Reaction mass of N,N,N',N'-tetrabutylmethylenediamine and dibutylamine

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 March - 11 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

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:

26 July 2013

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

Council Regulation (EC) No 440/2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N,N,N',N'-tetrabutylmethylenediamine

EC Number:

243-678-5

EC Name:

N,N,N',N'-tetrabutylmethylenediamine

Cas Number:

20280-10-8

Molecular formula:

C17H38N2

IUPAC Name:

dibutyl[(dibutylamino)methyl]amine

Constituent 2

Reference substance name:

Dibutylamine

EC Number:

203-921-8

EC Name:

Dibutylamine

Cas Number:

111-92-2

Molecular formula:

C8H19N

IUPAC Name:

1-Butanamine, N-butyl-

Test material form:

liquid

Details on test material:

Clear, light yellow liquid
Storage: 2 to 8 °C, in the dark
Expiry date: 01-Jan-19

Specific details on test material used for the study:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity:
N/A
- Specific activity:
N/A
- Locations of the label:
N/A
- Expiration date of radiochemical substance:
N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Room temperature, in darkness
- Stability under test conditions:
Asumed stable
- Solubility and stability of the test substance in the solvent/vehicle:
N/A
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:
No

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
N/A
- Preliminary purification step (if any):
N/A
- Final dilution of a dissolved solid, stock liquid or gel:
N/A
- Final preparation of a solid:
N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material)
Applied as supplied

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)
N/A

OTHER SPECIFICS: N/A

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Corneas from bovine eyes were obtained from a local abattoir
- Number of animals: 9 corneas were prepared
- Characteristics of donor animals (e.g. age, sex, weight): < 30 months old
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): The eyes were removed after slaughter, completely immersed in Hanks’ Balanced Salt Solution (containing penicillin at 100 IU/mL and streptomycin at 100 μg/mL) in a suitably sized container and transported on the same day to the testing facility.
- Time interval prior to initiating testing: Same day
- indication of any existing defects or lesions in ocular tissue samples: Only corneas free from such defects were used.
- Indication of any antibiotics used: During transport, eyes were immersed in Hanks’ Balanced Salt Solution (containing penicillin at 100 IU/mL and streptomycin at 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): 750 ¨¨
- Concentration (if solution): Applied as supplied

VEHICLE
- Amount(s) applied (volume or weight with unit): N/A
- Concentration (if solution): N/A
- Lot/batch no. (if required): N/A
- Purity: N/A

Duration of treatment / exposure:

10 mins

Duration of post- treatment incubation (in vitro):

After washing, the corneas were incubated (horizontally) for 2 hours and 2 minutes after which, corneal opacity was measured and then the anterior chamber emptied. For the permeability endpoint, 1 mL of sodium fluorescein (4 mg/mL solution in 0,9% sodium chloride) was added into the anterior chamber, while the posterior chamber was filled with fresh MEM, and the corneas were incubated in the vertical position for 1.5 hours at 32±1°C. Following this period, the media in the posterior chamber was removed, mixed and held in a labelled tube. Three 350 μL aliquots of this media (per cornea) were analysed for optical density at 490 nanometers (OD490) using a spectrophotometer.

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
:

On arrival at the test facility the eyes were carefully examined for defects including increased opacity, scratches and neovascularisation. Only corneas free from such defects were used.

QUALITY CHECK OF THE ISOLATED CORNEAS

Upon arrival at the test facility, the corneas were excised from the eyes and loaded onto the specifically designed holders that consist of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea. The holders were uniquely identified by a number written in indelible ink. Both chambers of each holder were filled with pre-warmed Minimal Essential Medium (MEM), with the posterior chamber filled first, ensuring that no bubbles were formed. The holders were incubated at 32±1°C for 1 hour. After the incubation, the media was removed from both the anterior and posterior chambers. Fresh media was added to the posterior chamber first and then the anterior chamber (this media replacement order ensured the cornea retained its natural curvature as much as possible). The opacity of each cornea was measured using an opacitometer. Any corneas found to have scratches or increased neovascularization or an opacity of >7 opacity units when examined prior to treatment were discarded.

NUMBER OF REPLICATES

3 per test group

NEGATIVE CONTROL USED

Yes, the negative control substance was 0.9% sodium chloride solution (batch number 15107BA1A, expiry date 06 April 2017), supplied by Baxter Healthcare Ltd., Thetford, UK.

SOLVENT CONTROL USED (if applicable)

N/A

POSITIVE CONTROL USED

Yes, the positive control substance was 100% dimethylformamide (DMF) (batch number SZBF1680V expiry date 26 October 2017), supplied by Sigma-Aldrich Company Ltd., Poole, UK.

APPLICATION DOSE AND EXPOSURE TIME

A volume of 750 μL of the test article was applied to each of three corneas followed by a 10 minute incubation at 32 °C.

TREATMENT METHOD:

Closed chamber

POST-INCUBATION PERIOD:

After opacity was measured, the corneas were incubated (horizontally) for 2 hours after which, corneal opacity was measured and then the anterior chamber emptied. For the permeability endpoint, 1 mL of sodium fluorescein (4 mg/mL solution in 0.9% sodium chloride) was added into the anterior chamber, while the posterior chamber was filled with fresh MEM, and the corneas were incubated in the vertical position for 1.5 hours at 32±1°C.

