IPEMA

Endpoint summary

Description of key information

Based on the results of two skin in vitro tests it was concluded that IPEMA fulfilles the CLP classification criteria as skin irritant category 2, H315

Based on the results of an eye in vitro tests it was concluded that IPEMA does not fulfill the CLP classification criteria as eye irritant

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-11-12 to 2019-12-25

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

June 18, 2019

GLP compliance:

yes

Test system:

human skin model

Cell type:

non-transformed keratinocytes

Cell source:

other: donor - All cells used to produce EpiDermTM are purchased or derived from tissue obtained by MatTek Corporation from accredited lnstitutions, ln all cases, consent was obtained by these Institutions from the donor or the donor's legal next of kin, for use

Justification for test system used:

EpiDerm SCT kit is recommended in the test method

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm SCT kit
- Tissue batch number: 32116
- Production date: 2019-11-07
- Delivery date: 2019-11-11
- Date of initiation of testing: 2019-11-12

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3-minutes exposure at room temperature; at 60-minute exposure each plate was placed into the inbubator
- Temperature of post-treatment incubation (if applicable): room temperature

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After the exposure, each tissue twenty times with PBS(-).
Inside and outside the tissue inserts were wiped with a sterile cotton swab. The tissue inserts were placed into a 24-well plate (Corning) filled with 300 µL/well of fresh medium

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
PRE-TEST
- MTT concentration: 1mg/ml MTT solution (MTT medium)
- 50 µl test substance + 1 ml MTT medium
- Incubation time: 60 minutes

NYLON MESH
- 50µl of test substance was dropped onto a nylon mesh on a glass slide. After 60 minutes at room
temp., the corrosion of the nylon mesh was evaluated microscopically. Corrosion was not observed.

MAIN TEST
- MTT concentration: 1mg/ml MTT solution (MTT medium); 0.9 mL/well
- 50µl test substance or 50 µl control substance
- Pre-incubation time: 60 +- 5 minutes (0.9 mL/well of the medium)
- Incubation time (after rinsing) : 180+- 5 minutes (with 300 µL/well of MTT medium); extraction at room temperature for 2 hours or more using a plate shaker
- Spectrophotometer: Multimode Microplate Reader (FlUOstar Omega, BMG LABTECH) at 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA - OD:
3 min exposure:
Negative control: 2.004 ± 0.166
Positive control: 0.157 ± 0.049
60 min exposure:
Negative control: 1.951 ± 0.152
Positive control: 0.053 ± 0.020

NUMBER OF REPLICATE TISSUES: 2 (Duplicate tissue inserts were used for the test substance, negative control substance and positive control substance. Duplicate tissue inserts were used to check the tissue-binding of the test substance
NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1
PREDICTION MODEL / DECISION CRITERIA
Mean cell viability Category
<25% (3-minute exposure) Corrosive ( UN GHS Cat. 1A)
>=25%, <50% (3-minute exposure) Corrosive ( UN GHS Cat. 1B and 1C)
>=50% (3-minutes exposure) and <15% (60-minutes exposure) Corrosive ( UN GHS Cat. 1B and 1C)
>=50% (3-minutes exposure) and >=15% (60-minutes exposure) Non-Corrosive (UN GHS Cat. 2 or not classified)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50µl

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50µl

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50µl
- Concentration (if solution): 8N KOH

Duration of treatment / exposure:

3 minutes
60 minutes

Duration of post-treatment incubation (if applicable):

180 minutes

Number of replicates:

2 tissue insert per treatment group

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes

Value:

97.4

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60-minutes

Value:

75.6

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes: The ODs in the negative control substance group in the 3-minute and 60-minute exposures were 1.848 and 1.689, respectively.
- Acceptance criteria met for positive control: Yes: The mean cell viability in the positive control substance group in the 60-minute exposure was 2.8%.
- Acceptance criteria met for variability between replicate measurements: The CVs between the two tissue inserts were = 30% for all substances which the cell viability was from 20% to 100%.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

It is concluded that IPEMA was "non-corrosive" (UN GHS category 2 or not classified) under the present test conditions

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

This study was not performed under GLP, but fulfils criteria set out in Annex XI 1.1.2 of REACH for studies not conducted to GLP. Old composition with Penothiazine at 5ppm was used. This will not influence the outcome of the assay.

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

Adopted: 2015-07-28

Deviations:

not specified

GLP compliance:

no

Remarks:

Study was performed in 2017 to comply with other (non-EU-REACH) requirements, before registration under EU REACH was relevant. The study fulfils criteria set out for studies not conducted under GLP and is considered valid for the purpose of registration.

