Reaction mass of potassium;sodium;2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetate;manganese, potassium;sodium;2-[bis[2-[bis(carboxymethyl)amino]ethyl]amino]acetate;manganese and potassium;sodium;2-[2-[bis(carboxymethyl)amino]ethyl-(2-hydroxyethyl)amino]acetate;manganese

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

REPORTING FORMAT FOR THE CATEGORY APPROACH

1. HYPOTHESIS FOR THE CATEGORY APPROACH (ENDPOINT LEVEL)
See the documentation in support for the category approach.

2. CATEGORY APPROACH JUSTIFICATION (ENDPOINT LEVEL
See the documentation in support for the category approach.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye 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 26 July 2013

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Version / remarks:

08 December 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of ammonium;potassium;sodium;2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetate;magnesium, ammonium;potassium;sodium;2-[bis[2-[bis(carboxymethyl)amino]ethyl]amino]acetate;magnesium and ammonium;potassium;sodium;2-[2-[bis(carboxymethyl)amino]ethyl-(2-hydroxyethyl)amino]acetate;magnesium

EC Number:

902-533-2

IUPAC Name:

Reaction mass of ammonium;potassium;sodium;2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetate;magnesium, ammonium;potassium;sodium;2-[bis[2-[bis(carboxymethyl)amino]ethyl]amino]acetate;magnesium and ammonium;potassium;sodium;2-[2-[bis(carboxymethyl)amino]ethyl-(2-hydroxyethyl)amino]acetate;magnesium

Test animals / tissue source

Species:

chicken

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source:
The eyes collected from chickens obtained from a slaughterhouse (Etablissement Brun, 33820 Etauliers, France) where they are killed for human consumption
- Number of animals: not specified
- Characteristics of donor animals (e.g. age, sex, weight):
The age and weight of the chickens used in this test method are that of spring chickens traditionally processed by a poultry slaughterhouse (i.e., approximately 7 weeks old, 1.5 - 2.5 kg).
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): see below
- Time interval prior to initiating testing:
Heads have been removed immediately after sedation of the chickens by electric shock, and incision of the neck for bleeding. The heads have been collected on 04 October 2017 at 8:25 am.
Because eyes were dissected in the laboratory, the intact heads were transported from the slaughterhouse at ambient temperature in plastic boxes humidified with towels moistened with physiological saline.
The eyes were enucleated at Phycher on 04 October 2017 at 09:55 am.
- indication of any existing defects or lesions in ocular tissue samples: no
- Indication of any antibiotics used: no

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): Immediately following the zero reference measurements, three eyes (in their holder) were removed from the superfusion apparatus, placed in a horizontal position, and 30 mg of the test item was applied, as supplied during 10 seconds, to the cornea such that the entire surface of the cornea was evenly covered with the test item.

Duration of treatment / exposure:

single application

Duration of post- treatment incubation (in vitro):

Treated corneas were evaluated pretreatment and starting at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse.

Number of animals or in vitro replicates:

three eyes

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
The eyelids were carefully excised, taking care not to damage the cornea. Then, the eye was further dissected from the skull, taking care not to damage the cornea. The eyeball was pulled from the orbit by holding the nictitating membrane firmly with surgical forceps, and the eye muscles were cut with a bent, blunt-tipped scissor. When the eye is removed from the orbit, a visible portion of the optic nerve should be left attached. Once removed from the orbit, the eye was placed on an absorbent pad and the nictitating membrane and other connective tissue were cut away.
The enucleated eye was mounted in a stainless steel clamp with the cornea positioned vertically. The clamp was then transferred to a chamber of the superfusion apparatus. The clamps were positioned in the superfusion apparatus such that the entire cornea was supplied with the physiological saline drip (in the range 0.1 to 0.15 mL/min). The chambers of the superfusion apparatus was temperature controlled between 31.4°C and 32.3°C.
After being placed in the superfusion apparatus, the eyes were examined with a slit-lamp microscope to ensure that they have not been damaged during the dissection procedure. Corneal thickness was also measured at this time at the corneal apex using the depth measuring device on the slit-lamp microscope. Eyes with; (i), a fluorescein retention score of > 0.5; (ii) corneal opacity > 0.5; or, (iii), any additional signs of damage were replaced. For eyes that are not rejected based on any of these criteria, individual eyes with a corneal thickness deviating more than 10% from the mean value for all eyes are to be rejected.

