1-benzyl-3-carbamoylpyridinium chloride

Endpoint summary

Description of key information

The potential of 1-Benzyl-3-carbamoyl-pyridinium, chloride to induce skin corrosion (OECD 431) and/or skin irritation (OECD 439) was tested in suitable in vitro test methods. Based on the results, 1-Benzyl-3-carbamoyl-pyridinium, chloride (~96% purity) can be considered non-irritant to the skin.

The eye irritancy potential of the target substance was assessed in a weight-of-evidence approach by using data from three in vitro eye irritating assays (OECD 437, OECD 438, OECD 491). Based on the results, 1-Benzyl-3-carbamoyl-pyridinium, chloride is considered to induce severe eye irritation and meets the classification criteria according to CLP (Eye Irrit. 2, H319).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-08-12 to 2016-09-22

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)

Version / remarks:

28 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Justification for test system used:

This test uses the EPISKIN-SM™ reconstructed human epidermis model (SkinEthic) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis. This test method is able to detect chemicals that cause skin irritation, i.e. produce reversible damage to the skin and allows for hazard identification in accordance with UN GHS "Category 2". Depending on the regulatory framework it can also be used to identify non-classified chemicals.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:
The test was carried out with the reconstituted three-dimensional human skin model EPISKIN-SM™ (SkinEthic). This skin model consists of normal human epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK are cultured on chemically modified, collagen-coated cell culture inserts. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
- Tissue batch number(s): 16-EKIN-035
- Expiration date: September 05, 2016
- Date of initiation of testing: August 17, 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (exposure duration: 15 min)
- Temperature of post-treatment incubation (if applicable):
a) 37 ± 1 °C (5.0% CO2 for 42 ± 1 h) in 2 ml pre-warmed fresh maintenance medium
b) 37 ± 1 °C (5.0% CO2 for 3 h ± 5 min) in 2 ml pre-warmed MTT medium

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps:
* washed with DPBS; excess DPBS was removed by blotting bottom with blotting paper;
* after post-incubation: excess medium was removed by blotting bottom on absorbent paper
- Observable damage in the tissue due to washing:
- Modifications to validated SOP:

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/ml
- Incubation time: 3 h ± 5 min
- Spectrophotometer: plate spectrophotometer
- Wavelength: 570 nm
- Filter: not specified
- Filter bandwidth: ± 30 nm
- Linear OD range of spectrophotometer: not reported

NUMBER OF REPLICATE TISSUES: 3

PREDICTION MODEL / DECISION CRITERIA
The test substance is considered to be not irritating (the relative mean tissue viability after 15 min of exposure and 42 h post-incubation was > 50%)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

PREPARATION AND APPLICATION OF THE TEST ITEM
Firstly, 5 µL distilled water (aqua dest.) was applied to the epidermal surface in order to improve further contact between the powder and the epidermis. The water was gently spread on the surface. Afterwards, approximately 10 ± 2 mg (26.3 mg/cm²) of the powder was applied to the epidermis surface. The test item was spread to match size of the tissue using a flat curved spatula. The volume of aqua dest. had to be increased by additional 5 µL to allow spreading of the test item.

NEGATIVE CONTROL: 10 µL DPBS (Gibco, Cat. No. 14040-091, Lot No.: 17371 07).
POSITIVE CONTROL: 10 µL 5% sodium dodecyl sulfate (SDS; AppliChem, Art.-No. A1112,0500, CAS No.: 151-21-3, Lot No.: 40015277) in aqua dest.
TEST ITEM: 10 ± 2 mg + 10 µL aqua dest.

Duration of treatment / exposure:

15 min +/- 0.5 min

Duration of post-treatment incubation (if applicable):

42 +/- 1 hour

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

main experiment

Value:

99.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Table 2: Result of the Test Item 1-Benzyl-3-carbamoyl-pyridinium, chloride

Name	Negative Control			Positive Control			Test Item
Tissue	1	2	3	1	2	3	1	2	3
Absolute OD570	0.742	0.816	0.676	0.069	0.086	0.086	0.786	0.695	0.766
	0.767	0.855	0.689	0.074	0.086	0.081	0.780	0.720	0.793
OD570 (Blank-Corrected)	0.699	0.773	0.634	0.027	0.044	0.044	0.744	0.653	0.724
	0.724	0.813	0.647	0.031	0.044	0.039	0.738	0.678	0.751
Mean OD570 of the Duplicates (Blank-Corrected)	0.712	0.793	0.641	0.029	0.044	0.041	0.741	0.666	0.737
Total mean OD570 of 3 Replicate Tissues (Blank-Corrected)	0.715*			0.038			0.715
SD OD570	0.076			0.008			0.043
Relative Tissue Viability [%]	99.5	110.9	89.6	4.1	6.1	5.8	103.6	93.0	103.1
Mean Relative Tissue Viability [%]	100.0			5.3**			99.9
SD Tissue Viability [%]***	10.6			1.1			5.9
CV [% Viability]	10.6			20.6			5.9

*      Corrected mean OD₅₇₀of the negative control corresponds to 100% absolute tissue viability.