Test system:

human skin model

Remarks:

LabCyte EPI-MODEL24 SIT

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
TEST KIT
- Source: Japan Tissue Engineering Co., Ltd. (J-TEC)
- Code number: 401124
- Receipt date: 2017-09-26
- Date of initiation of testing: 2017-09-26

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C
- Post-treatment incubation (if applicable): 42h

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Tissues were rinsed 15 times or more with PBS.
- Observable damage in the tissue due to washing: Not reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.5 mg/mL MTT solution
- Incubation time: 180 min
- Spectrophotometer: Multimode Microplate Reader
- Wavelength: 570 nm and 650 nm

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Method of calculation used:
OD of each extract was measured spectrophotometrically by Multimode Microplate Reader at 570 nm and 650 nm. The OD at 650nm was subtracted from OD at 570 nm. The blank OD was subtracted from the value was measurement value.
Cell viability (%) = (OD of each tissue insert of each treatment group : Mean OD of the negative control substance group) × 100
Standard deviation (SD) in each treatment group used three tissue inserts was calculated.

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the viability greater or equal than 50% (UN GHS Category: 1or 2)
- The test substance is considered to be non-irritant to skin if the viability is less than 50% (UN GHS Category: not classified)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL/NEGATIVE CONTROL/POSITIVE CONTROL:
- Amount applied:
25 µL of the control substances and the test substance were applied onto each tissue surface at 1 minute interval. Each tissue insert was placed until 15min±30 sec was completed.

Duration of treatment / exposure:

15 min ± 30 sec

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Value:

13.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

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 skin irritation test

Group	Tissue	Measurement value^a)		Cell viability^b,c)	SDd)	Category
	No.		Mean
Negative control (Distilled water)	1	0.851	0.851	100%	2.0%
	2	0.811
	3	0.832
Positive control (5w/v% SDS solution)	1	0.092	0.092	12.6%	3.0%
	2	0.144
	3	0.091
Test substance (IPEMA)	1	0.108	0.108	13.8%	1.0%	Irritant
	2	0.125
	3	0.127

1. Measurement value which the mean of blank OD ws subtracted from each tissue OD was


2. Cell viability in the negative control substance was regarded as 100%.


3. The cell viability was calculated from mean measurement value of each


4. The Standard deviation(SD) was calculated from the cell viability (n=3) of each

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

IPEMA was judged to be “Irritant” under the present test conditions.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (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:

2021-03-31

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:

2020-06-26

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: Abattoir Vion Beef B.V., Buchloe, Germany (isolated corneas obtained as by-product from animals freshly slaughered)
- Storage, temperature and transport conditions of ocular tissue: transported in HBSS containing Pen/Strep on ice to the laboratory.
- Time interval prior to initiating testing: Immediately after arrival of the eyes, cornea preparation was initiated.
- Selection and preparation of corneas: The eyes were carefully examined for defects and any defective eyes were discarded. 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 1640 medium (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI 1640 medium). The posterior chamber was always filled first. The corneas were incubated for one hour at 32 +-1 °C.

Vehicle:

physiological saline

Remarks:

0.9 % Sodium Chloride Solution (aq)

Controls:

yes

Amount / concentration applied:

750 µg/L of the test substance or the control substance was introduced into the anterior chamber.

Duration of treatment / exposure:

After 10 minutes incubation at 32 ± 1 °C 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 1640 medium (without phenol red).

Duration of post- treatment incubation (in vitro):

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 1640 medium. 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.

Number of animals or in vitro replicates:

3 corneas for the test item
3 corneas as negative controls treated with physiological saline 0.9% NaCl
3 corneas as positive control treated with ethanol 100%

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
On the test day, fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories. Immediately after arrival of the eyes, cornea preparation was initiated. The eyes were carefully examined for defects and any defective eyes were discarded. 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.

QUALITY CHECK OF THE ISOLATED CORNEAS:
Only corneas that had an initial illuminance reading I > I0/1.1651 lux were used for the assay. Eyes that were noted to have defects were discarded and not used on the study.

NUMBER OF REPLICATES: 3
VEHICLE CONTROL USED: yes
POSITIVE CONTROL USED: yes
APPLICATION DOSE AND EXPOSURE TIME: 750 µg/L and 10 minutes
REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: The epithelium was washed at least three times with MEM (containing phenol red). Once the medium was free of test item, the cornea was finally rinsed with complete RPMI (without phenol red).
- POST-EXPOSURE INCUBATION: 2 hours at 32 ± 1 °C; 90 minutes at 32 +- 1°C with 1mL of 4 mg/mL sodium fluorescein solution

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacimeter (BASF-OP3.0, Duratec GmbH)
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry
- Others: Pertinent observations
SCORING SYSTEM: In Vitro Irritancy Score (IVIS).
This was detailed in the study report as follows:
= 3 = no category
> 3; = 55 = no stand-alone 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.

