sodium;2-chloroprop-2-enoate;hydrate

Endpoint summary

Description of key information

Based on the results obtained with an OECD 431 compliant study the test item is considered as non-corrosive as the mean percentage tissue viability was greater than 50% after 3 minutes exposure and greater than 15% after 1 hour exposure of the negative control.

Based on the results obtained with an OECD 439 compliant study the test item is considered as not irritating to the skin. In conclusion the test item is not classified as skin irritating.

Under the conditions of an OECD 438 compliant study, the test item is considered to not require classification as eye irritant or eye damaging.

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:

weight of evidence

Study period:

2018-11-14

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:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

artificial membrane barrier model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: MatTek In Vitro Life Science Laboratories, s.r.o, MlynskéNivy 73, 821 05, Bratislava II, Slovak Republic

Justification for test system used:

As recommended in OECD Guideline No. 439, Reconstructed Human Epidermal Model EpiDerm™ (EPI-200-SCT) has been selected as test system for in vitro skin corrosion.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epidermal Model EpiDerm™ (EPI-200-SCT)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: rinsing with sterile PBS, fill and empty insert 20 times in a constant soft stream of (1xPBS)
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h +/- 15 min
- Wavelength: 570 or 540 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: approved
- Barrier function: approved
- Morphology: well
- Contamination: none

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
not applicable

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, 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%.
- 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%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

50 µL

Duration of post-treatment incubation (if applicable):

not applicable

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

test item

Value:

87

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
- Direct-MTT reduction: no
- Colour interference with MTT: 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.: 392.554.2938) using the ten Proficiency Chemicals according to OECD Test Guideline No. 439.

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 1 results of optical density (OD) measured at 570 nm of each replicate and the calculated % viability of the cells

Substance	Optical Density (OD)		Viability (%)
Negative Control 1x PBS	1	0.605	98
	2	0.601	98
	3	0.640	104
	Mean	0.616	100
	Standard deviation (SD)		3.48
Positive Control SDS (5% aq)	1	0.062	10
	2	0.056	9
	3	0.092	15
	Mean	0.070	11
	Standard deviation (SD)		3.15
Test item	1	0.590	96
	2	0.477	77
	3	0.532	86
	Mean	0.533	87
	Standard deviation (SD)		9.16

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results of this study the substance is not considered to be skin irritating.

Executive summary:

In this in vitro skin irritation test using the EPISKIN model, the test item 2-Chloroacrylic sodium salt did not show significantly reduced cell viability in comparison to the negative control (mean viability: 87 %). All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin.

Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered 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 2-Chloroacrylic sodium salt is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category).

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018-04-05 to 2018-05-23

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

artificial membrane barrier model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: MatTek In Vitro Life Science Laboratories, s.r.o, MlynskéNivy 73, 821 05, Bratislava II, Slovak Republic

Justification for test system used:

As recommended in OECD Guideline No. 431, Reconstructed Human Epidermal Model EpiDerm™ (EPI-200-SCT) has been selected as test system for in vitro skin corrosion.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epidermal Model EpiDerm™ (EPI-200-SCT)
- Tissue batch number(s): 25892

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: rinsing with sterile PBS, fill and empty insert 20 times in a constant soft stream of (1xPBS)
- Observable damage in the tissue due to washing: none
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h +/- 15 min
- Wavelength: 570 or 540 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: approved
- Barrier function: approved
- Morphology: well
- Contamination: none

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues : not applicable
- Procedure used to prepare the killed tissues: not applicable

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, 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%.
- 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%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

50 µL

Duration of treatment / exposure:

3 min and 1 h

Duration of post-treatment incubation (if applicable):

not applicable

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

test item - 3 min

Value:

98.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

test item - 60 min

Value:

80.4

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
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Prior to routine use the technical proficiency of the test method was established by using proficiency chemicals under Bioneeds Study No.: BIO-GT 1000, according to OECD Test Guideline No. 431.

