Reaction mass of Amines, N-tallow alkyltrimethylenedi-, mono(2-ethylhexanoates), Amines, N-tallow alkyltrimethylenedi-, acetates and n-tallow-1,3-diaminopropane ditallate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The test was performed on 04 November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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)

Remarks:

Date of inspection: 25 April 2012, 23 ,25,Date of inspection: 13-16 July 2015, Date of Signature: 14 September 2015

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of Amines, N-tallow alkyltrimethylenedi-, mono(2-ethylhexanoates), Amines, N-tallow alkyltrimethylenedi-, acetates and n-tallow-1,3-diaminopropane ditallate

EC Number:

947-476-4

Molecular formula:

Not applicable, UVCB

IUPAC Name:

Reaction mass of Amines, N-tallow alkyltrimethylenedi-, mono(2-ethylhexanoates), Amines, N-tallow alkyltrimethylenedi-, acetates and n-tallow-1,3-diaminopropane ditallate

impurity 1

Reference substance name:

Heptane

EC Number:

205-563-8

EC Name:

Heptane

Cas Number:

142-82-5

Molecular formula:

C7H16

IUPAC Name:

heptane

Test material form:

solid

Remarks:

paste

Details on test material:

Batch HJI16-040
Synthesis of sample for testing in heptane.
Residual heptane after drying: 0.5%
Acetic acid: 4.6 % (w/w)
2-ethylhexanoic acid: 10.6 % (w/w)
Tall oil fatty acids 32.2 % (w/w)
Amines, N-tallow alkyltrimethylenedi 52.2 % (w/w)

Test animals / tissue source

Species:

other: Freshly isolated bovine cornea (at least 9 month old donor cattle)

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Source: Schlachthof Aschaffenburg, 63739 Aschaffenburg, Germany

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

0.75 mL of 20% (w/v) suspension of the test item in saline was prepared using ultrasonic technique (5 minutes).

Duration of treatment / exposure:

240 minutes

Duration of post- treatment incubation (in vitro):

The incubation period was 240 minutes.

Number of animals or in vitro replicates:

3 corneae per group (test item, negative control, positive control)

Details on study design:

Three corneas were exposed to each 0.75 mL of the test item, the negative, and the positive control for 10 minutes.

SCORING SYSTEM:
Opacity Measurement:
The opacitometer determines changes in the light transmission passing through the corneae, and displays a numerical opacity value. The opacitometer OP_KiT opacitometer (Electro Design, 63-Riom France) was calibrated as described in the manual and the opacity of each of the corneae was determined by reading each holder placed in the photoreceptor compartment for treated cornea.
After exposure of the corneae to the test groups and after rinsing, the opacity value was determined again (t240).

Permeability Determination:
Following to the opacity readings, the permeability was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the incubation medium was removed from the anterior compartment and replaced by 1 mL of a 0.5% (w/v) sodium fluorescein solution in HBSS. Corneae were incubated again in a horizontal position for 90 ± 10 minutes in a water-bath at 32 ± 1 °C. Incubation medium from the posterior compartment were removed, well mixed and transferred into a 96 well plate and the optical density at 490 nm (OD490) was determined with a spectrophotometer.
The optical density was measured with a microplate reader (Versamax® Molecular Devices) at 490 nm (OD490). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).

Data Evaluation

Opacity:
The change of opacity value of each treated cornea or positive and negative control corneae is calculated by subtracting the initial basal opacity from the post treatment opacity reading (t240 – t0), for each individual cornea.
The average change in opacity of the negative control corneae is calculated and this value is subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.

Permeability:
The corrected OD490 value of each cornea treated with positive control and test item is calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.

IVIS Calculation:

The following formula is used to determine the IVIS of the negative control:
IVIS = opacity value + (15 x OD490 value)

The following formula is used to determine the IVIS of the positive control and the test item:
IVIS = (opacity value – opacity value mean negative control) + (15 x corrected OD490 value)

The mean IVIS value of each treated group is calculated from the IVIS values
.
Depending on the score obtained, the test item is classified in the following category according to OECD guideline 437:
IVIS: In vitro Irritancy Score (according to OECD 437):
≤ 3 No Category (according to GHS)
> 3; ≤ 55 No prediction can be made
> 55 CSerious eye damage according to CLP/EPA/GHS (category 1)


Criteria for Determination of a Valid Test:

The test will be acceptable if
• the positive control gives an IVIS that falls within two standard deviations of the current historical mean (updated every three months), and if
• the negative control responses result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean of three cornea

Value:

40.21

Vehicle controls validity:

not valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No prediction can be made

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Any other information on results incl. tables

Results after 240 Minutes Incubation Time

Test Group	Opacity value = Difference (t240-t0) of Opacity		Permeability at 490 nm (OD490)		IVIS	Mean IVIS	Proposed in vitro Irritancy Score
		Mean		Mean
	0		0.087		1.31
Negative Control	1	0.33	0.077	0.083	2.16	1.58	Not categorized
	0		0.085		1.28
	112.67*		0.105*		114.24
Positive Control	113.67*		0.184*		116.43	113.77	Category 1
	107.67*		0.199*		110.65
	21.67*		0.975*		36.29
Diamine-Fatty Acids mixture salt	14.67*		1.900*		43.17	40.21	No prediction
	11.67*		1.966*		41.16

*corrected values

Applicant's summary and conclusion

Interpretation of results:

other: Diamine-Fatty Acids mixture salt is not serious eye damaging (CLP/EPA/GHS (Cat 1) but a prediction for the damage hazard cannot be made (GHS).

Conclusions:

In conclusion, according to the current study and under the experimental conditions reported, Diamine-Fatty Acids mixture salt is not serious eye damaging (CLP/EPA/GHS (Cat 1) but a prediction for the damage hazard cannot be made (GHS).

Executive summary:

This in vitro study was performed to assess thecorneal damage potentialofDiamine-Fatty Acids mixture salt by means of the BCOP assay usingfresh bovine corneae.

After a first opacity measurement of the fresh bovine corneae (t0), the 20% (w/v)suspensionin saline (0.9% (w/v) NaCl in deionised water) of the test item Diamine-Fatty Acids mixture salt, the positive, and the negative controls were applied to corneae fixed in an incubation chamber in horizontal position and incubated for 240 minutes at 32 ± 1 °C. The posterior chamber containedincubation medium. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae andopacity was measured again (t240).

After the opacity measurements permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.

With the negative control (physiological saline) neither an increase of opacity nor permeability of the corneae could be observed.

The positive control (10% (w/v) Benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae corresponding to a classification as serious eye damage (CLP/EPA/GHS (Cat 1)).

Relative to the negative control, the test item Diamine-Fatty Acids mixture salt caused an increase of the corneal opacity and permeability compared with the values caused by the negative control. The calculated meanin vitroirritancy score was 40.21.According to OECD 437 the test item is not classified as serious eye damaging (CLP/EPA/GHS (Cat 1), but the test item’s hazard for eye damaging cannot be predicted.