Trimethyl[2-(phosphonooxy)ethyl]ammonium chloride, calcium salt (1:1)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

February 1, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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 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)

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

calcium tetrahydrate chloride [2-(trimethylazaniumyl)ethyl] phosphate

Cas Number:

72556-74-2

Molecular formula:

C5H21CaClNO8P

IUPAC Name:

calcium tetrahydrate chloride [2-(trimethylazaniumyl)ethyl] phosphate

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Test animals / tissue source

Species:

chicken

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Slaughterhouse (Etablissement Brun, 33820 Etauliers, France), the chickens were killed for human consumption
- Characteristics of donor animals: spring chickens (approximately 7 weeks old, 1.5 - 2.5 kg)
- Storage, temperature and transport conditions of ocular tissue: Heads were removed immediately after sedation of the chickens by electric shock, and incision of the neck for bleeding. Intact heads were transported from the slaughterhouse at ambient temperature in plastic boxes humidified with towels moistened with physiological saline.
- Time interval prior to initiating testing: The head collection was at 8:35 am and arrived at 9:45 am at the laboratory.
- indication of any existing defects or lesions in ocular tissue samples: No
- Indication of any antibiotics used: No

Test system

Vehicle:

unchanged (no vehicle)

Remarks:

The test item was applied as supplied

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 mg

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

4 hours

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
Dissection of the eyes: the eyelids were carefully excised and 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.
Selection of the eyes: 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 the entire cornea was supplied with the pysiological saline drip (in the range 0.1 to 0.15 mL/min). The chambers of the superfusion apparatus was temperature controlled between 32.3ºC and 32.7ºC. After being placed in the superfusion apparatus, the eyes were examined with a slit-lamp microscope to ensure that they have not been damage during the dissection procedure using sodium fluorescein. 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
The eyes examined and approved were incubated between 45 and 60 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 baselin (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: 30 µL Physiological saline - Dutscher Batch No. 3011922 (1 replicate)

POSITIVE CONTROL USED: 30 mg Sodium hydroxide - Sigma Batch No. MKBP7805V (3 replicates)

APPLICATION DOSE AND EXPOSURE TIME
30 mg of the test item was applied for 10 seconds

OBSERVATION PERIOD
Pretreatment and 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: 20 mL of physiological saline at ambient temperature
- Indicate any deviation from test procedure in the Guideline: no

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: It was measured using a HaagStreit BP900 slit-lamp microscope with depth-measuring device no. I. It was calculated using the area of the cornea that was most densely opacified for scoring.
- Damage to epithelium based on fluorescein retention: It was calculated for the 30-minute observation time point only, which was used for the overall category score given for each test or control item.
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: It was measured using a HaagStreit BP900 slit-lamp microscope with depth-measuring device no. I, the slit-width was set at 91/2, equalling 0.095 mm. The mean percentage of corneal swelling was calculated for all observation time points.
- Macroscopic morphological damage to the surface: include "pitting" of corneal epithelial cells, "loosening" of epithelium, "roughening" of the corneal surface and "sticking" of the test item to the cornea. The classification of these findings is subjective according to the interpretation of the investigator.

SCORING SYSTEM:
- Mean corneal swelling (%): (corneal thickness at time t -corneal thickness at time=0/corneal thickness at time=0)x100.
- Mean maximum opacity score: 0= No opacity; 0.5= Very faint opacity; 1= Scattered or diffuse areas, details of the iris clearly visible; 2= Easlily discernible translucent area, details of the iris are slightly obscured; 3= Severe corneal opacity, no specific details of the iris are visible, size of the pupil is barely discernible; 4= Complete corneal opacity, iris invisible.
- Mean fluorescein retention score at 30 minutes post-treatment: 0= No fluorescein retention; 0.5= Very minor single cell staining; 1= Single cell staining scattered throughout the treated area of the cornea; 2= Focal or confluent dense single cell staining; 3= Confluent large areas of the cornea retaining fluorescein.

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used. All endpoints were evaluated separately to generate an ICE class for each endpoint and then combined to generate an Irritancy Classification for the test item. The ICE classes were assigned based on a predetermined range.
ICE classification criteria for corneal thickness: 0 to 5 = class I; >5 to 12= class II; >12 to 18 (>75 min after treatment)=class II; >12 to 18 (≤75 min after treatment)=class III;>18 to 26= class III; >26 to 32 (>75 min after treatment)= class III; >26 to 32 (≤75 min after treatment)= class IV; >32=class IV.
ICE classification criteria for opacy: 0.0-0.5 = class I; 0.6-1.5= class II; 1.6-2.5 =class III; 2.6-4.0=class IV.
ICE classification criteria for mean fluorescein retention: 0.0-0.5 = class I; 0.6-1.5= class II; 1.6-2.5 =class III; 2.6-3.0=class IV.