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 1.           Summary of Optical Density (OD) and viability (%)

 

3 Minutes Exposure                                                                                        Refer Appendix - 1

Treatment		OD	Viability (%)	Classification
Negative Control (Sterile water)	Mean	1.729	100.0	NC
	±SD	0.018	1.4
	n	2	2
Positive Control (Glacial acetic acid)	Mean	0.030	1.7	C (Category 1A)
	±SD	0.000	0.0
	n	2	2
Test Item (2-Chloroacrylic Acid Sodium Salt)	Mean	1.704	98.6	NC
	±SD	0.040	3.2
	n	2	2

                                           

 

1 Hour Exposure

Treatment		OD	Viability (%)	Classification
Negative Control (Sterile water)	Mean	1.738	100.00	NC
	±SD	0.020	1.6
	n	2	2
Positive Control (Glacial acetic acid)	Mean	0.030	1.7	C (Category 1A)
	±SD	0.001	0.1
	n	2	2
Test Item (2-Chloroacrylic Acid Sodium Salt)	Mean	1.397	80.4	NC
	±SD	0.201	16.3
	n	2	2

NC = Non Corrosive; C = Corrosive; n = No. of tissues; SD = Standard Deviation.

Interpretation of results:

other: not Categroy 1 (GHS)

Conclusions:

Based on the results obtained under the conditions of this study the test item is considered as non-corrosive in accordance with UN GHS, as the mean percentage tissue viability was greater than 50% after 3 minutes exposure and greater than 15% after 1 hour exposure of the negative control. However, this test system does not allow discrimination between category 2 of skin irritation and no classification according to CLP.

Executive summary:

The objective of this study was to evaluate the in vitro skin corrosion potential of the test item by measurement of tissue viability in the Epidermal Model - Epiderm™ (EPI-200-SCT) as per OECD Guideline for the testing of chemicals No. 431, “In vitro skin corrosion: reconstructed human epidermis (RHE) test method”, adopted on 29th July 2016. The test item did not develop any colour when dissolved in distilled water/isopropanol and was considered as non-reducer of MTT as no purple colour was developed when mixed and incubated with MTT solution.

After receipt of the tissues, visual inspection was done to verify the defects. There were no tissue defects, air bubble or excess moisture observed. All tissue inserts were used for the study. Tissue inserts were transferred to upper row of 6 well plates prefilled with 0.9 mL of assay medium and incubated in CO2 incubator for 60 minutes. Exposure to the test item was performed for 1 hour and 3 minutes separately. All the treatments were maintained in duplicates. For 3 minutes treatment, quantity of 50 µL of sterile distilled water (NC) was dispensed into the first insert atop the tissue. After 60 seconds the procedure was repeated with second tissue and continued for all other tissues. Similar procedure was followed in the same manner until all the tissues were treated. Tissues were treated with 50 µL of test item or 50 µL of positive control (glacial acetic acid). For 1 hour treatment, quantity 50 µL of test item, 50 µL of negative control or50 µL of positive control were dispensed directly atop Epiderm™ tissues at 1 minute intervals to facilitate rinsing after exposure. The tissues were incubated at standard culture conditions for 1 hour.

At the end of treatment time tissue inserts were rinsed with sterile PBS (fill and empty insert 20 times in a constant soft stream of 1xPBS) to remove any residual test item. Post rinsing procedure, each insert was removed from the 6-well plate and gently blotted on absorbent material. The tissues were placed into 24-well plate containing 0.3 mL of MTT solution (1 mg/mL) and incubated for 3 hours at 37±1°C and 5±1% CO2. Post incubation, the tissue inserts were removed and blotted onto the tissue paper and transferred to a pre-labelled 24-well plate containing 2.0 mL of isopropanol in each designated well. The plates were placed on an orbital plate shaker and shaken (̴ 120 rpm/minute) for 5 hours (for 1 hour exposure) and 4 hours and 41 minutes (for 3 minutes exposure) at room temperature. At the end of the extraction period, the tissue was pierced with an injection needle and the extract was allowed to run into the well from which the insert was taken. The punctured inserts were discarded and solution was placed on mixer for 15 minutes until it became homogenous. The optical density of the extracted formazan was measured in 96-well plate spectrophotometer at 570 nm. Viability of tissues was calculated.

