4-[2-[4-[benzylmethyl(ethyl)amino]phenyl]vinyl]-1-(2-hydroxyethyl)pyridinium acetate

Endpoint summary

Description of key information

Six male rabbits were dermally exposed to the substance for 24 hours under occlusion. Erythema could not be scored due to staining of the skin. Based on the oedema scores the substance is considered to be irritating to the skin (Bayer 1980).

In a test which is similar to the OECD guidance, the substance as tested (chloride) is considered not irritating to the skin of 6 rabbits exposed (under occlusion) during 24 hours ( Laboratorium für Pharmakolgie und Toxikologie)

The substance was tested in the EpiDerm™ Human Skin Model using duplicate tissues during 3 and 60 minutes.

Additional controls were included for the observed color interference. The relative mean corrected viability of the test substance treated tissues was 50.7 % for the 3 minutes exposure and 24.2 % for the 60 minutes exposure. Therefore it can be concluded that the substance is not corrosive to the skin (Eurofins 2017).

In a test, similar to OECD 405, on 6 male rabbits, the substance was found to be severely irritating to the eyes. Based on the scores for opacity, and iritis, the substance needs to be classified as eye damaging category 1 (Bayer 1980).

The eye irritancy potential of the substance was investigated in the bovine corneal opacity and permeability assay (Eurofins 2017). All 3 corneas treated with the substance showed strong reddish discolouration of the tissue. The mean in vitro irritation score was calculated: 141.76. Therefore the substance needs to be classified as a category 1 substance.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: 16 CFR 1500.41 - Method of testing primary irritant substances.

Principles of method if other than guideline:

The exposure period was 24 hours.
A test on the abraded skin was included in the study design, but the results are not included in this summary

GLP compliance:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Lippischen Versuchstierzucht Extertal
- Weight at study initiation: average 2.1 kg
- Housing: individual
- Diet: "Mümmel z" (SNIFF Soest Germany) ad libitum
- Water: ad libitum
- Acclimation period: 7-14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16-18 °C
- Humidity (%): 40%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

occlusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

0.5 mL

Duration of treatment / exposure:

24 hours

Observation period:

8 days (observations after 30 min (=24 h), 48 and 72 hours and after 8 days)

Number of animals:

6 males

Details on study design:

TEST SITE
- Area of exposure: 2.5 x 2.5 cm
- Type of wrap if used: occulsive (chirugical gaze covered with PVC foil kept in place with elastic bandage (Leukoflex and Eloflex)

REMOVAL OF TEST SUBSTANCE
- Washing (if done): none

OBSERVATION TIME POINTS: 24, 48 and 72 hours and on day 8

SCORING SYSTEM:
- Method of calculation: similar to Draize

Irritation parameter:

erythema score

Basis:

mean

Time point:

24/48/72 h

Remarks on result:

not determinable

Remarks:

due to staining of the test site

Irritation parameter:

edema score

Basis:

mean

Time point:

24/48/72 h

Score:

>= 1.83 - <= 2.17

Max. score:

3

Reversibility:

not fully reversible within: 8 days

Remarks on result:

probability of weak irritation

Irritant / corrosive response data:

Indication of irritation which was reversible within 8 days in 2/6 rabbits
The averga oedema score in 2/6 rabbits was > 2.3 (see table)

Other effects:

no other effects reported

Animal no	1				2				3
	24	48	72	8	24	48	72	8	24	48	72	8
erythema	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA
oedema	2		2	2	2		1	0	2		2	1
Animal no	4				5				6
	24	48	72	8	24	48	72	8	24	48	72	8
erythema	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA
oedema	3		2	2	3		3	2	1		1	0

 

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

The substance is irritating to the skin

Executive summary:

Six male rabbits were dermally exposed to the substance for 24 hours under occlusion. Erythema could not be scored due to staining of the skin. Based on the oedema scores the substance is considered to be 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:

13 June 2017 to 27 July 2017

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:

2016

Qualifier:

according to guideline

Guideline:

other: Method B.40bis of Commission Regulation (EC) No 440/2008, of 30 May 2008,

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

other: EpiDerm™ Human Skin Mode

Cell source:

other: EpiDerm™ Human Skin Mode

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Human Skin Mode (MatTek)
- Tissue batch number(s):25822 and 25823
- Delivery date: 21 June 2017 and 26 July 2017
- Temperature used during treatment / exposure: 37 °C, 5% CO2
- Temperature of post-treatment incubation (if applicable): 37°C, 5% CO2

REMOVAL OF TEST MATERIAL AND CONTROLS: rinsed with PBS and blotted dry

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL MTT
- Incubation time: 1 h at 37 °C, 5% CO2
- Spectrophotometer: plate spectrophotometer using isopropanol as a blank.
- wavelength : 570 nm

TISSUES:
- Fresh tissues: for treatment, negative control and positive control
- N. of replicates : 2/treatment
- Method of calculation used:
TMT/UMT
TMT= test item treated viable
UMT = negative control viable

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2 experiments

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50% (H314 1A), or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15% (H314 1B or 1C)
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15% (not classified as corrosive)
:

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL as such

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 50 µL distilled water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL 8.0 N Potassium Hydroxide

Duration of treatment / exposure:

3 min and 60 min

Duration of post-treatment incubation (if applicable):

The plates was incubated (37 °C, 5% CO2) for 3 hours in presence of MTT. Thereafter tissues were placed in isopropanol for MTT extraction, which was measured in triplicate samples as optical density at 570 nm.

Number of replicates:

2/treatment

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

A 3 minutes exposure

Value:

50.7

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: colour corrected value

Remarks:

corrosive

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

B 60 minutes exposure

Value:

43

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: colour corrected value

Other effects / acceptance of results:

The results were in agreement with the quality criteria:

Negative Control
The absolute OD570 of the negative control treated tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. The mean OD570 of the two negative control tissues should be ≥ 0.8 and ≤ 2.8 for each exposure time, which ensures that the tissue viability meets the acceptance criteria.
Positive Control
Potassium Hydroxide 8.0N solution is used as a positive control. An assay meets the acceptance criterion if mean relative tissue viability of the 60 minute positive control is < 15%.
Coefficient of Variation
In the range 20 and 100% viability, the Coefficient of Variation between tissue replicates should be ≤ 30%.

 

Results of 3 min

Name	Negative Control	Test Item	Positive Control
Total Mean OD570of 2 Replicate Tissues (Blank Corrected)	1.238^*	0.984	0.184
SD OD570 of 2 Replicate Tissues	0.049	0.162	0.050
TODTT	-	0.628	-
Mean Relative Tissue Viability [%]	100.0	79.5	14.8
Coefficient Of Variation [%]^***	4.0	16.5	27.3
NSCliving– Corrected Mean Relative Tissue Viability [%]	-	50.7	-

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

^***           coefficient of variation (CV) (in the range of 20 – 100% viability) between two tissues treated identically is 30%.

 

Results of 60 min

Name	Negative Control	Test Item	Positive Control
Total Mean OD570of 2 Replicate Tissues (Blank Corrected)	1.376^*	0.592	0.120
SD OD570 of 2 Replicate Tissues	0.003	0.136	0.013
TODTT	-	0.333	-
Mean Relative Tissue Viability [%]	100.0	43.0	8.7^**
Coefficient Of Variation [%]^***	0.2	23.0	10.9
NSCliving– Corrected Mean Relative Tissue Viability [%]	-	24.2	-

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

^**            mean relative tissue viability of the 60 min positive control < 15%,

^***           coefficient of variation (CV) (in the range of 20 – 100% viability) between two tissues treated identically is 30%.

 

 

Interpretation of results:

other: non-corrosive

Conclusions:

The substance is considered to be not corrosive

Executive summary:

The substance was tested in the EpiDerm™ Human Skin Model using duplicate tissues during 3 and 60 minutes. Additional controls were included for the observed color interference. The relative mean corrected viability of the test substance treated tissues was 50.7 % for the 3 minutes exposure and 24.2 % for the 60 minutes exposure. Therefore it can be concluded that the substance is not corrosive to the skin.

Reference 3

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

January 1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: Federal Register: 38 Fed. Reg. 26885 (Sept. 27, 1973).

