Registration Dossier

Administrative data

Description of key information

Based on an in vitro and an in vivo skin irritation test it is concluded that Ethylene glycol dibenzoate is not irritating to skin.

Based on an in vitro and an in vivo eye irritation test it is concluded that Ethylene glycol dibenzoate is not irritating to the eyes.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 April 2017 - 24 April 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
28 July 2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
20 July 2012
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
- Correction for purity: no correction factor required
- Stability at higher temperatures: Yes, maximum temperature: 60°C for a maximum duration of 1 hour
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
skin obtained from plastic surgery 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)
Remarks:
Tissues were moistened with 5 μl Milli-Q water to ensure close contact.
Details on test system:
TEST SYSTEM
- EPISKIN Small Model (TM) (EPISKIN-SM (TM), 0.38 cm^2, Lot no.: 17-EKIN-016); a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded in 12-well plates on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen and cultured for 13 days.

SKIN DISC PREPARATION
- Procedure used: On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for 3.5 hours at 37°C

ENVIRONMENTAL CONDITIONS
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 36.2-37.4 °C
- Humidity (%): 73-91

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: 1, with phosphate buffered saline
- Observable damage in the tissue due to washing: no

DYE BINDING METHOD
- Dye used in the dye-binding assay: Interaction with MTT was tested before the study by adding 10 mg of the test item to 2 mL of MTT solution.
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader.
- Wavelength: 570 nm

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin 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.
- The test substance is considered to be non-irritant to skin 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.


SCORING SYSTEM:
- Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Skin irritation is expressed as the remaining cell viability after exposure to the test item.
- The test item was checked for possible direct MTT reduction and colour interference

ACCEPTABILITY 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 ≤40% 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.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST ITEM:
24.53 to 37.24 mg on skin moistened with 5 μl Milli-Q water

NEGATIVE CONTOL:
- Amount applied: 25 µl Phosphate buffered saline

POSITIVE CONTROL
- Amount applied: 25 µl
- Concentration: 5% (aq) Sodium dodecyl sulphate
- Re-spread after 7 minutes contact time
Duration of treatment / exposure:
15 ±0.5 minutes
Duration of post-treatment incubation (if applicable):
42 hours and 3 hours with MTT
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean of 3 replicates
Value:
96
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
8.8%
Remarks on result:
other: Test item SD: 0.8%
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the absolute mean OD570 of the three tissues of the negative control were within the laboratory historical control data range (i.e., a mean of 1.342 ± 0.180) and the SD of the % viability was <18% (i.e., 13%)
- Acceptance criteria met for positive control: yes, the mean relative tissue viability of the positive control was <50% (i.e., 8.8%) and the SD of the % viability was <18% (i.e., 1.9%).
- Acceptance criteria met for variability between replicate measurements: yes, the SD calculated from individual % tissue viabilities of the three identically treated replicates was <18% (i.e., 0.8%).

- Since the mean relative tissue viability for the test item was above 50% the test item is considered to be non-irritant.

Table 2 Individual OD measurements (570 nm)

 

A

(OD570)

B

(OD570)

C

(OD570)

Negative control

OD570measurement 1

OD570measurement 2

 

1.6283

1.5484

 

1.2915

1.2116

 

1.3597

1.2579

Test item

OD570measurement 1

OD570measurement 2

 

1.3449
1.2895

 

1.3995
1.2753

 

1.3624
1.3022

Positive control

OD570measurement 1

OD570measurement 2

 

0.1319

0.1385

 

0.1652

0.1534

 

0.2139

0.1567

OD = Optical density

Triplicate exposures are indicated by A, B and C.

Interpretation of results:
GHS criteria not met
Remarks:
Not classified according to Regulation (EC) 1272/2008
Conclusions:
An in vitro skin irritation test with Ethylene glycol dibenzoate was conducted according to OECD 439 guideline and GLP principles. It is concluded that this test is valid and that Ethylene glycol dibenzoate is not irritating in the in vitro skin irritation test. The substance is therefore not classified according to the GHS and CLP criteria.
Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 July 2017 - 27 July 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
in vivo required for non-EU registration
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Version / remarks:
2015
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2500 (Acute Dermal Irritation)
Version / remarks:
August 1998
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Version / remarks:
May 2008
Deviations:
no
Qualifier:
according to
Guideline:
other: Appendix to Director General Notification, No. 12-Nousan-8147. Agricultural Production Bureau, Ministry of Agriculture, Forestry and Fisheries of Japan (JMAFF)
Version / remarks:
November 2000, including the most recent revisions
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
dd. 3 November 2015
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River France, L’Arbresle, France
- Age at study initiation: Young adult animals (approximately 14-15 weeks old)
- Weight at study initiation: 3121 to 3192 g
- Housing: individually in labeled cages with perforated floors
- Diet: pelleted diet for rabbits (Global Diet 2030 Teklad, Mucedola, Milanese, Italy), provided once daily. In addition, hay was available during the study period.
- Water: municipal tap-water, available ad libitum
- Acclimation period: the animals were allowed to acclimate for at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20
- Humidity (%): 56 to 92
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 18 July 2017 To: 27 July 2017
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
water
Controls:
yes, concurrent no treatment
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 0.5 g

