Bisguanidinium phosphate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018/08/28 - 2018/08/28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Bisguanidinium phosphate

EC Number:

226-552-4

EC Name:

Bisguanidinium phosphate

Cas Number:

5423-23-4

Molecular formula:

CH5N3.1/2H3O4P

IUPAC Name:

Diguanidinium hydrogen phosphate

Test material form:

solid: particulate/powder

Test animals / tissue source

Species:

chicken

Strain:

other: ROSS 308

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: TARAVIS KFT. 9600 Sárvár, Rábasömjéni út 129. Hungary
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Head collection was performed by a slaughter house technician. Heads were removed immediately after sedation of the chickens (sedation was performed by electric current). The heads were transported to Toxi-Coop ZRT. at the earliest convenience for use approximately within 2 hours from collection. The ambient temperature was optimal (19.3ºC to 20.4ºC) during the transport. All eyes used in the assay were from the same groups of eyes collected on one specific day. After collection, the heads were inspected for appropriate quality and wrapped with paper moistened with saline, then placed in a plastic box that can be closed (4-5 heads/box). Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained. The prepared eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head). Again avoiding too much pressure on the eye by the clamp. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was applied to fix the eye properly. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with saline solution dripping from a stainless steel tube, at a rate of approximately 3 to 4 drops/minutes. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.
- Time interval prior to initiating testing: If the selected eyes were appropriate for the test, acclimatization started and was conducted for approximately 45 to 60 minutes. The temperature was verified to be in the range of 32 ± 1.5 °C in all chambers during the acclimatization and treatment periods.
- indication of any existing defects or lesions in ocular tissue samples: One small drop of fluorescein solution 2 (w/v) % was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL isotonic saline. Then the fluorescein-treated cornea was examined with hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected.

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): 30 mg

Duration of treatment / exposure:

10 s

Observation period (in vivo):

240 min

Duration of post- treatment incubation (in vitro):

30 min

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES

Eyes selection
After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of fluorescein solution 2 (w/v) % was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL isotonic saline. Then the fluorescein-treated cornea was examined with hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit.

Preparation of eyes
The eyeball was carefully removed from the orbit by holding the nictitating membrane with surgical forceps, while cutting the eye muscles with bent scissors without cutting off the optical nerve too short. The procedure avoided pressure on the eye in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.

EQUILIBRATION AND BASELINE RECORDINGS
At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t = 0) for each individual eye. The cornea thickness of the eyes should not change by more than ± 5-7 % within approximately 45 to 60 minutes before the start of application. Slight changes in thickness (0% to 2%) were observed in the eyes. This finding is considered as normal when maintaining enucleated eyes. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effects after treatment. The location of any minor findings was marked on the record sheet as a drawing, if applicable. If any eye was considered to be unsuitable following baseline assessment, it was discarded.

NUMBER OF REPLICATES
3 (positive control and test item)
1 (negative control)

NEGATIVE CONTROL USED
yes, saline solution

POSITIVE CONTROL USED
yes, Imidazole

APPLICATION DOSE AND EXPOSURE TIME
30 mg for 10 s

OBSERVATION PERIOD
240 min

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 mL saline solution at ambient temperature, while taking care not to damage the cornea but attempting to remove all the residual test item if possible.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: no details given
- Damage to epithelium based on fluorescein retention: no details given
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: 0.095 mm
- Macroscopic morphological damage to the surface: If morphological effects were observed the classification of these findings were interpretations of the study director (e.g., pitting or loosening of the epithelium).
- Others (e.g, histopathology): A histopathology of the corneas was not performed as not borderline results were obtained, for which histopathology could provide final clarification. Corneas are discarded 2 months after the final report.

SCORING SYSTEM:
- Mean corneal swelling (%) : 3 (30 min), 4 (75 min), 6 (120 min), 7 (180 and 240 min)
- Mean maximum opacity score : 0.7 (30 and 75 min), 1.2 (120, 180 and 240 min)
- Mean fluorescein retention score at 30 minutes post-treatment : 1.0

DECISION CRITERIA: The decision criteria as indicated in the TG was used.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

1

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: result for 30, 75, 120, 180 and 240 min post-exposure

Results 2

Irritation parameter:

cornea opacity score

Run / experiment:

2

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: result for 30 and 75 min post-exposure

Results 3

Irritation parameter:

cornea opacity score

Run / experiment:

2

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: result for 120, 180 and 240 min post-exposure

Results 4

Irritation parameter:

cornea opacity score

Run / experiment:

3

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: result for 30 and 75 min post-exposure

Results 5

Irritation parameter:

cornea opacity score

Run / experiment:

3

Value:

2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: result for 120, 180 and 240 min post-exposure

Results 6

Irritation parameter:

percent corneal swelling

Run / experiment:

1

Value:

3.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: result for 30, 75, 120, 180 and 240 min post-exposure

Results 7

Irritation parameter:

percent corneal swelling

Run / experiment:

2

Value:

1.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: result for 30 min post-exposure

Results 8

Irritation parameter:

percent corneal swelling

Run / experiment:

3

Value:

3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: result for 30 min post-exposure

Results 9

Irritation parameter:

percent corneal swelling

Run / experiment:

2

Value:

3.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: result for 75 min post-exposure

Results 10

Irritation parameter:

percent corneal swelling

Run / experiment:

2

Value:

5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: result for 120, 180 and 240 min post-exposure

Results 11

Irritation parameter:

percent corneal swelling

Run / experiment:

3

Value:

5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: result for 75 min post-exposure

Results 12

Irritation parameter:

percent corneal swelling

Run / experiment:

3

Value:

10

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: result for 120 min post-exposure

Results 13

Irritation parameter:

percent corneal swelling

Run / experiment:

3

Value:

1.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: result for 180 and 240 min post-exposure