 

For 3 minutes exposure, percentage viability of negative control, positive control and test item was 100±1.4, 1.7±0.0 and 98.6±3.2, respectively. The percentage viability of the test item was thus greater than 50% of the negative control after 3 min exposure. Further, the percentage viability of the positive control (PC) was less than 50% of the negative control and thus clearly represents the irritation potential of the positive control. For 1 hour exposure, percentage viability of negative control, positive control and test item was 100±1.6, 1.7±0.1 and 80.4±16.3, respectively. The percentage viability of the test item was thus greater than 15% of negative control after 1 h exposure. The percentage viability of the positive control (PC) was less than 50% of the negative control and thus clearly represents the irritation potential of the positive control.

 

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:

2018-04-13 to 2018-05-28

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)

Version / remarks:

2017

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Version / remarks:

2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

chicken

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: local slaughterhouse
- Storage, temperature and transport conditions of ocular tissue: The intact heads are transported from the slaughterhouse at ambient temperature (typically between 18°C and 25°C) in plastic boxes humidified with tissues moistened with isotonic saline. The procedure involving the collection of chicken heads and placing the eyes in the superfusion chamber following enucleation will be completed within two hours to minimize deterioration and/or bacterial contamination.
- Time interval prior to initiating testing: maximally 2 hours
- indication of any existing defects or lesions in ocular tissue samples: eyes applied in the test were unremarkeble
- Indication of any antibiotics used: no

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 30 µL

Duration of treatment / exposure:

10 seconds

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES :
Upon receipt of the chicken heads to the laboratory, first the eyelids were carefully excised, taking care not to damage the cornea. Corneal intergrity was asssessed with a drop of 2% (w/v) sodium fluorescein applied to the corneal surface for few seconds, and then rinsed with isotonic saline. Fluorescein-treated eyes were then examined with a slit lamp microscope to ensure that the cornea is undamaged (i.e., fluorescein retention and corneal opacity scores ≤ 0.5).

Only undamaged eyes were further dissected from the skull, taking care not to damage the cornea. The eyeball was pulled from the orbit by holding the nictitating membrane firmly with surgical forceps, and the eye muscles were cut with a bent, blunt-tipped scissor. All necessary precautions were taken to aviod any corneal damage due to excessive pressure (i.e, compression artifacts). The visible portion of the optic nerve was left attached to the eye when it was removed from the orbit. Immediately after removing the eye from the orbit, the eye was placed on an absorbent pad and the nictitaing membrane and other connective tissue was removed.

The enucleated eye was mounted in a stainless steel clamp with the cornea positioned vertically. The clamp was then transferred to a chamber of the superfusion appratus. The clamps were positioned in the superfusion appratus in such a way that the entire cornea was supplied with the isotonic saline drip (3 to 4 drops per minute or 0.1 to 0.15 mL/min). The temperature of the chambers of the superfusion appratus was maintained at 32 ± 1.5 °C.

After being placed in the superfusion appratus, the eyes were again examined with a slip-lamp microscope to ensure that they have not been damaged during the dissection procedure. Corneal thickness was also measured at this time at the corneal apex using the depth measuring device on the slit –lamp microscope. Eyes with (i), a fluorescein retention score of >0.5, (ii) corneal opacity >0.5 or (iii) any additional signs of damage were replaced. Out of the eyes that were not rejected based on the beforementioned criteria, individual eyes with a corneal thickness deviating more than 10% from the mean value for all eyes of this batch were rejected. During corneal thickness measurements, slit-width of slit-lamp microscope was set at 0.095 mm.

EQUILIBRATION AND BASELINE RECORDINGS : Immediately after examination and approval of all eyes, they were incubated for approximately 45 - 60 minutes to equilibrate them to the test system prior to dosing. Following the equilibration period, a zero reference measurement was recorded for corneal thickness and opacity to serve as baseline (i.e., time=0). The fluorescein score determined at dissection was used as the baseline measurement for that endpoint.

