Reaction mass of 2,2',2''-nitrilotriethanol and disodium succinate and sodium acetate and sodium bromide

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-02-07 to 2013-03-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: study according to OECD TG 437 (2009) and EU Method B.47 and under GLP

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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 Ocular Corrosives and Severe Irritants)

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction Mass of CXN1-55

IUPAC Name:

Reaction Mass of CXN1-55

Details on test material:

- Name of test material (as cited in study report): Reaction mass of CXN1-55- Substance type: multi-constituent substance- Physical state: Paste- Stability under test conditions: substance considered stable under normal ambient conditions- Storage condition of test material: at room temperature at 20 ± 5 °C, in the dark.

Test animals / tissue source

Species:

other: bovine eyes from at least 9 month old donor cattle

Strain:

other: strain of cattle not necessary to be specified in this study

Details on test animals or tissues and environmental conditions:

TEST ANIMALS- Source: Odenwaldschlachthof Brensbach, 64395 Brensbach, Germany- Age at study initiation: Freshly isolated bovine cornea (at least 9 month old donor cattle)- Acclimation period: Freshly isolated bovine eyes of at least 9 month old donor cattle were collected from the abattoir. Excess tissue was removed from the excised eyes. The isolated eyes were transported to the laboratory in Hank’s BSS supplemented with streptomycin / penicillin at ambient temperature. The corneae were isolated on the same day after delivery of the eyes. The corneae were inserted in pre-cooled preservation medium (Medium 199 supplemented with L-glutamine, Nabicarbonate and Taurine). Shortly before use, Dextran was added to the medium and stored in the refrigerator at 2 - 8 °C until use on the following day.Each isolated cornea was mounted in a specially designed cornea holder according to the description given in OEDC guideline 437, annex III, that consists of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea, respectively. For equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath.ENVIRONMENTAL CONDITIONS- Temperature (°C): 32 ± 1 °C- Humidity (%): not applicable for this study since the corneae are submersed in a solution within the cornea holder.- Air changes (per hr): not applicable for this study since the corneae are submersed in a solution within the cornea holder.- Photoperiod (hrs dark / hrs light): not applicable for this study.IN-LIFE DATES: 2013-02-07 to 2013-02-07

Test system

Vehicle:

physiological saline

Remarks:

0.9 % (w/v) NaCl (saline)

Controls:

yes

Amount / concentration applied:

731.17 mg of the test item were solved in 3.7 mL of saline to reach a weight/volume ratio of 20 %.Positive Control: 10 % (w/v) Benzalkonium chloride (purity not indicated by the producer) in 0.9 % (w/v) NaCl (saline) served as positive control.Negative Control: Saline served as negative control.The anterior compartment received the test item solution or negative or positive control at a volume of 0.75 mL each on the surface of the corneae.

Duration of treatment / exposure:

240 minutes

Observation period (in vivo):

At the end of the incubation period, the basal opacity was determined (t0).Each corneae with a value of the basal opacity > 7 was discarded. Sets of three corneae were used for treatment with the test item and the negative and positive controls.After the exposure time, the test item or control items, respectively, were rinsed off from the application side with saline. Afterwards, fresh incubation medium (MEM, supplemented with sodium bicarbonate and L-glutamine. Immediately before starting the test, MEM was supplemented with 1 % fetal calf serum (FCS)) was added into the anterior compartment and opacity was measured (t240). The opacitometer determines changes in the light transmission passing through the corneae, and displays a numerical opacity value. This value was recorded in a table. The opacitometer (OP_KiT opacitometer (Electro Design, 63-Riom, France)) was calibrated and the opacity of each of the corneae was determined by reading each holder placed in the photoreceptor compartment for treated cornea.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 (Hank’s Buffered Salt Solution). 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 was removed, well mixed and transferred into a 96 well plate and the optical density at 490 nm (OD490) was determined with a spectrophotometer (Versamax® Molecular Devices). The absorbance values will be determined using the software SoftMax Pro Enterprise (version 4.7.1).

Number of animals or in vitro replicates:

3 corneae each for the negative control, the positive control and the test item treatment

Details on study design:

REMOVAL OF TEST SUBSTANCE- Washing (if done): after the exposure period of 240 minutes- Time after start of exposure: 240 minutesSCORING SYSTEM: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 Optical density 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 (In Vitro Irritation Score): 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 was calculated from the IVIS values.TOOL USED TO ASSESS SCORE:Opacity: measurement of the light transmission passing through the corneae (OP_KiT opacitometer (Electro Design, 63-Riom, France)).Permeability: measurement of sodium fluorescein in the medium from the posterior part of the eye holder after incubation of the anterior part with 0.5 % (w/v) sodium fluorescein solution in HBSS for 90 ± 10 minutes in a water-bath at 32 ± 1 °C (spectrophotometer (Versamax® Molecular Devices), determination of absorbance values using the software SoftMax Pro Enterprise (version 4.7.1)).

Results and discussion

In vivo

Results

Irritation parameter:

cornea opacity score

Basis:

other: opacity value IVIS

Time point:

other: 240 minutes

Score:

4.08

Reversibility:

not specified

Any other information on results incl. tables

With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS = 2.71).

The positive control (10% (w/v) Benzalkonium chloride in saline) induced distinct opacity on the corneae (mean IVIS =229.67)corresponding to a classification as corrosive /severe irritant to the eye (CLP/EPA/GHS (Cat 1)).

Relative to the negative control, the test item Reaction mass of CXN1-55 caused only a slight increase of the corneal opacity. Permeability effects did not occur. The calculated mean IVIS was 4.08 (threshold for corrosivity / severe irritancy: IVIS ≥ 55.1) . According to OECD 437 the test item is classified as not corrosive / not severe irritant to the eye.

Applicant's summary and conclusion

Interpretation of results:

other: not corrosive

Remarks:

Criteria used for interpretation of results: OECD GHS

Conclusions:

In conclusion, according to the current study and under the experimental conditions reported, the test item Reaction mass of CXN1-55 is not corrosive / not severely irritating to the eye (CLP/EPA/GHS (Cat 1)).

Executive summary:

A valid, reliable and conclusive in vitro study was performed to assess the corneal irritation and damage potential of Reaction mass of CXN1-55 by means of the BCOP assay using fresh bovine corneae. Testing was performed compliant to OECD testing guideline no. 437 and EU Method B.47 and under GLP.

After a first opacity measurement of the fresh bovine corneae (t0), the 20 % (w/v) solution in saline (0.9 % (w/v) NaCl in deionised water) of the test item Reaction mass of CXN1-55, the positive, and the negative controls were applied to corneae and incubated for 240 minutes at 32 ± 1 °C. The posterior chamber contained incubation medium. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae and opacity 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) caused distinct opacity of the corneae corresponding to a classification as corrosive / severe irritant to the eye (CLP/EPA/GHS (Cat 1)).

Relative to the negative control, the test item Reaction mass of CXN1-55 caused a very slight increase of the corneal opacity. Permeability could not be observed. The calculated mean IVIS was 4.08. According to OECD 437 the test item is classified as not corrosive / not severe irritant to the eye.

In conclusion, according to the current study and under the experimental conditions reported, the test item Reaction mass of CXN1-55 is not corrosive / not severe irritant to the eye (CLP/EPA/GHS (Cat 1)).