VEHICLE
- Amount applied: 0.7 mL

NEGATIVE CONTROL: adjacent areas of the untreated skin

Duration of treatment / exposure:
4 hours
Observation period:
4 days
Number of animals:
3 animals in a stepwise manner, starting with treatment of 1 animal and followed by treatment of 2 other animals one week later.
Details on study design:
TEST SITE
- Area of exposure: dorsal area, 150 square centimeters (10 x 15 cm)
- % coverage: not indicated
- Type of wrap if used: Micropore taoe, wrapped around the abdomen and secured with Coban elastic bandage.

REMOVAL OF TEST SUBSTANCE
- Washing: after removing the dressing, the skin was cleaned with tap water.
- Time after start of exposure: four hours

OBSERVATION TIME POINTS:
- Post dose observations: once daily for four days (1, 24, 48 and 72 hours after removal of the dressings).

SCORING SYSTEM: Draize
- Method of calculation: visual scoring
Irritation parameter:
erythema score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Irritation parameter:
edema score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Irritant / corrosive response data:
- No skin irritation was observed
- No staining of the treated skin by the test item was observed
- No signs of systemic toxicity during the study period were observed and no mortality occured
Interpretation of results:
GHS criteria not met
Conclusions:
Based on the results of an in vivo skin irritation study, performed according to OECD 404 and GLP principles, Ethylene glycol dibenzoate does not have to be classified according to GHS and Regulation (EC) 1272/2008.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 May 2017 - 05 May 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Version / remarks:
28 July 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
dd 03 November 2015
Specific details on test material used for the study:
- Correction factor for purity: no correction required
- Stability at higher temperatures: yes, maximum temperature: 60°C for a maximum duration of 1 hour
Species:
human
Details on test animals or tissues and environmental conditions:
TEST SYSTEM:
The EpiOcular tissue construct is a nonkeratinized epithelium prepared from normal human keratinocytes. It models the cornea epithelium with progressively stratified, but not cornified cells. A cell suspension is seeded into the insert in specialized medium. After an initial period of submerged culture, the medium is removed from the top of the tissue so that the epithelial surface is in direct contact with the air. This allows the test material to be directly applied to the epithelial surface in a fashion similar to how the corneal epithelium would be exposed in vivo.
- Justification: In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro eye irritation tests is the EpiOcular test, which is recommended in international guidelines and scientific publications (e.g. OECD).
Vehicle:
unchanged (no vehicle)
Remarks:
Tissues were pre-wetted with 20 μL of Ca2+Mg2+-Free- DPBS.
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: at least 50 mg

NEGATIVE CONTROL
- Amount applied: 50 µl of sterile MilliQ water per tissue

POSITIVE CONTROL
Amount applied: 50 µl Methyl Acetate per tissue
Duration of treatment / exposure:
6 hours ± 15 minutes
Duration of post- treatment incubation (in vitro):
18 hours (after a post-soak period of 25 ± 2 minutes)
Number of animals or in vitro replicates:
Test item: 2 tissues
Negative control (MilliQ water): 2 tissues
Positive control (Methyl Acetate): 2 tissues
Details on study design:
TEST SYSTEM
- EpiOcular™ (OCL-200-EIT MatTek Corporation, Lot: 23479)
The EpiOcular tissue construct is a non-keratinized epithelium (0.6 cm2) prepared from normal human keratinocytes.

Interference of the test item with the MTT endpoint
- Test item was checked for possible colour intereference and direct MTT reduction before the study started by adding the test item to MTT medium.

- All incubations, with the exception the exposure and post-exposure immersion at room temperature, were carried out in a controlled environment:
- Humidity: 46 - 83%
- CO2: 5.0 ± 0.5%
- Temperature: 36.4 - 37.2°C.