Principles of method if other than guideline:

The test substance was applied to the intact and abraded skin of 6 rabbits. Only the results on the intact skin are reported in this summary.

GLP compliance:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: no data
- Weight at study initiation: 2.3-2.8 kg
- Housing: individually in V2A steel cages (0.4 m2)
- Diet/water: no data
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 0.5°C
- Humidity (%): 55 ± 5%
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

occlusive

Preparation of test site:

shaved

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 mL

Duration of treatment / exposure:

24 h

Observation period:

72 h

Number of animals:

3 males + 3 females

Details on study design:

TEST SITE
- Area of exposure: 2.5x2.5 cm
- Type of wrap if used: linen patch (covered with plastic foil and elastic bandage (Leukoplast, Beiersdorf)

REMOVAL OF TEST SUBSTANCE
- Washing (if done): none

OBSERVATION TIME POINTS: 24, 48 and 72 hours

SCORING SYSTEM: according to Draize

Irritation parameter:

erythema score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

fully reversible

Irritation parameter:

edema score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

fully reversible

Irritant / corrosive response data:

No signs of irritation

Other effects:

no other effects noted

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results of the test the substance is considered not irritant to the skin

Executive summary:

In a test which is similar to the OECD guidance, the substance as tested is considered not irritating to the skin of 6 rabbits exposed (under occlusion) during 24 hours.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (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:

key study

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)

GLP compliance:

yes (incl. QA statement)

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: from animals freshly slaughtered at the abattoir A. Moksel AG, Buchloe, Germany (no details)
- Storage, temperature and transport conditions of ocular tissue: transported in HBSS containing Pen/Strep on ice, stored for < 30 min in HBBS
- Time interval prior to initiating testing: incubated in corneal holders in RPMI (without phenol red) containing 1% FBS and 2 mM L-glutamine for 1 hour at 32 ± 1 °C
- indication of any existing defects or lesions in ocular tissue samples: any corneas with defects were discarded
- Indication of any antibiotics used: yes see above

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

750 uL in the anterior chamber of the holder (applied directly onto the cornea due to high viscosity)

Duration of treatment / exposure:

10 min at 32 ± 1 °C

Duration of post- treatment incubation (in vitro):

After 10 minutes incubation either the 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 after 2 hours incubation at 32 ± 1 °C .

Number of animals or in vitro replicates:

3 for test substance, 3 for negative controls and 3 for positive controls

Details on study design:

SELECTION AND PREPARATION OF CORNEAS: from slaughter house animals
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 for <30 min. Before the corneas were mounted in corneal holders 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.

QUALITY CHECK OF THE ISOLATED CORNEAS: at arrival in the laboratory, before use and after exposure visually inspection for defects

NUMBER OF REPLICATES: 3/group

NEGATIVE CONTROL USED: physiological saline

POSITIVE CONTROL USED: ethanol

APPLICATION DOSE AND EXPOSURE TIME: 750 uL during 10 min at 32 ± 1 °C

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: yes 2 hours at 32 ± 1 °C

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: washed at least three times with MEM (containing phenol red). The washing medium was thereafter free of coloring, but the corneas showed a strong reddish discoloration..
- POST-EXPOSURE INCUBATION: 2 hours at 32 ± 1 °C

METHODS FOR MEASURED ENDPOINTS:
Before exposure: An initial measurement was performed on each of the corneas using an 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.

After exposure: 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 after 2 hours incubation at 32 ± 1 °C. Also, each cornea was observed visually and pertinent observations were recorded.
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 4 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 at 490 nm (OD490) was determined, using a spectrophotometer

- Corneal opacity: BASF opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured at 490 nm (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: according to the guidance
IVIS UN GHS
≤ 3 No Category
> 3; ≤ 55 No prediction can be made
> 55 Category 1

Irritation parameter:

in vitro irritation score

Value:

141.46

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

the tissue treated with the substance showed strong reddish discolouration and therefore it cannot be excluded that these have interfered with the opacity readings

