Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs.

Endpoint summary

Description of key information

The skin irritation potential of Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs was evaluated in vitro in the EpiSkinTM Small Model (EpiSkinTMSM) in accordance to OECD 439. The  testing material (Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs) was  concluded not to have skin irritating properties, thus no classification applies.

The eye irritation potential of Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs was evaluated in vitro in the Bovine Corneal Opacity and Permeability test method (BCOP) in accordance to OECD 437. The  testing material (Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs) was  concluded not to eye irritating properties, thus no classification applies.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 - 06 Feb 2015

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

06 July 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

OGYÉI, Országos Gyógyszerészeti és Élelmezés-egészségügyi Intézet, Budapest, Hungary

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, closed container, protected from sunlight

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The EPISKIN model has been validated for irritation testing. It is used as a replacement for the Draize Skin Irritation test (OECD TG 404) for the purposes of distinguishing between skin irritating and non-skin irritating test substances.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTM Small Model (EpiSkinTMSM), manufactured by EPISKIN SNC Lyon, France
- Tissue batch number(s): 15-EKIN-005
- Date of initiation of testing: 09 Feb 2015

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume of washing steps: rinsed thoroughly with 25 mL PBS
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours (± 5 min)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 21.9 ± 0.4
- Barrier function: 2.2 mg/mL
- Morphology: Well-differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum
- Contamination: no
- Reproducibility: yes

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Method of calculation used:
Blank: The mean of the 6 blank optical density (OD) values is calculated
Negative control:
- Individual negative control OD values are corrected with the mean blank OD:
OD Negative Control (ODNC) = ODNCraw – ODblank mean
- The corrected mean OD of the 3 negative control values is calculated: this corresponds to 100% viability
Positive control:
- Individual positive control OD values are corrected with the mean blank OD: :
OD Positive Control (ODPC) = ODPCraw – ODblank mean
- The corrected mean OD of the 3 positive control values is calculated
- The % viability for each positive control replicate is calculated relative to the mean negative control:
% Positive Control 1 = (ODPC1 / mean ODNC) ×100
- The mean value of the 3 individual viability % for positive control is calculated:
Mean PC % = (%PC1 + %PC2 + %PC3) / 3
Test item:
- Individual test item OD values are corrected with the mean blank OD: :
OD Treated Tissue (ODTT) = ODTTraw – ODblank mean
- The corrected mean OD of the 3 test item values is calculated
- The % viability for each test item replicate is calculated relative to the mean negative control:
% Treated Tissue 1 = (ODTT1 / mean ODNC) ×100
% Treated Tissue 2 = (ODTT2 / mean ODNC) ×100
% Treated Tissue 3 = (ODTT3 / mean ODNC) ×100
- The mean value of the 3 individual viability % for test item is calculated
Mean TT % = (%TT1 + %TT2 + %TT3) / 3

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive or irritant to skin if the mean relative viability after 15 minutes exposure and 42 h post incubation is less or equal to 50% of the negative
control.
- The test substance is considered to be non-corrosive or non-irritant to skin if the tissue viability after exposure and post-treatment incubation is more than 50% of the negative control.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 10µL
- Concentration: 100%

NEGATIVE CONTROL
- Amount(s) applied: 10 μL

POSITIVE CONTROL
- Amount(s) applied:10 μL
- Concentration: 5%

Duration of treatment / exposure:

15 min

Duration of post-treatment incubation (if applicable):

42 ± 1 h

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean value of negative control (PBS)

Value:

100

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean value of positive control (5% SDS)

Value:

20

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean value of test item (100%)

Value:

99

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: yes

ACCEPTANCE OF RESULTS:
The mean OD value of the three negative control tissues should be between 0.6 and 1.5 and the standard deviation value (SD) of the % viability should be ≤ 18.
The acceptable mean percentage viability range for positive controls is 0-40% and the standard deviation value (SD) of the % viability should be ≤ 18.
For test chemicals, the standard deviation value (SD) of the % viability should be ≤ 18.

- 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: MTT assay after 3 hours (± 5 min) exposure

Substance	Tissue No.	OD570	OD570(mean)	Relative viability [%]	Relative viability (mean) [%]	Standard Deviation of relative viability [%]
Negative Control	1	0.854	0.850	100	100	1.67
	2	0.862		101
	3	0.835		98
Positive Control	1	0.212	0.171	25	20	6.32
	2	0.110		13
	3	0.192		23
Test Item	1	0.801	0.839	94	99	6.02
	2	0.897		106
	3	0.820		96

Interpretation of results:

GHS criteria not met

Conclusions:

The skin irritation potential of Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs was evaluated in vitro in the EpiSkinTM Small Model (EpiSkinTMSM) in accordance to OECD 439. The testing material (Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs) was concluded not to have skin irritating properties, thus no classification applies.

Executive summary:

The skin irritation potential of Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs was evaluated in vitro in the EpiSkinTM Small Model (EpiSkinTMSM) in accordance to OECD 439.

Acceptance criteria for negative and positive controls as well as variability between replicate measures was met and the study was therefore considered valid.

The relative viability scores for the testing material (Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs) was comparable to the negative control group and therefore concluded not to have skin irritating properties.

Thus, no classification applies in acordance to CLP (Regulation (EC) No 1272/2008).