**   Mean relative tissue viability of the three positive control tissues is ≤ 40%.

*** Standard deviation (SD) obtained from the three concurrently tested tissues is ≤ 18%.

Table 3: Test Acceptance Criteria

	Value	Cut off	pass/fail
Mean OD570 Blank	0.042	< 0.1	pass
Mean Absolute OD570 NK	0.757	0.6≤NK≤1.5	pass
Mean Relative Viability PC [%]	5.3	≤ 40%	pass
Max. SD of % Viability	10.6	≤ 18%	pass

 

Table 4: Historical Data

	OD570 ± 30 nmBlank	Absolute OD570 ± 30 nmNK	Relative Viability PC [%]	Max. Difference Viability [%]
Mean	0.043	0.866	11.7	8.47
SD	0.001	0.120	8.2	8.08
n	67	66	67	286

Historical control data were generated from 2008 - 2015.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item showed no irritant effects in an in vitro skin irritation study conducted according to OECD 439.

Executive summary:

In an In Vitro Skin Irritation Study according to OECD Guideline 439 (Reconstructed Human Epidermis Test), the potential of the 1-Benzyl-3-carbamoyl-pyridinium, chloride (~ 96 % purity) to induce skin irritation was analysed by using the three-dimensional human skin model EPISKIN-SM™ (SkinEthic) comprising a reconstructed epidermis with a functional stratum corneum.

In the present study the test item was applied topically to the EPISKIN-SM™ tissue for 15 min followed by a 42 h post-incubation period and immediate determination of cytotoxic effects via MTT reduction assay. Irritant potential of the test item was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with DPBS.

The test item showed no irritant effects and is therefore considered to be non-irritating to the skin.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-07-12 to 2016-08-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

28 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Details on animal used as source of test system:

Not applicable

Justification for test system used:

This test uses the EPISKIN-SM™ reconstructed human epidermis model (SkinEthic) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.

Vehicle:

physiological saline

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: reconstituted three-dimensional human skin model EPISKIN-SM™ (SkinEthic);
This skin model consists of normal human epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK are cultured on chemically modified, collagen-coated cell culture inserts. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
- Tissue batch number(s): 16-EKIN-030
- Expiration date: August 1, 2016
- Date of initiation of testing: July 25, 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (MTT incubation): 37 ± 1 °C (for 3 h ± 15 min, 5.0% CO2 / 95% air)

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps:
* Tissue was gently rinsed about 15 times with 25 ml PBS to remove any residual test item
* Excess PBS was removed by gently shaking the insert and blotting bottom with blotting paper.
* The surface was swept with a cotton bud. The insert was placed in a prepared 12-well "holding plate" containing 2.2 ml prewarmed assay medium per well. All inserts were treated in the same manner.
* Then the inserts were transferred into a prepared 12-well "MTT assay plate" containing 2 ml prewarmed MTT solution.
* After the 3 h MTT incubation period the tissues were placed on blotting paper to remove excess liquid.
- Observable damage in the tissue due to washing:
- Modifications to validated SOP:

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/ml
- Incubation time: 3 h ± 15 min ( at 37 ± 1 °C)
- Spectrophotometer: plate spectrophotometer
- Wavelength: 570 nm
- Filter: not specified
- Filter bandwidth: max. ± 30 nm
- Linear OD range of spectrophotometer: not reported

FOR FURTHER DETAILS ON TEST METHOD AND EXPERIMENTAL PROCEDURE PLEASE REFER TO THE SECTION "ANY OTHER INFORMATION ON MATERIALS AND METHODS INCL. TABLES"

PREDICTION MODEL / DECISION CRITERIA:
The test substance is considered to be non-corrosive to skin as the relative mean tissue viability after 4 h treatment was not decreased to less than 35% of the corresponding negative control tissues.

Control samples:

yes, concurrent vehicle

yes, concurrent positive control

Amount/concentration applied:

1. Negative control: 50 µL 0.9% NaCI solution
2. Positive control: 50 µL glacial acetic acid
3. Test Item: 20 ± 2 mg + 100 ± 5 µL 0.9% NaCI

Duration of treatment / exposure:

- Test item and negative control: 3 min, 60 min and 4 h exposure time
- Positive control: 4 h exposure time

Duration of post-treatment incubation (if applicable):

3 h ± 15 min

Number of replicates:

- Test item and negative control: 2 replicates for each treatment period
- Positive control: 2 replicates

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min Experiment

Value:

94

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not examined

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 min Experiment

Value:

88

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not examined

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

4 h Experiment

Value:

106

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control : yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Results of Pre-Experiments:

20 mg test item per 2 ml MTT medium showed no reduction of MTT as compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%.

The mixture of 10 mg test item per 300 µL Aqua. dest. and per 90 µL isopropanol showed no colouring as compared to the solvent. Therefore NSC equalled 0%.

