C10-13-alkyl(branched, unsaturated)-succinic acid-4-(3-methyl-butyl)-ester

Endpoint summary

Description of key information

In order to evaluate the skin irritating properties of the registration substance and to avoid animal testing a tiered approach in

two in vitro test systems was performed (in vitro skin corrosion test (OECD 431, Reconstructed human epidermis (RHE) test method (adopted 28 July 2015)) and an in vitro skin irritation test (OECD 439, Reconstructed Human Epidermis Test Method (adopted 28 July 2015)).
In order to evaluate the eye irritating properties of the registration substance and to avoid animal testing a tiered approach was performed with one in vitro test (OECD 437, Bovine Corneal Opacity and permeability Test method). As no prediction was possible with the in vitro BCOP-test an in vivo eye irritation test (OECD 405) was performed in one rabbit due to a stepwise manner.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-02-16 to 2016-02-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline and GLP conform well documented scientific study report.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 28 July 2015

GLP compliance:

yes

Specific details on test material used for the study:

Identification Tetrapropylene succinic acid monoisobutylester
Appearance Light brown, viscous liquid
Batch ESD0018639
Purity/Composition 93.0% (w/w)
Test item storage At room temperature
Stable under storage conditions until 30 November 2016 (expiry date)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from multiple donors

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2).
- Tissue batch number: Lot no.: 16-EKIN-007
- Production date: 2016-02-16
- Shipping date: n.a.
- Delivery date: n.a.
- Date of initiation of testing: 2016-02-16

TEMPERATURE USED FOR TEST SYSTEM
- On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for 23 hours at 37°C
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: n.a.
- Observable damage in the tissue due to washing: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 3 mg/mL in PBS; final concentration 0.3 mg/mL
- Incubation time: 3 h at 37°C
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm


NUMBER OF REPLICATE TISSUES:
- Test was performed on a total of 3 tissues per test item together with negative and positive controls.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Tetrapropylene succinic acid monoisobutylester was checked for possible direct MTT reduction and colour interference in the Skin corrosion test using EpiDerm as a skin model and (project 511530). Because solutions did not turn blue / purple and a blue / purple precipitate was not observed it was concluded that Tetrapropylene succinic acid monoisobutylester did not interfere with the MTT endpoint

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

PREDICTION MODEL / DECISION CRITERIA
Relative mean viability of 3 individual tissues after 15 minutes of exposure and 42 hours of post incubation:
- The test substance is considered to be irritiant to skin Category 1 or Category 2 (additional information on corrosion needed) if the test item has ≤ 50% of the mean viability of the negative controls
- The test substance is considered to be non-irritatnt to skin (no category) if the test item has > 50% of the mean viability of the negative controls

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25µL
- Concentration (if solution): undiluted

NEGATIVE CONTROL: physiological saline
- Amount(s) applied (volume or weight): 25 µL
- Concentration (if solution): 0.9%

POSITIVE CONTROL: SDS
- Amount(s) applied (volume or weight): 25µL
- Concentration (if solution): 5%

Duration of treatment / exposure:

15 ± 0.5 minutes at room temperature
The positive control was re-spread after 7 minutes contact time.

Duration of post-treatment incubation (if applicable):

42 hours at 37°C

Number of replicates:

3

Species:

human

Strain:

other: not relevant

Details on test animals or test system and environmental conditions:

EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Lot no.: 16-EKIN-007
Rationale
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

4.9

Vehicle controls validity:

other: no vehicle used, test item used undiluted

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

Acceptability of the assay
The in vitro skin irritation test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 (optical density at 570 nm) of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the Standard Deviation value (SD) of the % viability should be ≤18.
b) The mean relative tissue viability of the positive control should be ≤50% relative to the negative control and the Standard Deviation value (SD) of the % viability should be ≤18.
c) The SD calculated from individual % tissue viabilities of the three identically treated replicates should be ≤18.