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:

30 Jan 2015

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:

see above

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, closed container, away from sunlight

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Schlachthof Bensheim, Bensheim, Germany
- Characteristics of donor animals: at least 9 month old donor cattle
- Storage, temperature and transport conditions of ocular tissue: The isolated eyes were transported in HBSS (with streptomycin / penicillin) at ambient temperature.
- Time interval prior to initiating testing: The comea were isolated on the same day after delivery of the eyes and were directly used in the BCOP test.
- indication of any existing defects or lesions in ocular tissue samples: no
- Indication of any antibiotics used: streptomycin / penicillin

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 750 µL
- Concentration: undiluted

Duration of treatment / exposure:

10 min at 32 ± 1 °C

Observation period (in vivo):

not applicable

Duration of post- treatment incubation (in vitro):

2 h at 32 ± 1 °C

Number of animals or in vitro replicates:

Number of eyes for the test item/ negative control/ positive control: 3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS: A careful macroscopic examination was performed on all eyes to detect the presence of any defects (opacity, scratches, pigmentation, vascularization etc). Eyes with defects were discarded. The cornea was carefully removed from the eye using a scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea. The cornea were directly used in the BCOP test on the same day.

QUALITY CHECK OF THE ISOLATED CORNEAS: yes, eyes were free of defects

TREATMENT METHOD: corneas were incubated horizontally, in a water-bath

REMOVAL OF TEST SUBSTANCE
After incubation the test item or control item, respectively, was rinsed off from the application side with saline.

- POST-EXPOSURE INCUBATION: The incubation medium consisted of MEM (supplemented with 1.1 g / 500 mL sodium bicarbonate, 5 mL / 500 mL L-glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin). Immediately before starting the test, MEM was supplemented with 1 % fetal calf serum (FCS)). The OECD foresees the use of EMEM which is in composition and osmolarity equivalent to the
MEM, thus MEM can be used without restriction.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was determined by the amount of light transmission through the cornea using an opacitometer (OP_KiT opacitometer, Electro Design, 63-Riom France).
- Corneal permeability: Passage of sodium fluorescein dye measured with the aid of spectrophotometry (OD490) (Versamax® Molecular Devices). Prior to application of the test item or controls, the corneae in the holder were incubated in a vertical position for about 1 h at 32 ± 1 °C in a water-bath. At the end of the incubation period, the basal opacity was determined (to). After exposure of the corneae to the test groups, after rinsing and further incubation of the corneae for two hours, the opacity value was determined again (t130).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The following formula is used to determine the IVIS of the positive control and the test item:
IVIS = (opacity value - opacity value mean negative control) + (15 x corrected OD490 value)

Depending on the score obtained, the test item is classified into one of the categories according to OECD 437.

DECISION CRITERIA: The decision criteria as indicated in the TG (OECD 437) was used.

VALIDATION CRITERIA: The test will be acceptable if
- the positive control gives an IVIS that falls within two standard deviations of the current historical mean (updated every three months), and if
- the negative control responses result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control

Irritation parameter:

cornea opacity score

Run / experiment:

negative control - mean out of all 3 eyes

Value:

0.33

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

cornea opacity score

Run / experiment:

positive control - mean out of all 3 eyes

Value:

55.67

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: value is corrected

Irritation parameter:

cornea opacity score

Run / experiment:

test item - mean out of all 3 eyes

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: value is corrected

Irritation parameter:

fluorescein leakage

Remarks:

permeability

Run / experiment:

negative control - mean out of all 3 eyes

Value:

0.054

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

fluorescein leakage

Remarks:

permeability

Run / experiment:

positive control - mean of all 3 eyes

Value:

0.812

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: value is corrected

Irritation parameter:

fluorescein leakage

Remarks:

permeability

Run / experiment:

test item - mean out of all 3 eyes

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: value is corrected

Irritation parameter:

in vitro irritation score

Remarks:

(IVIS)

Run / experiment:

negative control - mean out of all 3 eyes

Value:

1.15

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

in vitro irritation score

Remarks:

(IVIS)

Run / experiment:

positive control - mean out of all 3 eyes

Value:

67.84

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

in vitro irritation score

Remarks:

(IVIS)

Run / experiment:

test item - mean out of all 3 eyes

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values if different from the ones specified in the test guideline: yes

Table 1: In-Vitro Irritancy Score (IVIS) values

	IVIS	Mean IVIS
Negative Control	0.78	1.15
	0.77
	1.90
Test substance	-1.43	0.0
	-0.43
	-0.41
Positive control	70.61	67.84
	66.52
	66.38

Table 2: Opacity and Permeability values

	Difference (t130-to) of Opacity
Parameter	Opacity 1	Opacity 2	Opacity 3	Mean opacity value	Permeability at 490 nm	Mean Permeability at 490 nm
Negative control	0	0	1	0.33	0.052	0.054
					0.051
					0.060
Test substance	-1.33*	-0.33*	-0.33*	-	-0.006*	-
					-0.006*
					-0.005*
Positive control	54.67*	55.67*	56.67*	-	1.063*	-
					0.724*
					0.648*

* = corrected values

Interpretation of results:

GHS criteria not met

Conclusions:

The eye irritation potential of Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs was evaluated in vitro in the Bovine Corneal Opacity and Permeability test method (BCOP) in accordance to OECD 437. The testing material (Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs) was concluded not to eye irritating properties, thus no classification applies.

Executive summary:

The eye irritation potential of Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs was evaluated in vitro in the Bovine Corneal Opacity and Permeability test method (BCOP) in accordance to OECD 437.

Acceptance criteria for negative and positive controls as well as variability between replicate measures was met and the study was therefore considered valid.

The scores for cornes opacity and permeability for the testing material (Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs) was comparable to the negative control group and the In-Vitro Irritancy Score (IVIS) values did not indicate any eye irritative properties. Therefore, it was concluded that Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs does not to have eye irritating properties.

Thus, no classification applies in acordance to CLP (Regulation (EC) No 1272/2008).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The skin and eye irritation he acute oral toxicity of Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs was evaluated in accordance to OECD 439 and OECD 437, respectively. No skin and eye irritating properties was concluded.

Thus, no classification applies in acordance to CLP (Regulation (EC) No 1272/2008).

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