Results of Main Experiment:

Table 2: Blank values

Name	Blank
absolute OD570-values (raw data)	0.029	0.043	0.043
	0.043	0.043	0.044
mean OD570 (mean of 2 aliquots)	0.041

Table 3: 3 min Experiment

Name	Negative Control		Test Item
Tissue	1	2	1	2
Absolute OD570-values (raw data)	1.049	1.059	0.990	1.004
	1.135	1.133	1.051	1.064
Mean OD570 (mean of 2 aliquots per tissue)	1.092	1.096	1.020	1.034
Mean OD570 (blank corrected)	1.051	1.055	0.979	0.993
Total mean OD570 (mean of 2 aliquots per tissue, blank corrected)	1.053*		0.986
Relative tissue viability [%]	99.8	100.2	93.0	94.3
Mean relative tissue viability [%]	100		94
Difference of relative tissue viability [%]***	0.3		1.3

* corrected mean OD₅₇₀ of the negative control corresponds to 100% absolute tissue viability;

*** difference between each two replicates is ≤ 30% (in the range of 20-100% viability and for ODs > 0.3).

Table 4: 60 min Experiment 

Name	Negative Control		Test Item
Tissue	1	2	1	2
Absolute OD570-values (raw data)	1.147	1.100	1.002	1.002
	1.179	1.173	1.027	1.020
Mean OD570 (mean of 2 aliquots per tissue)	1.163	1.136	1.014	1.011
Mean OD570 (blank corrected)	1.122	1.095	0.973	0.970
Total mean OD570 (mean of 2 aliquots per tissue, blank corrected)	1.109*		0.972
Relative tissue viability [%]	101.2	98.8	87.8	87.5
Mean relative tissue viability [%]	100		88
Difference of relative tissue viability [%]***	2.4		0.3

*   corrected meanODs1oof the negative control corresponds to 100% absolute tissue viability.

*** difference between each two replicates is ≤ 30% (in the range of 20 - 100% viability and for ODs > 0.3).

Table 5: 4 h Experiment 

Name	Negative Control		Test Item		Positive Control
Tissue	1	2	1	2	1	2
Absolute OD570-values (raw data)	1.010	1.032	1.086	1.079	0.083	0.110
	1.066	1.031	1.093	1.130	0.078	0.088
Mean OD570 (mean of 2 aliquots per tissue)	1.038	1.031	1.089	1.104	0.080	0.099
Mean OD570 (blank corrected)	0.997	0.990	1.048	1.063	0.039	0.058
Total mean OD570 (mean of 2 aliquots per tissue, blank corrected)	0.994*		1.056		0.049
Relative tissue viability [%]	100.3	99.7	105.5	107.0	3.9	5.8
Mean relative tissue viability [%]	100		106		5**
Difference of relative tissue viability [%]***	0.7		1.5		1.9

*   corrected meanODs1oof the negative control corresponds to 100% absolute tissue viability.

** mean relative tissue viability of the two positive control tissues of the 4 h treatment period iss20%.

*** difference between each two replicates is≤30% (in the range of 20 - 100% viability and for ODs>0.3).

Table 6: Test Acceptance Criteria

	Value	Cut off	pass/fail
Mean OD570Blank	0.041	< 0.1	pass
Mean Absolute OD570NK (4h experiment)	1.035	0.6-1.5	pass
Mean Absolute OD570NK (60 min experiment)	1.150	0.6-1.5	pass
Mean Absolute OD570NK (3 min experiment)	1.094	0.6-1.5	pass
Mean Relative Viability PC [%] (4h experiment)	5	≤ 20%	pass
Max. Difference of Tissue Viability [%]	2.4	≤ 20%	pass

Historical Data

Table 7: Historical Data

	OD570 ± 30 nm Blank	Absolute OD570 ± 30 nmNK			Relative Viability PC [%]	Max. Difference Viability [%]
		4 h	60 min	3 min
Mean	0.044	0.875	0.926	0.904	5.0	8.0
SD	0.002	0.120	0.075	0.089	1.9	7.4
n	21	21	21	21	21	197

Historical control data were generated from 2014- 2015.

Interpretation of results:

GHS criteria not met

Conclusions:

In an in vitro skin corrosion test (OECD 431) using the human skin model EPISKIN-SM™ the test item was tested as non-corrosive.

Executive summary:

The potential of 1-Benzyl-3-carbamoyl-pyridinium, chloride (~96% purity) to induce skin corrosion was analysed by using the three-dimensional human skin model EPISKIN-SM™, comprising a reconstructed epidermis with a functional stratum corneum (OECD 431). The test item was applied topically to the EPISKIN-SM™ tissue for 3 min, 60 min and 4 h followed by immediate determination of cytotoxic effects via MTT reduction assay.

Corrosivity potential of the test item was predicted from the relative mean tissue viabilities compared to the corresponding negative control tissues concurrently treated with 0.9% NaCI (= 100% tissue viability). The positive control did induce the appropriate response. The controls confirmed the validity of the study. In this study under the given conditions the test item showed no corrosive effects. The relative mean tissue viability after 4 h treatment was ≥35% (106%). Relative mean tissue viability was reduced to 88% after 60 min treatment and to 94% after 3 min treatment. The test item is therefore considered to be non-corrosive.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2016-10-04 to 2016-12-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability 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:

26 July 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: obtained as a by-product from animals freshly slaughtered at the abattoir A. Moksel AG, Buchloe, Germany
- Number of animals: no data
- Characteristics of donor animals (e.g. age, sex, weight): no data
- Storage, temperature and transport conditions of ocular tissue: On the test day, fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories.
- Time interval prior to initiating testing: Immediately after arrival of the eyes, cornea preparation was initiated.
- Indication of any existing defects or lesions in ocular tissue samples: The eyes were carefully examined for defects and any defective eyes were discarded.
- Indication of any antibiotics used: no data