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

Irritation parameter:

in vitro irritation score

Value:

0.16

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Other effects / acceptance of results:

The eye irritancy potential of 3-Methyl-3-butenyl 2-methyl-2-propenoate was investigated in the bovine corneal opacity and permeability assay.
The test item was tested as provided by the sponsor.
None of the corneas treated with 3-Methyl-3-butenyl 2-methyl-2-propenoate showed any opacity of the tissue.
The following mean in vitro irritation score was calculated:
0.16
Therefore, the test item was classified into UN GHS No Category.
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.

Opacity

Cornea No.	Test Item	Initial Opacity	Final Opacity	Change of Opacity Value	Corrected Opacity Value
1		2.66	4.52	1.86
2	Negative	3.03	5.41	2.38
3	Control	2.88	3.89	1.02
MV		2.85	4.61	1.75
4		5.20	27.15	21.94	20.19
5	Positive	1.42	26.07	24.65	22.90
6	Control	1.53	21.34	19.81	18.06
MV		2.72	24.85	22.13	20.38
7		2.41	4.88	2.47	0.72
8	Test Item	2.55	5.41	2.86	1.11
9		3.59	4.76	1.17	-0.58
MV		2.85	5.01	2.17	0.41

MV = mean value

 

Permeability

Cornea No.	Test Item	OD490	Corrected OD490 Value
1		0.021
2	Negative	0.028
3	Control	0.036
MV		0.028
4		0.696	0.668
5	Positive	1.173	1.145
6	Control	1.292	1.264
MV		1.054	1.025
7		0.008	-0.020
8	Test Item	0.010	-0.018
9		0.017	-0.011
MV		0.012	-0.017

MV = mean value

 

In Vitro Irritation Score

Cornea No.	Test Item	Corrected Opacity	Corrected OD490 Value	IVIS
1		1.86	0.021
2	Negative	2.38	0.028
3	Control	1.02	0.036
MV		1.75	0.028	2.18
4		20.19	0.668
5	Positive	22.90	1.145
6	Control	18.06	1.264
MV		20.38	1.025	35.76
7		0.72	-0.020
8	Test Item	1.11	-0.018
9		-0.58	-0.011
MV		0.41	-0.017	0.16

MV = mean value

 

Historical Mean In Vitro Irritation Score of the Positive Control

	IVIS Positive Control - Ethanol 100 %
Mean Value (MV)	47.24
Standard Deviation (SD)	8.86
MV- 2xSD	29.52
MV+2xSD	64.95
Number of Replicates providing Historical Mean: 74

Positive controls are updated after every single experiment or at least every 3 months

Interpretation of results:

GHS criteria not met

Remarks:

According to the evaluation criteria the test item 3-Methyl-3-butenyl 2-methyl-2-propenoate is classified into UN GHS No Category.

Conclusions:

According to the evaluation criteria the test item 3-Methyl-3-butenyl 2-methyl-2-propenoate is classified into UN GHS No Category.

Executive summary:

The eye irritancy potential of 3-Methyl-3-butenyl 2-methyl-2-propenoate was investigated in the bovine corneal opacity and permeability assay. None of the corneas treated with 3-Methyl-3-butenyl 2-methyl-2-propenoate showed any opacity of the tissue. The mean in vitro irritation score was 0.16. Therefore IPEMA was not classified as eye irritant

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

As a result of an in vitro skin irritation test (OECD guidance 439), the cell viability treated by IPEMA was 13.8%. This value met the judgement criteria of “Irritant” (¿50%). As a result of an in vitro skin corrostion test (OECD guidance 431) in the 3-minutes and 60-minutes exposure the cell viabilities treated by the IPEMA were 97.4% and 75.6% respectively, leading to the conclusion that IPEMA is non-corrosive. Therefore IPEMA was classified as skin irritant category 2.

The eye irritancy potential of 3-Methyl-3-butenyl 2-methyl-2-propenoate was investigated in the bovine corneal opacity and permeability assay. None of the corneas treated with 3-Methyl-3-butenyl 2-methyl-2-propenoate showed any opacity of the tissue. The mean in vitro irritation score was 0.16. Therefore IPEMA was not classified as eye irritant.