NUMBER OF REPLICATES 3

NEGATIVE CONTROL USED : physilogical saline, Sodium Chloride injection IP, 0.9 % w/v

POSITIVE CONTROL USED : Benzalkonium chloride (5 % in saline solution [9 g NaCl/L)]

APPLICATION DOSE AND EXPOSURE TIME : 30 µL for 10 seconds

OBSERVATION PERIOD : approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse (+/- 5 min)

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: isotonic saline (approximately 20 mL) at ambient temperature

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: scroing the area of the cornea that is most densely opacified
- Damage to epithelium based on fluorescein retention: Fluorescein retention was evaluated at the 30 minutes observation time point
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: 0.095 mm
- Macroscopic morphological damage to the surface: none

SCORING SYSTEM:
- Mean corneal swelling (%)
- Mean maximum opacity score
- Mean fluorescein retention score at 30 minutes post-treatment

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used.

Irritation parameter:

percent corneal swelling

Run / experiment:

test item

Value:

>= 4.51 - <= 17.89

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

cornea opacity score

Run / experiment:

test item

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

fluorescein retention score

Run / experiment:

test item

Value:

0

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: roughening of the corneal surface observed

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Prior to routine use the technical proficiency of the test method was established by using proficiency chemicals under Bioneeds Study No.: BIO-GT 1046 according to OECD Test Guideline No. 438.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Table 1 Corneal swelling/thickness and isolated chicken eye (ICE) classification

Treatment	Eye No. and Sw%	Corneal Thickness in Instrument Units (µm) at t = (mins)							ICE Class
		Selection	0	30	75	120	180	240
Negative Control	1	281	291	288	289	288	290	284	I
	Sw%	NA	NA	-1.03	-0.69	-1.03	-0.34	-2.41
Positive Control	2	289	296	351	352	463	468	481	IV
	Sw%	NA	NA	18.58	18.92	56.42	58.11	62.50
	3	279	289	338	353	465	472	492
	Sw%	NA	NA	16.96	22.15	60.90	63.32	70.24
	4	299	288	335	360	455	477	490
	Sw%	NA	NA	16.32	25.00	57.99	65.63	70.14
	Mean sw% ±SD	NA	NA	17.29 1.17	22.02 3.04	58.43 2.27	62.35 3.85	67.63 4.44
Test Item	5	269	266	292	320	322	328	324	I
	Sw%	NA	NA	9.77	20.30	21.05	23.31	21.80
	6	267	278	295	317	317	326	326
	Sw%	NA	NA	6.12	14.03	14.03	17.27	17.27
	7	279	298	291	316	322	337	328
	Sw%	NA	NA	-2.35	6.04	8.05	13.09	10.07
	Mean Sw% ±SD	NA	NA	4.51 6.22	13.46 7.15	14.38 6.51	17.89 5.14	16.38 5.92

Table 2 Data of corneal opacity scores and isolated chicken eye (ICE) Classification

Treatment	Eye No.	Corneal Opacity Scores at t =							ICE Class
		Selection	0	30	75	120	180	240
Negative Control	1	0	0	0	0	0	0	0	I
Positive Control	2	0	0	2	2	3	4	4	IV
	3	0	0	2	2	3	4	4
	4	0	0	1	2	2	4	4
	Mean	0	0.0	1.7	2.0	2.7	4.0	4.0
Test Item	5	0	0	0	0	0	0	0	I
	6	0	0	0	0	0	0	0
	7	0	0	0	0	0	0	0
	Mean	0	0.0	0.0	0.0	0.0	0.0	0.0

Table 3 Data of fluorescein retention, morphological effects and isolated chicken eye (ICE) classification

Treatment	Eye No.	Fluorescein retention at t = (Min)			Morphological effects	ICE Class
		-50	0	30
Negative Control	1	0	0	0	No morphological effects observed	I
Positive Control	2	0	0	3	Loosening of epithelium	IV
	3	0	0	3	Loosening of epithelium
	4	0	0	3	Loosening of epithelium
	Mean	0	0.0	3.0	NA
Test Item	5	0	0	0	Roughening of the corneal surface	I
	6	0	0	0	Roughening of the corneal surface
	7	0	0	0	Roughening of the corneal surface
	Mean	0	0.0	0.0	NA

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of this study, the test item is identified to not require classification as eye damaging or eye irritant.