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µl of test item preparation
- Concentration (if solution): 20 % concentration in 0.9 % physiological saline NaCl

VEHICLE
- Amount(s) applied (volume or weight with unit): 750 µl
- Concentration (if solution): 0.9 % physiological saline NaCl
- Lot/batch no. (if required): B. Braun Melsungen, lot no. 1406804, expiry date: 05/2017

Duration of treatment / exposure:

4 hours ± 5 minutes incubation at 32 +/- 1 °C

Duration of post- treatment incubation (in vitro):

90 minutes at 32 +/- 1 °C

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS + QUALITY CHECK OF THE ISOLATED CORNEAS
The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing HBSS. Before the corneas were mounted in corneal holders (Duratec GmbH) with the endothelial side against the O-ring of the posterior chamber, they had been visually examined for defects and any defective cornea had been discarded. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with RPMI (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI). The posterior chamber was always filled first. The corneas were incubated for one hour at 32 +/- 1 °C.

NUMBER OF REPLICATES: 3 per test group (test item, positive and negative control)

NEGATIVE CONTROL USED: yes, physilogical saline 0.9 % NaCl

SOLVENT CONTROL USED (if applicable): see negative control

POSITIVE CONTROL USED: yes, imidazole 20% in physiological saline 0.9% NaCl; imidazole from Sigma, lot no. SLBK9670V, expiry date: 01/2017

TREATMENT METHOD: open chamber
After the equilibration period, the medium was removed from both chambers and replaced with fresh complete RPMI. An initial measurement was performed on each of the corneas using the opacitometer. Three corneas with illuminance readings approximately equivalent to the median illuminance of all corneas were selected as negative-control corneas. The illuminance of each cornea was read and recorded. Only corneas that had an initial illuminance reading I > I0/1.1651 lux were used for the assay. The medium was removed from the anterior chamber and replaced with the test item or control.

APPLICATION DOSE AND EXPOSURE TIME
750 µL of the test item preparation or the control substance was introduced into the anterior chamber (closed-chamber method). Corneas were exposed for 4 hours ± 5 minutes incubation at 32 +/- 1 °C.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: Test substance or the control substance was removed and the epithelium washed at least three times with MEM (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red). The anterior chamber was refilled with complete RPMI and an illuminance measurement was performed. Also, each cornea was observed visually and pertinent observations were recorded.

POST-EXPOSURE INCUBATION:
After the illuminance measurement was performed, the medium was removed from both chambers of the holder. The posterior chamber was refilled with fresh complete RPMI. 1 mL of a 5 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes at 32 +/- 1 °C. Then the medium from the posterior chamber was removed and its optical density was determined.

METHODS FOR MEASURED ENDPOINTS:
- The optical density was determined at 490 nm (OD490) using a spectrophotometer (Jenway 6405 UV/VIS).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
- IVIS ≤ 3: UN GHS No Category
- IVIS > 3 to ≤ 55: No prediction can be made
- IVIS > 55: UN GHS Category 1

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean score of 3 replicates

Value:

69.38

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks:

The negative control responses should result in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

Positive controls validity:

valid

Remarks:

The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid.

Evaluation of Results

The following formula was used to calculate the opacity, whereas the values a and b are equipment-specific variables empirically determined by the manufacturer:

 

Opacity = (I₀/I - b) / a

with a = 0.025 and b = 0.9894

 

The value I₀ is the illuminance through a holder without cornea, but with windows and liquid. This value is determined by taking the mean for a set of cornea holders and is reevaluated periodically.

The change in opacity for each cornea was calculated by subtracting the initial opacity reading from the final opacity reading. These values were corrected by subtracting from each the average change in opacity observed for the negative-control corneas. The mean opacity value for each treatment was calculated by averaging the corrected opacity values of each cornea for a given treatment.

The mean OD490 for the blank cuvettes was calculated. The mean blank OD490 was subtracted from the OD490 of each cuvette (corrected OD490). Any dilutions that were made to bring the OD490 values into the linear range of the spectrophotometer (OD490 should be less than 1.500), were taken into account by multiplying the OD490 value of the dilution by the dilution factor. The final-corrected OD490 of the test article and the positive control were calculated by subtracting the average-corrected OD490 of the negative-control corneas from the corrected OD490 value of each treated cornea:

Final-corrected OD490 = (OD490 – mean blank OD490) – average-corrected negative control OD490

The mean OD490 value of each treatment group was calculated by averaging the final corrected OD490 values of the treated corneas for that treatment condition.

The following formula was used to determine the in vitro irritation score (IVIS):

IVIS = mean opacity value + (15 x mean permeability OD490 value)

The IVIS cut-off values for identifying test substances as inducing serious eye damage (UN GHS Category 1) and test substances not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given in Table 1:

Table 1:  Evaluation of the BCOP Assay

IVIS	UN GHS
≤ 3	No Category
> 3; ≤ 55	No prediction can be made
> 55	Category 1

An identification of test substances that should be classified as irritating to eyes (UN GHS Category 2 or Category 2A) or test substances that should be classified as mildly irritating to eyes (UN GHS Category 2B) cannot be made.For this purpose further testing with another suitable method is required.