Overall in vitro classifications:
No category= 3 x I / 2 x I, 1 x II
No prediction can be made= Other combinations
Category 1 (Corrosive/Severe Irritant)= 3 x IV / 2 x IV, 1 x III / 2 x IV, 1 x II / 2 x IV, 1 x I / Corneal opacity ≥ 3 at 30 min (in at least 2 eyes) / Corneal opacity = 4 at any time point (in at least 2 eyes) / Severe loosening of the epithelium (in at least 1 eye)

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

Maximal mean

Value:

ca. 0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class I

Results 2

Irritation parameter:

fluorescein retention score

Run / experiment:

Mean

Value:

ca. 2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class III

Results 3

Irritation parameter:

percent corneal swelling

Run / experiment:

Maximal mean

Value:

ca. 1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class I

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No. No morphological effects were noted, whatever the examination time.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes, 12 of the 13 chemicals tested obtained the same category as OECD 438. There was a borderline chemical (DMSO) over-classified ("No prediction can be made" vs. "No Category") in three separated tests. It should be tested with other adequately validated in vitro test.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, the combination of the 3 endpoints was 3 x I and classified as "No category"
- Acceptance criteria met for positive control: Yes, the combination of the 3 endpoints was 3 x IV and classified as "Corrosive/Severe Irritant"

Any other information on results incl. tables

Table 1. Individual and average values for evaluation of corneal lesions after treatment with the negative control (Physiological saline)

 

Endpoint measured	Eye No.	Time (min)
		0	30	75	120	180	240
Corneal opacity	13	0.0	0.0	0.0	0.0	0.0	0.0
ICE class		I
Fluorescein retention	13	0.5	0.5	-	-	-	-
ICE class			I	-	-	-	-
Corneal thickness	13	0.60	0.60	0.60	0.60	0.60	0.6
Corneal swelling (%)	13	-	0	0	0	0	0
ICE class		I
Combination of the 3 endpoints		3 x I
CLASSIFICATION		No Category

Note: No morphological effects were noted, whatever the examination time.

 

 

Table 2. Individual and average values for evaluation of corneal lesions after treatment with the positive control (Sodium hydroxyde)

 

Endpoint measured	Eye No.	Time (min)
		0	30	75	120	180	240
Corneal opacity	1	0	4	4	4	4	4
	2	0	4	4	4	4	4
	3	0	4	4	4	4	4
Mean		0.0	4.0	4.0	4.0	4.0	4.0
ICE class		IV
Fluorescein retention	1	0.5	3	-	-	-	-
	2	0.5	3	-	-	-	-
	3	0.5	3	-	-	-	-
Mean		0.5	3.0	-	-	-	-
ICE class			IV	-	-	-	-
Corneal thickness	1	0.59	(-)	(-)	(-)	(-)	(-)
	2	0.56	(-)	(-)	(-)	(-)	(-)
	3	0.56	(-)	(-)	(-)	(-)	(-)
Corneal swelling (%)	1	(-)	(-)	(-)	(-)	(-)	(-)
	2	(-)	(-)	(-)	(-)	(-)	(-)
	3	(-)	(-)	(-)	(-)	(-)	(-)
Mean		-	-	-	-	-	-
ICE class		IV
Combination of the 3 endpoints		3 x IV
CLASSIFICATION		Category 1: Corrosive/Severe irritant

Note:

 (-): evaluation of corneal swelling not possible (Corneal opacity=4 at each examination time, leading to a marked refraction of the light preventing from the evaluation of the corneal swelling with the biomicroscope).

Severe loosening of the corneal epithelium noted from 30 minutes post-dose in eyes No. 1, 2 & 3.

 

 

Table 3. Individual and average values for evaluation of corneal lesions after treatment with the test item.

 

Endpoint measured	Eye No.	Time (min)
		0	30	75	120	180	240
Corneal opacity	10	0	0	0	0	0	0
	11	0	0	0	0	0	0
	12	0	0	0	0	0	0
Mean		0.0	0.0	0.0	0.0	0.0	0.0
ICE class		I
Fluorescein retention	10	0.5	2	-	-	-	-
	11	0.5	2	-	-	-	-
	12	0.5	2	-	-	-	-
Mean		0.5	2.0	-	-	-	-
ICE class			III	-	-	-	-
Corneal thickness	10	0.58	0.59	0.59	0.59	0.59	0.59
	11	0.62	0.62	0.62	0.62	0.62	0.62
	12	0.56	0.56	0.56	0.56	0.57	0.57
Corneal swelling (%)	10	-	2	2	2	2	2
	11	-	0	0	0	0	0
	12	-	0	0	0	0	0
Mean		-	1	1	1	1	1
ICE class		I
Combination of the 3 endpoints		1 x III, 2 x I
CLASSIFICATION		No prediction can be made

Note: No morphological effects were noted, whatever the examination time.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

The combination of the 3 endpoints fot the test item was 1 x III, 2 x I, according to OECD 438. Therefore, no prediction can be made.

Executive summary:

The eye irritation of the test item has been evaluated using the tests Isolated Chicken Eye test method, according to OECD 438 and EU method B.48, following GLP. The test item was applied, as supplied, at the dose of 30 mg to 3 enucleated chicken eyes during 10 seconds. Then the eyes were rinsed twice with 10 mL of physiological saline. Three eyes were treated in the same manner with a positive control (sodium hydroxide) and one eye with a negative control (physiological saline). Damages by the tests item were assessed by determination of corneal swelling, opacity, and fluorescein retention were evaluated pretreatment and at 30, 75, 120, 180 and 240 minutes post-dose. Fluorescein retention, corneal opacity and corneal swelling were evaluated, then the results of each endpoint were assigned to ICE classes according to OECD guideline 438. Positive control and negative control results were valid. The mean corneal opacity for the test item trated corneas was 0, corresponding to ICE category class I; the mean score of fluorescein retention was 2, corresponding to ICE category class III; and the mean corneal swelling was 1%, corresponding to ICE class I. The combination of the three endpoints for the test item was 1 x III, 2 x I. Therefore, no prediction can be made.