Data evaluation and statistical procedures
A test item is considered irritant in the skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
A test item is considered non-irritant in the in vitro skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

Table 1. Data interpretation of test items

Relative mean viability of 3 individual tissues after 15 minutes of exposure and 42 hours of post incubation	Prediction to be considered
≤50% of the mean viability of the negative controls	Category 1 or Category 2 (additional information on corrosion needed)
> 50% of the mean viability of the negative controls	No category

 

Table 2. Mean absoption in the in vitro skin irritation test with Tetrapropylene succinic acid monoisobutylester

	Mean (OD570)
Negative control	0.892 ± 0.109
Tetrapropylene succinic acid monoisobutylester	0.043 ± 0.006
Positive control	0.086 + 0.045

Table 3. Mean tissue viability in the in vitro skin irritation test with Tetrapropylene succinic acid monoisobutylester

	Mean tissue viability (percentage of control)
Negative control	100
Tetrapropylene succinic acid monoisobutylester	4.9
Positive control	10

Interpretation of results:

irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

It is concluded that Tetrapropylene succinic acid monoisobutylester is irritant in the in vitro skin irritation test under the experimental conditions described in this report.

Executive summary:

In order to assess the possible skin irritation potential, Tetrapropylene succinic acid monoisobutylester was tested in vitro through topical application for 15 minutes on a human three dimensional epidermal in vitro skin model (EPISKIN Standard model). The study procedures described in this report were based on the most recent OECD 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method (adopted 28 July 2015)). The test item was applied undiluted directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Skin irritation is expressed as the remaining cell viability after exposure to the test item.

The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 4.9%. Since the mean relative tissue viability for Tetrapropylene succinic acid was below 50% after 15 ± 0.5 minutes treatment it is considered to be irritant.The positive control had a mean cell viability of 10% after 15 minutes exposure.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-01-11 to 2016-01-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline and GLP conform well documented scientific study report.

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

adopted 28 July 2015

Deviations:

no

GLP compliance:

yes

Remarks:

Statement of compliance within report

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: n.a.

Justification for test system used:

Recommended test system in international guidelines (OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model (EPI-200)
The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.
- Tissue batch number(s): Lot no.: 23280 kits X and J
- Production date: January 2016
- Shipping date: n.a.
- Delivery date: n.a.
- Date of initiation of testing: 2016-01-11

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: n.a.
- Observable damage in the tissue due to washing: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT concentrate (5 mg/ml) diluted (1:5) with MTT diluent (supplemented DMEM). Both supplied by MatTek Corporation.
- Incubation time: 3 h at 37°C
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES:
The test was performed on a total of 4 tissues per test item together with a negative control and positive control. Two tissues were used for a 3-minute exposure to the test item and two for a 1-hour exposure.

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

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%, 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:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50µL
- Concentration (if solution):

NEGATIVE CONTROL: Milli-Q water
- Amount(s) applied (volume or weight): 50µL
- Concentration (if solution):

POSITIVE CONTROL: Potassium hydroxide (KOH)
- Amount(s) applied (volume or weight): 50µL

Duration of treatment / exposure:

3 minutes (two tissues) and 1 hour (two tissues)

Duration of post-treatment incubation (if applicable):

Rinsed tissues were kept in 24 well plates on 300 µl DMEM medium until 6 tissues (= one application time) were dosed and rinsed.

Number of replicates:

The test was performed on a total of 4 tissues per test item together with a negative control and positive control. Two tissues were used for a 3-minute exposure to the test item and two for a 1-hour exposure.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min

Value:

113

Vehicle controls validity:

other: no vehicle used, testitem is used undeluted

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: not corrosive

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour

Value:

100

Vehicle controls validity:

other: no vehicle used, test item used undeluted

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: not corrosive

Table 1           Data interpretation and sub-categorisation of test items

Viability measured after 3-minutes and 1 hour	Prediction to be considered
< 50% after 3 minute exposure	Corrosive: sub-category 1A (optional)
≥50% after 3 minute exposure AND < 15% after 1 hour exposure	Corrosive: sub-category 1B and 1C (optional)
≥50% after 3 minute exposure AND ≥15% after 1 hour exposure	Non-corrosive

 

Table 2           Mean absorption in the in vitro skin corrosion test with the test item

	3-minute application					1-hour application
	A (OD570)	B (OD570)	Mean (OD570)		SD	A (OD570)	B (OD570)	Mean (OD570)		SD
Negative control	1.624	1.593	1.609	±	0.022	1.828	1.962	1.895	±	0.094
Test item	1.778	1.863	1.820	±	0.060	1.825	1.956	1.890	±	0.093
Positive control	0.195	0.289	0.242	±	0.066	0.168	0.339	0.254	±	0.121

SD = Standard deviation

Duplicate exposures are indicated by A and B.

