemicals were correctly categorised.

ACCEPTANCE OF RESULTS: (refer to Table 1 under "Any other information on results incl. tables")
- Acceptance criteria met for negative control: yes, the opacity and permeability values of the negative control were less than the upper limit of the historical control range of opacity and permeability values.
- Acceptance criteria met for positive control: yes, the IVIS of the positive control was within 2 standard deviations of the latest historical control mean of the IVIS of the testing facility.

Any other information on results incl. tables

Table 1: Summary of results

Test group	Number of corneas	Opacity	Permability	IVIS
		Mean corrected final opacity^#	Mean corrected OD490 change^##
Negative control (distilled water)	3	1.0x	0.000xx	1.0
Test item	3	5.0	0.043	5.6
Positive control (Dimethylformamide)	3	109.3	1.221	127.6
x mean final opacity; xx: mean OD490 change ^#: (Mean final opacity) - (Mean final opacity of negative control) ^##: (OD490 change) - (Mean OD490 change of negative control) IVIS: in vitro irritation score (= mean opacity value + (15 x mean permeability OD490 value))

 

Table 2: Historical control data generated in the testing facility from Jul 2012 - Feb 2021

Control		No. of data	IVIS			Permeability			Opacity
			Mean ± SD	Range	Mean ± 2 SD	Mean ± SD	Range	Mean ± 2 SD	Mean ± SD	Range	Mean ± 2 SD
Negative	Distilled water	176	0.4 ± 0.8	-2.0 - 2.4	-1.2 - 2.0	0.005 ± 0.005	-0.003 - 0.030	-0.005 - 0.015	0.4 ± 0.8	-2.0 - 3.0	-1.2 - 2.0
Positive	Dimethylformamide	115	101.3 ± 15.1	42.3 - 143.4	71.1 - 131.5	0.953 ± 0.346	0.171 - 1.966	0.261 - 1.645	87.0 ± 11.6	38.0 - 120.0	63.8 - 110.2

 

 

 

 

Applicant's summary and conclusion

Interpretation of results:

other: not corrosive

Conclusions:

Based on the experimental results, the test item produced an IVIS score of 5.6, which is classified in "no stand-alone prediction is possible" based on GHS criteria.
There is regulatory acceptance in the EU that a substance can be considered corrosive (Eye Corrosive Cat. 1) based on a positive result in the Bovine corneal opacity and permeability test method. A result for which no prediction can be made is not conclusive with respect to non-classification or classification as an eye irritant and therefore requires further evaluation and/or data generation.