REMOVAL OF TEST SUBSTANCE

- Number of washing steps after exposure period:
Each cornea was washed with media containing phenol red (as a pH indicator, to identify and monitor the rinsing of acidic or alkaline materials) until this indicator showed no pH effect occurring, demonstrating that the test article had been removed successfully. The corneas were then washed once in media without phenol red and the opacities measured.

- POST-EXPOSURE INCUBATION:

After opacity was measured, the corneas were incubated (horizontally) for 2 hours after which, corneal opacity was measured and then the anterior chamber emptied. For the permeability endpoint, 1 mL of sodium fluorescein (4 mg/mL solution in 0.9% sodium chloride) was added into the anterior chamber, while the posterior chamber was filled with fresh MEM, and the corneas were incubated in the vertical position for 1.5 hours at 32±1°C. Following this period, the media in the posterior chamber was removed, mixed and held in a labelled tube. Three 350 μL aliquots of this media (per cornea) were analysed for optical density at 490 nanometers (OD490) using a spectrophotometer.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opaciometer used
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry (OD490)
- Others: Not conducted

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: As per OECD 437 guidance.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

Test item mean IVIS

Value:

6.68

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

No prediction can be made if the IVIS is >3 but ≤55

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No changes to the appearance of the corneas treated with the test article were noted.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Not reported.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Opacity and permeability values less than the established upper limits for background opacity and permeability values were reported.
- Acceptance criteria met for positive control: Positive controls IVIS fell within two standard deviations of the current historical mean (June 2009-May 2016, n=91)
- Range of historical values if different from the ones specified in the test guideline: Historical values reported.

Any other information on results incl. tables

Table 1      Corneal Opacity

 

Test Substance	Cornea Number	Initial Opacity	Post Incubation Opacity	Change in Opacity	Mean Change in Opacity	Corrected Opacity	Mean Corrected Opacity
Test article	8	4	8	4	N/A	4.3	6.3
	9	3	12	9		9.3
	10	4	9	5		5.3
Negative	2	4	4	0	-0.33	0.3	0.0
	3	4	3	-1		-0.7
	4	3	3	0		0.3
Positive	14	4	61	57	N/A	57.3	69.0
	15	4	88	84		84.3
	16	3	68	65		65.3

 

Table 2      Corneal Permeability

 

Test Substance	Cornea Number	Mean Blank OD490	OD490	Corrected OD490	Mean Corrected OD490	Final Corrected OD490	Mean Group Corrected OD490
Test article	8	N/A	0.059	0.059	N/A	0.052	0.023
	9		0.014	0.014		0.007
	10		0.017	0.017		0.010
Negative	2	0.000	0.012	0.012	0.007	0.004	0.000
	3		0.004	0.004		-0.003
	4		0.006	0.006		-0.002
Positive	14	N/A	0.395	0.395	N/A	0.388	0.755
	15		1.052	1.052		1.044
	16		0.840	0.840		0.833

 

Table 3      Calculated IVIS

 

Test Chemical	Mean Opacity	Mean Permeability	IVIS (Mean Opacity + (15 x Mean Permeability))
Test article	6.3	0.023	6.68
Negative control	0	0	0
Positive control	69	0.755	80.33

 

 

Table 4: Historical Opacity and Permeability ranges

 

Control Article	Parameter	Mean	Upper Limit
Negative Control  (0.9% NaCl)	Opacity	0.9	9.0
	Permeability	0.014	0.560
	IVIS	0.0	NA

 

NA = not applicable

Upper limit = established upper limit for corneas treated at the test facility

 

Table 5: Historical Calculated IVIS ranges

Control Article	Parameter	Mean	Lower Limit	Upper Limit
Positive Control (dimethylformamide)	Opacity	43.4	3.1	83.8
	Permeability	0.363	-0.283	1.008
	IVIS	48.7	10.3	87.2

 

Lower limit = Mean value - 2 standard deviations of data set

Upper limit = Mean value + 2 standard deviations of data set

n = 91 (June 2009 to May 2016)

 

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the conditions of this study Reaction mass of N,N,N',N'-tetrabutylmethylenediamine and dibutylamine produced an IVIS score of 6.68 and it was concluded that no prediction can be made in respect of its potential to cause eye irritation.

The assay was considered valid as the assay acceptance criteria were met.

Executive summary:

OECD 437 (2017) - The Bovine Corneal Opacity and Permeabilty (BCOP) test was conducted using Reaction mass of N,N,N',N'-tetrabutylmethylenediamine and dibutylamine in accordance with 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" (2013).

 

Undiluted test item was applied evenly to the surface of three corneas before being washed off with media solution after 10 minute test item contact time. A negative and positive control group, each containing 3 corneas, were also prepared.

 

Measurements for corneal opacity were made after 2 h incubation in the horizontal position with fresh media. Measurements for corneal permeability were made following 1 h and 30 mins incubation in the vertical position with sodium fluorescein.

 

Corneal opacity and corneal permeability media solutions were analysed by a spectrophotometer at 490 nanometers (OD₄₉₀).

 

The mean corrected opacity reading and permeability readings for the test item were 6.3 and 0.023, resulting in anIn VitroIrritation Score (IVIS) of 6.68.

 

It was concluded that under the condition of this study the test item produced an IVIS score of 6.68 and therefore, no prediction could be made in respect of its potential to cause eye irritation.