Individual Results Data

Table 2: Opacity

Cornea No.	Test item	Initial Opacity	Final Opacity	Change of Opacity Value	Corrected Opacity Value
1	Negative Control	0.84	1.60	0.76
2		0.74	2.01	1.27
3		0.81	1.64	0.83
MV		0.80	1.75	0.95
4	Positive Control	1.42	99.72	98.30	97.35
5		2.05	116.82	114.77	113.82
6		2.05	118.43	116.39	115.43
MV		1.84	111.66	109.82	108.87
7	Test item	-0.06	69.57	69.63	68.68
8		0.42	52.87	52.45	51.50
9		1.38	78.87	77.49	76.53
MV		0.58	67.11	66.52	65.57

MV = Mean value

 

Table 3: Permeability

Cornea No.	Test item	OD490	Corrected OD490 Value
1	Negative Control	0.015
2		0.026
3		0.014
MV		0.018
4	Positive Control	1.975	1.957
5		2.175	2.157
6		2.245	2.227
MV		2.132	2.113
7	Test item	0.191	0.173
8		0.358	0.340
9		0.267	0.249
MV		0.272	0.254

MV = Mean value

 

Table 4: In Vitro Irritation Score

Cornea No.	Test item	Corrected Opacity	Corrected OD490 Value	IVIS
1	Negative Control	0.76	0.015
2		1.27	0.026
3		0.83	0.014
MV		0.95	0.018	1.23
4	Positive Control	97.35	1.957
5		113.82	2.157
6		115.43	2.227
MV		108.87	2.113	140.57
7	Test item	68.68	0.173
8		51.50	0.340
9		76.53	0.249
MV		65.57	0.254	69.38

MV = Mean value

 

Table 5: Historical mean In Vitro Irritation Score of the Positive Control

	IVIS Positive Control
Mean Value (MV)	126.72
Standard Deviation (SD)	17.00
MV – 2xSD	92.72
MV + 2xSD	160.72
Number of Replicates providing Historical Mean: 17

 

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

Based on the mean in vitro irritation score of 69.38 obtained in the bovine corneal opacity and permeability assay (BCOP, OECD 437), the test substance needs to be classified as Eye Dam. 1 (H318) in accordance with Regulation (EC) No 1272/2008 (CLP).

Executive summary:

The eye irritancy potential of 1-Benzyl-3-carbamoyl-pyridinium, chloride was investigated in the bovine corneal opacity and permeability assay (OECD 437). The test item was suspended with physiological saline 0.9% NaCl to give a 20% concentration. All three corneas treated with 1-Benzyl-3-carbamoyl-pyridinium, chloride showed strong opacity of the tissue. The mean in vitro irritation score in this test was 69.38. The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid. The negative control responses should result in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control. Based on the results obtained the test substance needs to be classified as Eye Dam. 1 (H318) in accordance with Regulation (EC) No 1272/2008 (CLP).

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-09-04 to 2017-10-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 491 (Short Time Exposure In Vitro 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:

July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

TEST MATERIAL
- Identification: 1-Benzyl-3-carbamoyl-pyridinium, chloride
- Batch No.: RR757
- CAS No.: 5096-13-9
- Purity: ~ 96% (LC-UV), correction for purity was not made
- Appearance: Solid, white powder
- Expiry Date: 14 October 2017
- Storage Conditions: RT, in the dark (only valid for storage)

Species:

rabbit

Details on test animals or tissues and environmental conditions:

The rabbit corneal cell line SIRC was used for performing the STE test method. SIRCs are growing as confluent monolayers. Large stocks of the SIRC cell line (supplied by ATCC) were stored in liquid nitrogen in the cell bank of Envigo CRS GmbH allowing the repeated use of the same cell culture batch in experiments. Therefore, the parameters of the experiments remain similar, because of the reproducible characteristics of the cells. Thawed stock cultures were propagated at 37 ± 1.5 °C in plastic flasks (e.g. NUNC) containing a culture medium comprising Eagle’s minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine and 100 units/mL penicillin and 100 µg/mL streptomycin. The cells were sub-cultured twice weekly. The cell cultures were incubated at 37 ± 1.5 °C and 5.0 ± 0.5% carbon dioxide atmosphere. Cells were propagated 2 to 3 passages in a culture flask before being employed for testing and did not exceed 25 passages from thawing.

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 200 µL
- Concentration (if solution): 0.5 % (v/v), 0.05% (v/v)

Duration of treatment / exposure:

5 min

Observation period (in vivo):

N.A.