In this table the values are corrected for background absorption (0.0433). Isopropanol was used to measure the background absorption.

Table 3           Mean tissue viability in the in vitro skin corrosion test withthe test item

	3-minute application viability (percentage of control)	1-hour application viability (percentage of control)
Negative control	100	100
Tetrapropylene succinic acid monoisobutylester	113	100
Positive control	15	13

Interpretation of results:

GHS criteria not met

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Because the mean relative tissue viability for the test item was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment the test item is considered to be not corrosive.

Executive summary:

The mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues. Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 113% and 100% respectively. Because the mean relative tissue viability for the test item was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment the test item is considered to be not corrosive

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-02-08 to 2016-02-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline and GLP conform well documented scientific study report.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Identification Tetrapropylene succinic acid monoisobutylester
Appearance Light brown, viscous liquid
Batch ESD0018639
Purity/Composition 93.0% (w/w)
Test item storage At room temperature
Stable under storage conditions until 30 November 2016 (expiry date)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

Species: Albino rabbit, New Zealand White, (SPF-Quality). Recognized by international guidelines as the recommended test system (e.g. EC, OECD)
Source: Charles River France, L’Arbresle Cedex, France
Number of animals: 1 Male
Age and body weight: At start of dosing, the animal was 18 weeks old and the body weight was at least 1.5 kg.
Identification: Earmark
Health inspection: At least prior to dosing. It was ensured that the animal was healthy and that eyes were free from any abnormality.

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

instillation of 0.1 mL of the undiluted test item

Duration of treatment / exposure:

The animal was treated by instillation of 0.1 mL of the test item, in the conjunctival sac of one of the
eyes after gently pulling the lower lid away from the eyeball. The lids were then gently held together for
about one second to prevent loss of the test item. The other eye remained untreated and served as
the reference control.

Observation period (in vivo):

1, 24, 48, 72 hours after treatment and 7, 14 and 21 days

Number of animals or in vitro replicates:

The study was performed in a stepwise manner and was started by treatment of a single rabbit
(sentinel). Based on the duration of the ocular lesions of the first animal, the two further rabbits
assigned to the study were not treated.

Details on study design:

The study was performed in a stepwise manner and was started by treatment of a single rabbit (sentinel).

Preemptive Pain management:
One hour prior to instillation of the test item, buprenorphine (Buprenodale®, Dechra Ltd., Stoke-on-Trent, United Kingdom) 0.01 mg/kg was administered by subcutaneous injection in order to provide a therapeutic level of systemic analgesia.

Five minutes prior to instillation of the test item, two drops of the topical anesthetic alcaine 0.5% (SA Alcon-Couvreur NV, Puurs, Belgium) were applied to both eyes.

Treatment
The animal was treated by instillation of 0.1 mL of the test item, in the conjunctival sac of one of the eyes after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second to prevent loss of the test item. The other eye remained untreated and served as the reference control.

Immediately after the 24-hour observation, a solution of 2% fluorescein (Merck, Darmstadt, Germany) in water (adjusted to pH 7.0) was instilled into both eyes of each animal to quantitatively determine corneal epithelial damage. This procedure was repeated to assess recovery. Any bright green stained area, indicating epithelial damage, was estimated as a percentage of the total corneal area.

Immediately after fluorescein examination on Day 2, in order to provide a continued level of systemic analgesia, buprenorphine 0.01 mg/kg and meloxicam (Metacam®, Boehringer Vetmed GmbH, Ingelheim/Rhein, Germany) 0.5 mg/kg were administered by subcutaneous injection.

Additional injections were supplied during the observation period to reduce pain and distress

After the final observation, the animal was sacrificed by intra-venous injection of Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands).

Based on the duration of the ocular lesions of the first animal, the two further rabbits assigned to the study were not treated.

