Bisguanidinium phosphate

Endpoint summary

Description of key information

skin irritation

OECD TG 439, RhE, GLP, negative

eye irritation

OECD TG 438, Isolated Chicken Eye Test, GLP, neither UN GHS Classification Category I (an ocular corrosive or severe eye irritant) nor No Category.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018/11/07 - 2018/11/09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

isolated skin discs

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: not specified

Source strain:

other: not applicable (human)

Justification for test system used:

The EPISKIN model has been validated for irritation testing in an international trial. After a review of scientific reports and peer reviewed publications on the EPISKIN method, it showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404 and Method B.4 of Annex V to Directive 67/548/EEC) for the purposes of distinguishing between skin irritating and no- skin irritating test substances (STATEMENT OF VALIDITY OF IN-VITRO TESTS FOR SKIN IRRITATION; ECVAM; Institute for Health & Consumer Protection; Joint Research Centre; European Commission; Ispra; 27 April 2007).

Type of coverage:

other: not applicable (in vitro test system)

Preparation of test site:

other: The epidermal surface was first moistened with 5 μL deionised water

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): 10 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL
- Concentration (if solution): 5 %

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL

Duration of treatment / exposure:

15 min

Observation period:

42 h

Number of animals:

not applicable (in vitro test system)

Details on study design:

Exposure (day 0)
The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min) at room temperature.

Rinsing (day 0)
After the incubation time the EpiSkinTMSM units were removed and rinsed thoroughly with approximately 25 mL PBS 1x solution to remove all of the test material from the epidermal surface. The rest of the PBS was removed from the epidermal surface with suitable pipette tip linked to a vacuum source (care was taken to avoid the damage of epidermis).

Post-incubation (day 0-2)
After rinsing the units were placed into the plate wells with fresh pre-warmed “maintenance medium” (2 mL/well) below them and then incubated for 42 hours (± 1h) at 37±1°C in an incubator with 5±1 % CO2, ≥95% humidified atmosphere.

MTT test after 42 hours incubation (day 2)
After the 42 hours (± 1h) incubation the EpiSkinTMSM units were transferred into the MTT solution filled wells (2 mL of 0.3 mg/mL MTT per well) and then incubated for 3 hours (± 5 min) at 37±1°C in an incubator with 5±1 % CO2 protected from light, ≥95% humidified atmosphere.

Formazan extraction (day 2)
At the end of incubation with MTT a formazan extraction was undertaken:
A disk of epidermis was cut from the unit (this involves the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube of 500 μL acidified isopropanol (one tube corresponding to one well of the tissue culture plate).
The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material with the acidified isopropanol then incubated for four hours at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction. At the middle and at the end of the incubation period, each tube was additionally mixed using a vortex mixer to help extraction.

Cell viability measurements (day 2)
Following the formazan extraction, 2×200 μL sample from each tube was placed into the wells of a 96-well plate (labelled appropriately) and read the OD (Absorbance / Optical Density) of the samples in a 96-well plate spectrophotometer (Thermo Scientific; Multiscan FC) at 570 nm (±10nm; Read out range: 0-3.5 Abs, Linearity range: 0.2136 – 3.1752) using acidified isopropanol solution as the blank (6×200 μL).

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

103

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

110

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3

Value:

105

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

106

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: No colour change was observed after three hours of incubation. The test material did not interact with the MTT, therefore additional controls and data calculations were not necessary. A false estimation of viability can be precluded.
- Colour interference with MTT: The test item showed no ability to become coloured in contact with water. The intrinsic colour of test item is white and therefore considered to be not able to significantly stain the tissues and lead false estimate of viability. Furthermore, the test item was completely removed from the epidermal surface at rinsing period. Additional controls and data calculations were not necessary. A false estimation of viability can be precluded.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Prior to routine use of the method Toxi-Coop ZRT. demonstrated the technical proficiency in a separate study (study no.: 392.554.2938) using the ten Proficiency Chemicals according to OECD Test Guideline No. 439.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, the mean OD value of the three negative control tissues was 0.880.
- Acceptance criteria met for positive control: Yes, the mean OD value obtained for the positive control was 0.088 and this result corresponds to 10 % viability when compared to the results obtained from the negative control.
- Acceptance criteria met for variability between replicate measurements: Yes, each calculated standard deviation value (SD) for the % viability was below 18.