Duration of post- treatment incubation (in vitro):

After exposure, cells were washed twice with 0.2 mL of PBS and 0.2 mL MTT solution (0.5 mg MTT/mL of MEM) was added. After a two-hour reaction time at 37 ± 1.5 °C and 5.0 ± 0.5% carbon dioxide atmosphere the MTT solution was decanted. MTT formazan was extracted by adding 0.2 mL of 0.04 N hydrochloric acid – isopropanol for approximately 60 minutes in the dark at room temperature and the absorbance of MTT formazan solution was measured with a microplate reader

Number of animals or in vitro replicates:

triplicates per treatment group, three independent runs

Details on study design:

CELL CULTURE:
Large stocks of the SIRC cell line (supplied by ATCC) were stored in liquid nitrogen in the cell bank of Envigo CRS GmbH allowing the repeated use of the same cell culture batch in experiments. Therefore, the parameters of the experiments remain similar, because of the reproducible characteristics of the cells. Thawed stock cultures were propagated at 37 ± 1.5 °C in plastic flasks (e.g. NUNC) containing a culture medium comprising Eagle’s minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine and 100 units/mL penicillin and 100 µg/mL streptomycin. The cells were sub-cultured twice weekly. The cell cultures were incubated at 37 ± 1.5 °C and 5.0 ± 0.5% carbon dioxide atmosphere. Cells were propagated 2 to 3 passages in a culture flask before being employed for testing and did not exceed 25 passages from thawing.

SEEDING THE CULTURES:
Exponentially growing stock cultures more than 50% confluent were rinsed with PBS and treated with Trypsin at 37 ± 1.5 °C for 5 minutes. Then the enzymatic digestion was stopped by adding complete medium and a single cell suspension was prepared. Individual wells of a 96-well tissue-culture microtiter plate were inoculated with 0.2 mL complete medium containing approximately 3 x 10^4 cells/mL in case that the cells were seeded four days prior to the treatment and 1.5 x 10^4 cells/mL in case that the cells were seeded 5 days prior to the treatment. The plates were incubated at 37 ± 1.5 °C and 5.0 ± 0.5% carbon dioxide atmosphere for 4 days and 5 days, respectively. Cells should reached a confluence of more than 80% at the time of testing.

TREATMENT:
The test item was tested in three independent runs (with different cell cultures and on different days). On the day of the experiments right before application, the test item was dissolved in 0.9% NaCl to reach a final concentration of 5% (w/w). Following, this solution was diluted by serial 10-fold dilution with the respective solvent to reach final concentrations of 0.5% (v/v) and 0.05% (v/v). The test item was prepared freshly prior to each experiment. For the treatment the complete medium was removed and the cells were re-fed with 200 µL treatment solution containing medium, solvent and positive control as well as the two different concentrations of the test item (5% and 0.05%) and the medium blank, respectively. All dose groups were tested 3 times. The exposure period was 5 minutes at room temperature.

CELL VIABILITY MEASUREMENT:
After exposure, cells were washed twice with 0.2 mL of PBS and 0.2 mL MTT solution (0.5 mg MTT/mL of MEM) was added. After a two-hour reaction time at 37 ± 1.5 °C and 5.0 ± 0.5% carbon dioxide atmosphere the MTT solution was decanted. MTT formazan was extracted by adding 0.2 mL of 0.04 N hydrochloric acid – isopropanol for approximately 60 minutes in the dark at room temperature and the absorbance of MTT formazan solution was measured with a microplate reader (Versamax® Molecular Devices) at 570 nm (without a reference). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).

DATA EVALUATION:
The optical density (OD) value obtained from the test item was used to calculate cell viability relative to the solvent control, which is set at 100%. The relative cell viability is expressed as a percentage and obtained by dividing the OD of the test item by the OD of the solvent control after subtracting the OD of blank from both values. Similarly, the relative cell viability of each solvent control is expressed as a percentage and obtained by dividing the OD of each solvent control by the OD of the medium control after
subtracting the OD of blank from both values. The arithmetic mean of the three wells of the test item and solvent control in each independent repetition was used to calculate the arithmetic mean of relative cell viability. The final arithmetic mean of the cell viability was calculated from the three independent runs.

Irritation parameter:

other: Relative cell viability [%]

Run / experiment:

5% test item, mean of three experiments

Value:

42.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: Relative cell viability [%]

Run / experiment:

0.05% test item, mean of three experiments

Value:

94.9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Table 2: Summary of the results

Test Group		Cell Viability [%] per Test			Standard Deviation [%]		Mean Cell Viability [%]		Predicted Eye Irritation Potential
		Test 1	Test 2	Test 3
Medium control		103.9	96.3	110.9	7.3		103.7		No category
Solvent control		100.0	100.0	100.0	-		100.0		No category
Positive control		29.6	33.2	24.8	4.3		29.2		Category 1
Test item, 5%		48.2	38.3	41.9	5.0		42.8		No prediction can be made
Test item, 0.05%		88.5	94.8	101.5	6.5		94.9
Solvent control		96.2	104.0	90.2

Interpretation of results:

other: no prediction can be made

Conclusions:

In conclusion, no prediction can be made for the eye irritation potential of 1-Benzyl-3-carbamoyl-pyridinium, chloride under the experimental conditions reported.

Executive summary:

The eye irritancy potential of 1-Benzyl-3-carbamoyl-pyridinium, chloride (purity 96 %) was investigated in the Short Time Exposure in vitro test method for identifying eye irritation (OECD 491). The test item was diluted with physiological saline 0.9% NaCl to give a 5 and 0.05 % concentration. The mean relative cell viabilities were 42.8% (5 %) and 94.9% (0.05 %). Based on the results obtained, no prediction can be made regarding the classification of the substance.