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

other: 24, 48, 72 hours after application

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 14 days

Irritation parameter:

iris score

Basis:

animal #1

Time point:

other: 24, 48, 72 hours after application

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 14 days

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #1

Time point:

other: 24,48, 72 hours after application

Score:

3

Max. score:

3

Reversibility:

not fully reversible within: 21 days after application

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

other: 24, 48, 72 hours after application

Score:

1.7

Max. score:

2

Reversibility:

fully reversible within: 14 days after application

The study was performed in a stepwise manner and was started by treatment of a single rabbit (sentinel). Based on the duration of the ocular lesions of the first animal, the two further rabbits assigned to the study were not treated.

Table1: Individual eye irritation scores

							Cornea							Iris				Conjunctivae									Comments
animal		Time after dosing					Opacity (0-4)		Area (0-4)		Fluor area (%)^1			(0-2)				Redness (0-3)			Chemosis (0-4)		Discharge (0-3)
994		1 hour					1		1					1				2			2		3				-
		24 hours					1		2		50			1				3			2		2				-
		48 hours					1		1					1				3			2		2				f
		72 hours					1		2		50			1				3			1		1				f
		7 days					1		1		25			1				2			1		1				f, p
		14 days					0		0		0			0				1			0		1				f
		21 days					0		0					0				1			0		0				-

 ¹  Green staining after fluorescein treatment (percentage of total corneal area).

 

Comments:

p         Pannus, neovascularization of the cornea.

f          Reduced elasticity of the eyelids.

Table 2:       Mean value eye irritation scores

Animal		Mean 24, 48 and 72 hours
		Corneal		Iris		Conjunctivae
		opacity				Redness		Chemosis
994		1.0		1.0		3.0		1.7

 

 

Table 3:       Animal specifications

Animal		Sex		Age at start		Body weights (grams)
				(weeks)		prior to application	after the final observation
994		♂		18		3201	3512

Interpretation of results:

Category 1 (irreversible effects on the eye)

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Based on the persistence of the ocular lesions until 21 days after instillation the test substance is considered to be irritant to the eye.

Executive summary:

Instillation of 0.1 mL of Tetrapropylene succinic acid monoisobutylester (unchanged) into an eye of one rabbit resulted in severe effects on the cornea, iris and conjunctivae. The corneal injury consisted of opacity and epithelial damage. As a result of the corneal injury, pannus (neovascularization of the cornea) was apparent 8 days after instillation. The corneal injury resolved within 15 days. Iridial irritation was observed and also resolved within 15 days. The irritation of the conjunctivae consisted of redness, chemosis and discharge. Chemosis and discharge had completely resolved within 15 days and 22 days, respectively. Redness was still present at 21 days after instillation.

There was no evidence of ocular corrosion. No staining of (peri) ocular tissues by the test item was observed and no test item remnants were seen. No signs of systemic toxicity were observed in the animal during the test period and no mortality occurred. Based on the persistence of the ocular lesions until 21 days after instillation the test substance is considered to be irritant to the eye.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-12-24 to 2015-12-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study is well documented, guideline conform and performed under GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

adopted July 26, 2013

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

Official Journal of the European Union No. L324; Amended by EC No. 1152/2010 No. L142, 09 December 2010

Deviations:

no

GLP compliance:

yes

Remarks:

Statement of compliance within report

Specific details on test material used for the study:

Identification Tetrapropylene succinic acid monoisobutylester
Appearance Light brown, viscous liquid
Batch ESD0018639
Purity/Composition 93.0% (w/w)
Test item storage At room temperature
Stable under storage conditions until 30 November 2016 (expiry date)

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.
The isolated corneas were stored in a petri dish with cMEM (Eagle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1°C. The corneas were incubated for the minimum of 1 hour at 32 ± 1°C.
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations will be performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea will be read against a cMEM filled chamber, and the initial opacity reading thus determined will be recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

yes, concurrent negative control

Amount / concentration applied:

The medium from the anterior compartment was removed and 750 μl of either the negative control, positive control (10% (w/v) Benzalkonium Chloride) or an excess amount of test item using a cotton swab was introduced onto the epithelium of the cornea.

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

120 ± 10 minutes at 32 ± 1°C.