In Vitro Irritation Score

Group	Cornea no.	Corrected opacity	Corrected OD490	IVIS
Negative control	1	-0.25	0.004
	2	0.80	0.005
	3	1.17	0.008
	Mean value	0.57	0.006	0.66
Positive control	1	23.92	1.436
	2	31.18	1.102
	3	29.69	1.749
	Mean value	28.26	1.429	49.70
Test substance	1	153.43	0.344
	2	127.34	0.369
	3	129.19	0.308
	Mean value	136.65	0.341	141.76

 

historical controls

	IVIS positive control ethanol
Mean value	48.40
SD	9.98
Mean value + 2*SD	28.44
Mean value – 2*SD	68.37
Based on 45 replicates

 

 

Interpretation of results:

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

Conclusions:

Based on the results of the test the substance is considered to be severely irritant to the eyes

Executive summary:

The eye irritancy potential of the substance was investigated in the bovine corneal opacity and permeability assay.

All 3 corneas treated with the substanceshowed strong reddish discolouration of the tissue.

The following mean in vitro irritation score was calculated: 141.76

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

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Qualifier:

according to guideline

Guideline:

other: 16 CFR 1500.42 - Test for eye irritants.

GLP compliance:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Lippischen Versuchstierzucht Extertal 1
- Weight at study initiation: average 2.1 kg
- Housing: individual
- Diet: "Mümmel z" (SNIFF Soest Germany) ad libitum
- Water: ad libitum
- Acclimation period: 7-14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16-18 °C
- Humidity (%): 40%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

100 uL

Observation period (in vivo):

8 days

Number of animals or in vitro replicates:

6 male rabbits

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): none

SCORING SYSTEM: similar to Draize

TOOL USED TO ASSESS SCORE: ophthalmoscope (fluorescein on day 8)

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

3.33

Max. score:

4

Reversibility:

not reversible

Remarks on result:

probability of severe irritation

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

1.83

Max. score:

2

Reversibility:

not reversible

Remarks on result:

probability of severe irritation

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

mean

Time point:

24/48/72 h

Score:

1.17

Max. score:

2

Reversibility:

not reversible

Remarks on result:

other: at the 24 hour observation scoring was not posible due to discoloration due to the substance

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Score:

4

Max. score:

4

Reversibility:

not reversible

Remarks on result:

probability of severe irritation

Irritation parameter:

conjunctivae score

Remarks:

discharge

Basis:

mean

Time point:

24/48/72 h

Score:

2.5

Max. score:

3

Reversibility:

not reversible

Irritant / corrosive response data:

Indication of severe eye damage
The scores are based on 6 rabbits

Animal no	1				2				3
	24	48	72	8	24	48	72	8	24	48	72	8
cornea	4	4	4	4	3	3	4	4	3	3	4	4
iris	2	2	1	2	2	1	1	2	2	2	2	2
conjunctivae
redness	NA	1	1	1	NA	1	1	3	NA	1	1	1
chemosis	4	4	4	4	4	4	4	4	4	4	4	4
discharge	2	3	3	3	2	3	3	3	2	3	3	3
Animal no	4				5				6
	24	48	72	8	24	48	72	8	24	48	72	8
cornea	4	4	4	4	2	3	3	3	2	3	3	3
iris	2	2	2	2	2	2	2	2	2	2	2	2
conjunctivae
redness	NA	1	1	1	NA	2	2	3	NA	1	1	1
chemosis	4	4	4	4	4	4	4	3	4	4	4	4
discharge	2	3	3	2	2	2	2	2	2	2	2	2

Interpretation of results:

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

Conclusions:

The substance formulation is consideed to cause severe eye damage

Executive summary:

In a test on 6 male rabbits similar to OECD 405, the substance was found to be severely irritating to the eyes. Based on the scores for opacity, and iritis, the substance needs to be classified as eye damaging category 1.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The in vivo skin irritation study with the substance is considered to represent a worst case (24 hours exposure under occlusion). In a similar in vivo study with the chloride, no skin irritation was seen. The amount of acetic acid present in the substance could give rise to classification as skin irritant 2. Therefore in view of the inconsistent study results and the presence of acetic acid, the substance will be classified for skin irritation as a precautionary measure.

Justification for classification or non-classification

Based on the available information, the substance needs to be classified for as H315: causes skin irritation (category 2) and H318: Causes serious eye damage (category 1) according to EC Regulation No 1272/2008 (CLP).