Interpretation of results:

other: EU GHS criteria not met

Conclusions:

In this in vitro skin irritation test using the EPISKIN model, the test item bisguanidinium phosphate did not show significantly reduced cell viability in comparison to the negative control (mean value: 106 %). All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control. Therefore the test item is considered to be non-irritant to skin. Positive and negative controls showed the expected cell viability values within acceptable limits. Thefore the experiment is evaluated to be valid. The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. The test item bisguanidinium phosphate is considered to be non-irritant to skin and has not to be classified for skin irritation (UN GHS No Category).

Executive summary:

EpiSkin^TM SM test of bisguanidinium phosphate was performed to predict its irritation potential by measurement of its cytotoxic effect, as reflected in the MTT assay, according to the OECD Test Guideline No. 439, 28 July 2015. Disks of EPISKIN (three units) were treated with test item and incubated for 15 minutes (± 0.5 min) at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. Epidermis units were then incubated at 37±1°C for 42 hours (± 1h) in an incubator with 5±1% CO₂, ≥ 95% humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours (± 5 min) with MTT solution at 37±1°C in 5±1% CO₂, ≥ 95% humidified atmosphere, protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically. SDS (5% aq.) and 1×PBS treated (three units / positive and negative control) epidermis were used as positive and negative controls respectively. For each treated tissue, viability was expressed as a percentage relative to negative control. The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control. In this in vitro skin irritation test using the EPISKIN model, the test item bisguanidinium phosphate did not show significantly reduced cell viability in comparison to the negative control (mean value: 106 %). All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control. Therefore the test item is considered to be non-irritant to skin. Positive and negative controls showed the expected cell viability values within acceptable limits. Thefore the experiment is evaluated to be valid. The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. The test item bisguanidinium phosphate is considered to be non-irritant to skin and has not to be classified for skin irritation (UN GHS No Category).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

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

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)

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.

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.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

skin irritation

Reconstructed human epidermis model (OECD TG 439)

EpiSkin^TMSM test of bisguanidinium phosphate was performed to predict its irritation potential by measurement of its cytotoxic effect, as reflected in the MTT assay, according to the OECD Test Guideline No. 439, 28 July 2015. Disks of EPISKIN (three units) were treated with test item and incubated for 15 minutes (± 0.5 min) at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. Epidermis units were then incubated at 37±1°C for 42 hours (± 1h) in an incubator with 5±1% CO₂, ≥ 95% humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours (± 5 min) with MTT solution at 37±1°C in 5±1% CO₂ , ≥ 95% humidified atmosphere, protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically. SDS (5% aq.) and 1×PBS treated (three units / positive and negative control) epidermis were used as positive and negative controls respectively. For each treated tissue, viability was expressed as a percentage relative to negative control. The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control. In this in vitro skin irritation test using the EPISKIN model, the test item bisguanidinium phosphate did not show significantly reduced cell viability in comparison to the negative control (mean value: 106 %). All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control. Therefore the test item is considered to be non-irritant to skin. Positive and negative controls showed the expected cell viability values within acceptable limits. Thefore the experiment is evaluated to be valid. The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. The test item bisguanidinium phosphate is considered to be non-irritant to skin and has not to be classified for skin irritation (UN GHS No Category).

eye irritation

Isolated Chicken Eye Test (OECD TG 438)

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 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.

Justification for classification or non-classification

According to the clear negative result of the in vitro skin irritation assay with the test item, bisguanidinium phosphate does not warrant classification for skin irritation/corrosion in accordance with Regulation (EC) No 1272/2008. The isolated chicken eye test does not allow a conclusive prediction indicating that the overall classification is neither UN GHS Classification Category I (an ocular corrosive or severe eye irritant) nor No Category. So taking this uncertainty into account and based on experience in handling of the slight acidic test item, a classification as Eye Irrit. 2 "H319 Causes serious eye irritation" is justified and does also ensure adequate risk measures without the need for further testing.