Number of animals or in vitro replicates:

three cormeas (selected randomly) for each treatment group

Irritation parameter:

in vitro irritation score

Run / experiment:

one

Value:

7.1

Vehicle controls validity:

other: no vehicle used , test item is used undiluted

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Irritation parameter:

other: in vitro occular irritancy scores (IVIS)

Basis:

mean

Time point:

other: after 10 minutes of treatment

Score:

7.1

Max. score:

10.2

Remarks on result:

other: Since Tetrapropylene succinic acid monoisobutylester induced an IVIS > 3 ≤ 55, no prediction on the classification can be made

Irritant / corrosive response data:

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (10% (w/v) Benzalkonium Chloride) was 120 and was within two standard deviations of the current historical positive control mean. (APPENDIX 3, Table 6). It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Tetrapropylene succinic acid monoisobutylester induced ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 7.1 after 10 minutes of treatment.

Since Tetrapropylene succinic acid monoisobutylester induced an IVIS > 3 ≤ 55, no prediction on the classification can be made.

Interpretation - In vitro irritancy score:

In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)

Additionally the opacity and permeability values were evaluated independently to determine whether the test item induced irritation through only one of the two endpoints.

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

In vitroscore range	UN GHS
≤3	No Category
> 3;≤55	No prediction can be made
>55	Category 1

Acceptability of assay:

The assay is considered acceptable if:

 -     The positive control gives an in vitroirritancy score that falls within two standard deviations of the current historical mean.

-     The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.

Interpretation of results:

other: Since Tetrapropylene succinic acid monoisobutylester induced an IVIS > 3 ≤ 55, no prediction on the classification can be made

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Since Tetrapropylene succinic acid monoisobutylester induced an IVIS > 3 ≤ 55, no prediction on the classification can be made. Further testing is necessary

Executive summary:

For evaluation of the eye hazard potential of Tetrapropylene succinic acid monoisobutylester the Bovine Corneal Opacity and Permeability test (BCOP test) according to OECD 437 was performed. The aim of this study was to evaluate the eye hazard potential of Tetrapropylene succinic acid monoisobutylester as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea.

The test consists of topical application of Tetrapropylene succinic acid monoisobutylester on the epithelium of the bovine cornea for 10 minutes. Tetrapropylene succinic acid monoisobutylester was applied undiluted.After exposure the cornea was thoroughly rinsed to remove the test item and incubated for 2 hours with fresh medium followed by opacity measurement and the permeability of the corneas was determined after a 90 minutes incubation period with sodium fluorescein.

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. Benzalkonium Chloride (10% (w/v) serves as positive control. The mean in vitro irritancy score of the positive control Benzalkonium Chloride was 120 and was within two standard deviations of current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Tetrapropylene succinic acid monoisobutylester induced ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 7.1 after 10 minutes of treatment.

Since Tetrapropylene succinic acid monoisobutylester induced an IVIS > 3 ≤ 55, no prediction on the classification can be made.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In order to evaluate the skin irritating properties of the registration substance and to avoid animal testing a tiered approach in two in vitro test systems was performed. Based on a negative in vitro skin corrosion test (OECD 431, Reconstructed human epidermis (RHE) test method (adopted 28 July 2015)) an in vitro skin irritation test (OECD 439, Reconstructed Human Epidermis Test Method (adopted 28 July 2015)) was performed, which was positiv in result.

In order to evaluate the eye irritating properties of the registration substance and to avoid animal testing a tiered approach was performed with one in vitro test (OECD 437, Bovine Corneal Opacity and permeability Test method) for which no prediction on classification could be made. As no prediction was possible with the in vitro BCOP-test an in vivo eye irritation test (OECD 405) was performed in one rabbit due to a stepwise manner. Due to the persistency of conjunctivae redness the test substance is determined to cause serious eye damage.

Justification for classification or non-classification

Skin irritation:

Tetrapropylene succinic acid monoisobutylester is irritant in the in vitro skin irritation test. According to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments) and based on the negative in vitro skin corrosion test, Tetrapropylene succinic acid monoisobutylester should be classified skin irritant category 2.

Eye irritation:

According to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments), Tetrapropylene succinic acid

monoisobutylester should be classified as: having irreversible effects on the eyes (Category 1);