Reference 3

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-05-31 to 2017-10-19

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)

Specific details on test material used for the study:

TEST MATERIAL
- Name: 1-Benzyl-3-carbamoyl-pyridinium, chloride
- CAS No.: 5096-13-9
- Batch: RR757
- Purity: ~96%
- Appearance: white powder
- Expiry Date: 2017-10-14
- Storage Conditions: RT, in the dark

Species:

chicken

Details on test animals or tissues and environmental conditions:

Multiple chicken heads (Spring chickens (Gallus Gallus e.g. Ross 308 Broiler)), were supplied by Baileys Turkeys Ltd., Cheshire, UK. The chickens weighed approximately 3 kg and were approximately 56 days old prior to being humanely killed for human consumption. Heads were removed immediately after the chickens had been humanely killed at the source, for use on the same day. The time interval between collection of chicken heads and placing the eyes in the superfusion chamber following enucleation was minimized although all eyes had to fall within the acceptance criteria identified in the test guideline. Following slaughter, the intact chicken heads were placed into individual plastic compartments within a plastic box in order to minimize any damage to the eyes. The base of each compartment was lined with a paper towel moistened with isotonic saline. The heads were transported to the test facility at ambient temperature.

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): 0.03 g applied to cornea

POSITIVE CONTROL
- Amount: 0.03 g

NEGATIVE CONTROL
- Amount: 0.03 mL

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

Treated corneas were evaluated prior to treatment and at 30, 75, 120, 180 and 240 minutes (± 5 minutes) after the eyes had been decontaminated with the isotonic saline.

Number of animals or in vitro replicates:

test item & positive control: three eyes
negative control: two eyes

Details on study design:

SELECTION AND PREPARATION OF CORNEAS + QUALITY CHECK OF THE ISOLATED CORNEAS
Acceptable eyes were dissected from the skull and pulled from the orbit by holding the nictitating membrane firmly with surgical forceps. The tissue behind the eye was carefully removed with bent, blunt-tipped scissors. Once the eye was removed from the orbit a portion of the optic nerve remained. Other connective tissue was removed from the eye on an absorbent tray liner. Enucleated eyes were transferred to an appropriate clamp keeping the cornea vertical. They were then transferred to chambers within the superfusion apparatus ensuring the corneas received sufficient isotonic saline from the saline drip. The temperature of the chambers was at 32.0 ± 1.5 °C. Once all eyes were placed in the superfusion apparatus, the eyes were examined again with the Haag-Streit BQ 900 to ensure the eyes had not been damaged by the dissection procedure. Corneal thickness measurements are taken with a depth measuring device no. 1 on the Haag-Streit BQ 900 slit-lamp microscope at the center of each cornea. Eyes were replaced when: (i) the fluorescein score was > 0.5; (ii) the corneal opacity score was > 0.5; or (iii) there was any additional signs of damage, (iv) the corneal thickness measurements for individual eyes deviated more than 10% from the mean value for all eyes. After the approval process the eyes were incubated for 45 minutes for equilibrium purposes. Time zero measurements for corneal thickness and opacity were taken to serve as a baseline. The baseline for the fluorescein measurements were taken at dissection.

NUMBER OF REPLICATES: test item & positive control: three eyes; negative control: two eyes

NEGATIVE CONTROL USED: yes, sodium chloride 0.9 % w/v
- Batch No.: 3012488
- Purity: 0.9 %
- Appearance: clear colorless liquid
- Expiry date: 2018-11-01
- Storage conditions: RT

POSITIVE CONTROL: yes, imidazole
- Batch No.: 163571
- Purity: ≥99 %
- Appearance: white solid
- Expiry date: 2019-05-01
- Storage conditions: RT

TREATMENT METHOD
Immediately following the zero reference measurements, each test eye (including clamp) was removed from the superfusion apparatus and placed horizontally (cornea facing upwards) into a petri dish.

APPLICATION DOSE AND EXPOSURE TIME
0.03 g of test item was applied to the cornea. The entire surface of the cornea was evenly covered. The test item remained in place for 10 seconds and was then rinsed from the eye using 20 mL of isotonic saline. The treated eye (including clamp) was subsequently returned to the superfusion apparatus in the original upright position.
0.03 g of the positive control item, Imidazole, was similarly applied to the cornea of each positive control eye and 0.03 mL of the negative control item was applied to the cornea of each negative control eye.

REMOVAL OF TEST SUBSTANCE
- yes, after 10 seconds the eyes were rinsed with 20 mL isotonic saline

POST-EXPOSURE INCUBATION
- Treated corneas were evaluated prior to treatment and at 30, 75, 120, 180 and 240 minutes (± 5 minutes) after the eyes had been decontaminated with the isotonic saline. After final examination, eyes were preserved in neutral buffered formalin for possible histopathology.