EQUILIBRATION AND BASELINE RECORDINGS
Once all eyes had been examined and approved (see table in appendix 4), the eyes were incubated between 45 and 57 minutes to equilibrate them to the test system prior to dosing. Following the equilibration period, a zero reference measurement was recorded for corneal thickness and opacity to serve as a baseline (i.e., time = 0). The fluorescein score determined at dissection was used as the baseline measurement for that endpoint.

NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
physiological saline – 1 replicate
SOLVENT CONTROL USED (if applicable)
not applicable
POSITIVE CONTROL USED
sodium hydroxide –- 3 replicates
APPLICATION DOSE AND EXPOSURE TIME
Immediately following the zero reference measurements, three eyes (in their holder) were removed from the superfusion apparatus, placed in a horizontal position, and 30 mg of the test item was applied, as supplied during 10 seconds, to the cornea such that the entire surface of the cornea was evenly covered with the test item.

OBSERVATION PERIOD
Treated corneas were evaluated pretreatment and starting at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: Tthe eyes were rinsed twice with 10 mL of physiological saline at ambient temperature. The eye (in its holder) was subsequently returned to the superfusion apparatus in the original upright position.
- Indicate any deviation from test procedure in the Guideline
No deviation was registered during the study.

METHODS FOR MEASURED ENDPOINTS:
See any other information on materials and methods.

SCORING SYSTEM:
See any other information on materials and methods.

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used.
See any other information on materials and methods.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Value:

<= 2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

fluorescein retention score

Value:

3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

percent corneal swelling

Value:

<= 3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The combination of the three endpoints for the test item Reaction mixture of MgEDTA, MgDTPA and MgHEEDTA was 1 x IV, 1 x III, 1 x I.

The combination of the three endpoints for the positive control, sodium hydroxide, was 3 x IV. Therefore, the positive control is classified as “Corrosive/Severe Irritant”, as expected.

The combination of the three endpoints for the negative control, physiological saline, was 3 x I. Therefore, the negative control is classified as “No Category”, as expected.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

In accordance with the Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions lead to the category “no prediction can be made”, as defined by the OECD guideline No.438. Therefore, the test item Reaction mixture of MgEDTA, MgDTPA and MgHEEDTA is not predicted as causing serious eye damage (Category 1) or as not classified for eye irritation/serious eye damage (No category) with the Isolated Chicken Eye test method. Additional testing (in vitro and/or in vivo) are required to establish a definitive classification.

Executive summary:

The aim of the study was to evaluate the possible ocular corrosive or severe irritating effects of the test item after administration on enucleated chicken eyes.

The test item Reaction mixture of MgEDTA, MgDTPA and MgHEEDTA was applied, as supplied, at the dose of 30 mg, to 3 enucleated chicken eyes, during 10 seconds. Then eyes were rinsed twice with 10 mL of physiological saline. Three eyes were treated in the same manner with a positive control and one eye with a negative control. Damages by the test item were assessed by determination of corneal swelling, opacity, and fluorescein retention at 30, 75, 120, 180 and 240 minutes post-dose. The experimental protocol was established in accordance with theO.E.C.D. Test Guideline No. 438 adopted 26 July 2013 and the test method B.48–Commission Regulation (EU) No. 1152/2010 dated 08 December 2010 (EU Journal L324) - ATP Council regulation No. 440/2008 of 30 May 2008 (E.U. Journal L142).

The ocular reactions observed in eyes treated with the test item were:

• -  maximal mean score of corneal opacity: 2.0, corresponding to ICE class III;

• -  mean score of fluorescein retention: 3.0, corresponding to ICE class IV;

• -  maximal mean corneal swelling: 3%, corresponding to ICE class I.

  The combination of the three endpoints for the test item Reaction mixture of MgEDTA, MgDTPA and MgHEEDTA was 1 x IV, 1 x III, 1 x I.

  The combination of the three endpoints for the positive control, sodium hydroxide, was 3 x IV.Therefore, the positive control is classified as “Corrosive/Severe Irritant”, as expected.

  The combination of the three endpoints for the negative control, physiological saline, was 3 x I. Therefore, the negative control is classified as “No Category”, as expected.

  In accordance with the Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions lead to the category “no prediction can be made”, as defined by the OECD guideline No.438. Therefore, the test item Reaction mixture of MgEDTA, MgDTPA and MgHEEDTA is not predicted as causing serious eye damage (Category 1) or as not classified for eye irritation/serious eye damage (No category) with the Isolated Chicken Eye test method. Additional testing (in vitro and/or in vivo) are required to establish a definitive classification.