Executive summary:

The test item was evaluated in the “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” as per OECD Guideline for the Testing of Chemicals, Section 4, No. 438, adopted on 9th October 2017.

Heads of chickens approximately 6 to 7 weeks old and weighing around 1.8 to 2.8 kg were collected at poultry slaughterhouse. Within 2 hours of killing, enucleated eyes were placed in a susperfusion apparatus and maintained at 32 ± 1.5oC for 50 minutes. Before dosing, the eyes were incubated for 50 minutes to equilibrate them to the test system prior to treatment. Following the equilibration period, a zero reference measurement was recorded for corneal thickness and opacity to serve as baseline (i.e., time=0). Additionally, the fluorescein retention score was also recorded at 0 hour as baseline measurement value. Eyes with corneal opacity <0.5 and fluorescein retention score <0.5 were selected. Quantity of 30 µL of test item and Benzalkonium chloride [5% in saline solution (9 g NaCl/L)] was applied onto the cornea of three eyes for 10 seconds. Similarly, 30 µL of Sodium Chloride solution, 0.9 % w/v was used as negative control and was applied onto the cornea of a eye for 10 seconds. 

The control and test eyes were examined for corneal thickness and corneal opacity at 0, 30, 75, 120, 180 and 240 min after treatment and results were recorded according to a fixed scoring system. Fluorescin retention by damaged epithelial cells was scored at 30 min post-treatment. Additionally, the eyes treated with vehicle, positive control and test item were evaluated for morphological effects. All examinations were carried out with a slit-lamp microscope and Pachymeter.

For the test item the combination of ICE categories obtained for corneal swelling, corneal opacity, and fluorescein retention was 3 x I (ICE class of I observed in all 3 endpoints). This test is considered acceptable as the concurrent negative control and the positive control were identified as UN GHS Non-Classified and UN GHS Category 1, respectively.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation in vitro

OECD 431

The objective of this study was to evaluate the in vitro skin corrosion potential of the test item by measurement of tissue viability in the Epidermal Model - Epiderm™ (EPI-200-SCT) as per OECD Guideline for the testing of chemicals No. 431, “In vitro skin corrosion: reconstructed human epidermis (RHE) test method”, adopted on 29th July 2016. The test item did not develop any colour when dissolved in distilled water/isopropanol and was considered as non-reducer of MTT as no purple colour was developed when mixed and incubated with MTT solution.

After receipt of the tissues, visual inspection was done to verify the defects. There were no tissue defects, air bubble or excess moisture observed. All tissue inserts were used for the study. Tissue inserts were transferred to upper row of 6 well plates prefilled with 0.9 mL of assay medium and incubated in CO2 incubator for 60 minutes. Exposure to the test item was performed for 1 hour and 3 minutes separately. All the treatments were maintained in duplicates. For 3 minutes treatment, quantity of 50 µL of sterile distilled water (NC) was dispensed into the first insert atop the tissue. After 60 seconds the procedure was repeated with second tissue and continued for all other tissues. Similar procedure was followed in the same manner until all the tissues were treated. Tissues were treated with 50 µL of test item or 50 µL of positive control (glacial acetic acid). For 1 hour treatment, quantity 50 µL of test item, 50 µL of negative control or50 µL of positive control were dispensed directly atop Epiderm™ tissues at 1 minute intervals to facilitate rinsing after exposure. The tissues were incubated at standard culture conditions for 1 hour.