MEASUREMENT of ENDPOINTS:
Endpoints used during the evaluation procedure were corneal opacity, swelling, fluorescein retention and morphological effects (e.g. pitting, sloughing or roughening of the epithelium). All of the endpoints, with the exception of fluorescein retention (which is only determined at 30 minutes after test substance exposure) were determined at each of the above time points. After the final examinations at the 240 minute time point, the eyes were preserved in neutral buffered formalin for possible histopathology. Eyes were retained until finalization of the final report. Percentage corneal swelling was assessed from corneal thickness measurements. The calculation was expressed in the following formula: ((corneal thickness at time (t) - corneal thickness at time (0)) / corneal thickness at time (0)) x 100.
Mean percentage of corneal swelling for all test eyes was calculated for all the time points. The overall category score was determined from the highest mean score for epithelial swelling as observed at any time point. Corneal opacity was calculated with the most densely opacified areas for scoring. The mean value for all test eyes was calculated for all time points. The highest mean score, as observed at any time point was given an overall category for each test item. The mean fluorescein retention scores for all test eyes are calculated at the 30 minute time interval only. These measurements are used for the overall classification for each test item. Pitting, sloughing, roughening of the corneal surface, and adhering of test item are all
morphological effects that can be noted on the cornea. The classification of these findings was subject to interpretation.

DATA EVALUATION:
Once each endpoint had been established, ICE classes were determined based on a predetermined range. Interpretation of the endpoints was classified as described in OECD 438 testing guideline. A test was considered acceptable if the concurrent negative or vehicle/solvent items and the concurrent positive controls were identified as GHS Non-Classified and GHS Category 1, respectively.

Irritation parameter:

fluorescein retention score

Run / experiment:

maximal mean score

Value:

0.3

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class I

Irritation parameter:

percent corneal swelling

Run / experiment:

75 minutes

Value:

15.32

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class III

Irritation parameter:

cornea opacity score

Run / experiment:

maximal mean score

Value:

2

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class III

Table 1: Ocular reactions

Test item
Maximal mean score for corneal opacity	:	2.0	ICE Class III
Mean score of fluorescein retention	:	0.3	ICE Class I
Maximal mean corneal swelling compared to time zero	:	15.32 %	ICE Class III
Positive control item
Maximal mean score for corneal opacity	:	4.0	ICE Class IV
Mean score of fluorescein retention	:	3.0	ICE Class IV
Maximal mean corneal swelling compared to time zero	:	45.87 %	ICE Class IV
Negative control item
Maximal mean score for corneal opacity	:	0.5	ICE Class I
Mean score of fluorescein retention	:	0.3	ICE Class I
Maximal mean corneal swelling compared to time zero	:	2.78 %	ICE Class I

Corneal opacity scores

Easily discernible translucent area; details of the iris are slightly obscured of the cornea was noted in all test item treated eyes. Complete corneal opacity; iris invisible was noted in all positive control treated eyes. Very faint opacity was noted in the negative control treated eyes. No morphological effects were noted in the test item or negative control item treated eyes. Sloughing was noted in two positive control treated eyes.

Fluorescein Retention Scores

Very minor single cell staining was noted in two of the test item treated eyes. Confluent large areas of the cornea retaining fluorescein were noted in all positive control treated eyes. Very minor single cell staining was noted in one of the negative control treated eyes.

Interpretation of results:

other: no prediction can be made

Conclusions:

No prediction can be made following assessment of the data for all endpoints.

Executive summary:

The eye irritancy potential of 1-Benzyl-3-carbamoyl-pyridinium, chloride (96% purity) was investigated in the Isolated Chicken Eye Test (OECD 438) in vitro. The chicken eyes were treated for 10 seconds with the test substance. Afterwards, the eyes were rinsed with physiological saline. Irritation scores were taken at 0, 30, 75, 120, 180 and 240 minutes after rinsing. Based on the results obtained, no prediction can be made for classification of the test substance.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The potential of 1-Benzyl-3-carbamoyl-pyridinium, chloride to induce skin corrosion was tested in accordance with OECD Guideline 431. The test item was applied topically to the EPISKIN-SM™ tissue for 3 min, 60 min and 4 h followed by immediate determination of cytotoxic effects via MTT reduction assay. The relative mean tissue viability after 4 h treatment was ≥35% (106%). Relative mean tissue viability was reduced to 88% after 60 min treatment and to 94% after 3 min treatment. The test item is therefore classified as non-corrosive. Thus, the test item had to be tested for its potential for skin irritation. This test was conducted in accordance with OECD 439 using a reconstructed human epidermis with a functional stratum corneum. The test item was applied topically for 15 min followed by a 42 h post-incubation period and immediate determination of cytotoxic effects via MTT reduction assay. The test item showed no irritant effects as the mean relative tissue viability (% negative control) was > 50% (99.9%) after 15 min treatment and 42 h post-incubation.

The eye irritancy potential of 1-Benzyl-3-carbamoyl-pyridinium, chloride was assessed in a weight-of-evidence approach by using data from three in vitro eye irritation studies. 1-Benzyl-3-carbamoyl-pyridinium, chloride is considered to induce serious eye damage in the bovine corneal opacity and permeability assay (OECD 437). In two further in vitro eye irritation tests conducted in accordance with OECD test guidelines 438 and 491 contradictory results were obtained. In both cases, the conditions for classification for serious eye damage were not fulfilled. But, also the conditions to justify a non-classification were not fulfilled. Based on the available data and assessing them in a weight-of-evidence approach, classification for serious eye irritation (Eye Irrit. 2, H319) in accordance with the CLP regulation is warranted.

Justification for classification or non-classification

Based on the available data and assessing them in a weight-of-evidence approach, classification for serious eye irritation (Eye Irrit. 2, H319) in accordance with the CLP regulation is warranted. No classification for skin irritation is warranted based on the negative OECD 439 test result.