Results 14

Irritation parameter:

fluorescein retention score

Run / experiment:

1 and 2

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: result for 30 min post-exposure

Results 15

Irritation parameter:

fluorescein retention score

Run / experiment:

3

Value:

2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: result for 30 min post-exposure

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Prior to routine use of the method Toxi-Coop ZRT. demonstrated the technical proficiency in a separate study (study no.: 392.549.3229) using the ten Proficiency Chemicals according to OECD Test Guideline No. 438.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Negative control values were within the corresponding historical control data ranges (please refer to table 2.1)
- Acceptance criteria met for positive control: Positive control values were within the corresponding historical control data ranges (plese refer to table 2.2)
- Range of historical values if different from the ones specified in the test guideline: please refer to tables 2.1 and 2.2

Any other information on results incl. tables

Table 1.1 Results for Test Item Bisguanidiniumphosphate
Observation	Value	ICE Class
Mean maximum corneal swelling up to 75 min	4%	I
Mean maximum corneal swelling up to 240 min	7%	II
Mean maximum corneal opacity	1.2	II
Mean fluorescein retention	1.0	II
Other Observations	None
Overall ICE Class	3xII

Table 1.2: Results for Positive Control Imidazole
Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	34%	IV
Mean maximum corneal swelling at up to 240 min	39%	IV
Mean maximum corneal opacity	4.0	IV
Mean fluorescein retention	2.8	IV
Other Observations	Corneal opacity score 4 was observed in three eyes at 30 minutes after the post-treatment rinse.
Overall ICE Class	3xIV

Table 1.3 Results for NegativeControl NaCl (9 g/L saline)
Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	0%	I
Mean maximum corneal swelling at up to 240 min	2%	I
Mean maximum corneal opacity	0.0	I
Mean fluorescein retention	0.0	I
Other Observations	None
Overall ICE Class	3xI

Table 2.1: Historical control data of Positive control Imidazole (Period of 2011 - 2017)
Dose level: 30 mg / eye
n=276	Relative observation time (min)—	Corneal thickness					Corneal opacity score						Fluorescein retention
		30	75	120	180	240		30	75	120	180	240		AFR
Maximum swelling(%):		34	45	49	54	55	Max. OS:	4.0	4.0	4.0	4.0	4.0	Max. FR:	3.0
Minimum swelling(%):		3	9	12	14	15	Min. OS:	2.8	3.3	3.5	3.5	3.5	Min. FR:	2.7
Average:		21	28	32	35	37	Average:	3.6	3.9	3.9	4.0	4.0	Avarage:	3.0

Table 2.2: Historical control data of Negative control (Period of 2011 - 2017)
NaCl (9 g/L saline) Dose level: 30µL / eye
n=188	Relative observation time (min)—	Corneal thickness					Corneal opacity score						Fluorescein retention
		30	75	120	180	240		30	75	120	180	240		AFR
Maximum swelling(%):		3	5	5	5	5	Max. OS:	0.5	0.5	0.5	0.5	0.5	Max. FR:	0.5
Minimum swelling(%):		0	0	0	0	0	Min. OS:	0	0	0	0	0	Min. FR:	0.0
Average:		0.2	0.4	0.5	0.5	0.5	Average:	0.0	0.1	0.1	0.1	0.1	Average:	0.0
Remark: n= number of eyes AFR= Difference between fluorescein retention and fluorescein retention reference value OS= Opacity score FR= Fluorescein retention

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

In this ICET, bisguanidinium phosphate did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. The overall ICE classes were II based on corneal swelling, opacity and fluorescein retention. Positive and negative controls showed the expected results. The experiment is considered to be valid. According to the guideline OECD 438, bisguanidinium phosphate overall in vitro classification is neither UN GHS Classification Category I (an ocular corrosive or severe eye irritant) nor No Category. Thus, according to the guideline OECD 438, the test item has been categorized as “No prediction can be made” based on this test.

Executive summary:

The purpose of this Isolated Chicken Eye Test (ICET) was to evaluate the potential ocular corrosivity and irritancy of the test item bisguanidinium phosphate by its ability to induce toxicity in enucleated chicken eyes. The test compound was applied in a single dose (30 mg/eye) onto the cornea of isolated chicken eyes in order to potentially classify the test compound as either 1: causing "serious eye damage" (category 1 of the Globally Harmonised System for the Classification and Labelling of Chemicals (GHS)), or 2: not requiring classification for eye irritation or serious eye damage according to the GHS. Tested corneas were evaluated per pre-treatment and at approximately 30, 75, 120, 180, and 240 minutes after the post-treatment rinse. The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects. All of the endpoints, with the exception of fluorescein retention (which was determined only at pre-treatment and 30 minutes after test substance exposure) were determined at each of the above time points. Imidazole (positive control) was grounded before use in the study. The test item and positive control were applied in an amount of 30 mg/eye by powdering the entire surface of the cornea attempting to cover the cornea surface uniformly with the test substance or positive control. Three test item treated eyes and three positive control eyes were used in this study. One negative control eye was treated with 30 μL saline solution. After an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with ~20 mL saline solution at ambient temperature and this procedure was repeated for each eye. Imidazole was stuck on the corneas’ surface in all eyes at 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Imidazole treated cornea surfaces were not totally cleared at 240 minutes after the post-treatment rinse. In this ICET, bisguanidinium phosphate did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. The overall ICE classes were II for corneal swelling opacity and fluorescein retention. Positive and negative controls showed the expected results. The experiment is considered to be valid. According to the guideline OECD 438, bisguanidinium phosphate overall in vitro classification is neither UN GHS Classification Category I (an ocular corrosive or severe eye irritant) nor No Category. Thus, according to the guideline OECD 438, the test item has been categorized as “No prediction can be made” based on this test.