At the end of treatment time tissue inserts were rinsed with sterile PBS (fill and empty insert 20 times in a constant soft stream of 1xPBS) to remove any residual test item. Post rinsing procedure, each insert was removed from the 6-well plate and gently blotted on absorbent material. The tissues were placed into 24-well plate containing 0.3 mL of MTT solution (1 mg/mL) and incubated for 3 hours at 37±1°C and 5±1% CO2. Post incubation, the tissue inserts were removed and blotted onto the tissue paper and transferred to a pre-labelled 24-well plate containing 2.0 mL of isopropanol in each designated well. The plates were placed on an orbital plate shaker and shaken (̴ 120 rpm/minute) for 5 hours (for 1 hour exposure) and 4 hours and 41 minutes (for 3 minutes exposure) at room temperature. At the end of the extraction period, the tissue was pierced with an injection needle and the extract was allowed to run into the well from which the insert was taken. The punctured inserts were discarded and solution was placed on mixer for 15 minutes until it became homogenous. The optical density of the extracted formazan was measured in 96-well plate spectrophotometer at 570 nm. Viability of tissues was calculated.

 

For 3 minutes exposure, percentage viability of negative control, positive control and test item was 100±1.4, 1.7±0.0 and 98.6±3.2, respectively. The percentage viability of the test item was thus greater than 50% of the negative control after 3 min exposure. Further, the percentage viability of the positive control (PC) was less than 50% of the negative control and thus clearly represents the irritation potential of the positive control. For 1 hour exposure, percentage viability of negative control, positive control and test item was 100±1.6, 1.7±0.1 and 80.4±16.3, respectively. The percentage viability of the test item was thus greater than 15% of negative control after 1 h exposure. The percentage viability of the positive control (PC) was less than 50% of the negative control and thus clearly represents the irritation potential of the positive control.

 

OECD 439

In this in vitro skin irritation test using the EPISKIN model, the test item 2-Chloroacrylic sodium salt did not show significantly reduced cell viability in comparison to the negative control (mean viability: 87 %). All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin.

Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered 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 2-Chloroacrylic sodium salt is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category).

Eye irritation in vitro

OECD 438

The test item was evaluated in the “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” as per OECD Guideline for the Testing of Chemicals, Section 4, No. 438, adopted on 9th October 2017.

Heads of chickens approximately 6 to 7 weeks old and weighing around 1.8 to 2.8 kg were collected at poultry slaughterhouse. Within 2 hours of killing, enucleated eyes were placed in a susperfusion apparatus and maintained at 32 ± 1.5oC for 50 minutes. Before dosing, the eyes were incubated for 50 minutes to equilibrate them to the test system prior to treatment. Following the equilibration period, a zero reference measurement was recorded for corneal thickness and opacity to serve as baseline (i.e., time=0). Additionally, the fluorescein retention score was also recorded at 0 hour as baseline measurement value. Eyes with corneal opacity <0.5 and fluorescein retention score <0.5 were selected. Quantity of 30 µL of test item and Benzalkonium chloride [5% in saline solution (9 g NaCl/L)] was applied onto the cornea of three eyes for 10 seconds. Similarly, 30 µL of Sodium Chloride solution, 0.9 % w/v was used as negative control and was applied onto the cornea of a eye for 10 seconds. 

The control and test eyes were examined for corneal thickness and corneal opacity at 0, 30, 75, 120, 180 and 240 min after treatment and results were recorded according to a fixed scoring system. Fluorescin retention by damaged epithelial cells was scored at 30 min post-treatment. Additionally, the eyes treated with vehicle, positive control and test item were evaluated for morphological effects. All examinations were carried out with a slit-lamp microscope and Pachymeter.

For the test item the combination of ICE categories obtained for corneal swelling, corneal opacity, and fluorescein retention was 3 x I (ICE class of I observed in all 3 endpoints). This test is considered acceptable as the concurrent negative control and the positive control were identified as UN GHS Non-Classified and UN GHS Category 1, respectively.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on eye damaging, the test item does not require classification as eye damaging or eye irritant according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776. Regarding skin irritation the test item does not require classification according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.