- Before the assay was started all the tissues were pre-wetted with 20 μL of Ca2+Mg2+-Free-DPBS and incubated at standard culture conditions for 30 ± 2 minutes.
- Exposure: 6 hours ± 15 minutes at 37.0 ± 1.0°C
- Removal test item: rinsing with Ca2+Mg2+-free D-PBS (brought to room temperature)
- Post-exposure immersion: After rinsing the cell culture inserts were each dried carefully and immediately transferred to and immersed in 5 ml of previously warmed Assay Medium (room temperature) in a pre-labeled 12-well plate for a 25 ± 2 minute immersion incubation at room temperature (Post-Soak).
- Post-exposure incubation: After the Post-Soak period cell culture inserts were each dried carefully and transferred to the 6-well plate containing 1.0 ml of warm Assay Medium and were incubated for 18 hours ± 15 minutes at 37°C.

CELL VIABILITY MEASUREMENT
After incubation, cell culture inserts were dried carefully to remove excess medium. The cell culture inserts were transferred into a 24-wells plate prefilled with 0.3 ml MTT-medium (1.0 mg/ml). The tissues were incubated for 180 ± 10 minutes at 37°C. After incubation with MTT-medium the tissues were placed on blotting paper to dry the tissues. Formazan was extracted with 2 ml isopropanol for 2 - 3 hours at room temperature with gentle shaking. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.
Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Eye hazard potential of the test item was classified according to remaining cell viability following exposure of the test item.

Acceptability of the assay
The in vitro eye irritation test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 of the two tissues of the negative control should reasonably be > 0.8 and < 2.5.
b) The mean relative tissue viability of the positive control should be <50% relative to the negative control.
c) The SD calculated from individual % tissue viabilities of the two identically treated replicates should be <20%.

Data evaluation:
* The test chemical is identified as not requiring classification and labelling according to UN GHS (No Category) if the mean percent tissue viability after exposure and post-exposure incubation is more than (>) 60%. In this case no further testing in other test methods is required.
* The test chemical is identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and postexposure incubation is less than or equal (≤) to 60%.
Irritation parameter:
other: Mean tissue viability (% of negative control)
Run / experiment:
Single run
Value:
96
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
SD: 7.9%
Positive controls validity:
valid
Remarks:
29% (SD: 6.8%)
Remarks on result:
other: SD: 2.1%
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, the OD570 measurements of the negative control tissues were between 0.8 and 2.5 (i.e., a mean of 1.586 ± 0.125). The standard deviation value of the percentage viability of two tissues treated identically was 7.9%, indicating that the test system functioned properly.
- Acceptance criteria met for positive control: Yes, the positive control tissues had a mean tissue viability <50% (i.e., 29%).
- The standard deviation from individual % tissue viabilities of the two identically treated replicates was <20% (i.e., 2.1%).
Interpretation of results:
GHS criteria not met
Remarks:
Not classified according to Regulation (EC) 1272/2008
Conclusions:
Based on the evaluation of the eye hazard potential with Ethylene glycol dibenzoate using the EpiOcular cornea epithelial model and performed according to OECD guideline and GLP principles, it is concluded that Ethylene glycol dibenzoate is not irritant for the eye.
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 September 2017 - 27 September 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
in vivo required for non-EU registration
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
2012
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2400 (Acute Eye Irritation)
Version / remarks:
August 1998
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Version / remarks:
May 2008
Deviations:
no
Qualifier:
according to
Guideline:
other: Appendix to Director General Notification, No. 12-Nousan-8147. Agricultural Production Bureau, Ministry of Agriculture, Forestry and Fisheries of Japan (JMAFF)
Version / remarks:
November 2000, including the most recent revisions
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
dd. 03 November 2015
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Charles River France, L’Arbresle, France
- Age at study initiation: approx. 20-22 weeks old
- Weight at study initiation: 3480-3855 g
- Housing: individually in labeled cages with perforated floors
- Diet: Pelleted diet for rabbits (Global Diet 2030 Teklad®, Mucedola, Milanese, Italy), once daily and hay, ad libitum.
- Water: municipal tap-water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19
- Humidity (%): 72-76
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 07 September 2017 To: 27 September 2017
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 88.6-89.6 mg
The test item was ground to a powder prior to weighing.
Duration of treatment / exposure:
1 hour
Observation period (in vivo):
24 hours
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing: with 50 mL tepid tap water, using a velocity of flow
- Time after start of exposure: 1 hour

SCORING SYSTEM: Draize

TOOL USED TO ASSESS SCORE: where standard lighting was considered inadequate, an ophthalmic examination lamp was used.
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #1
Time point:
24/48/72 h
Score:
1.3
Max. score:
3
Reversibility:
fully reversible within: 7 days
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #2
Time point:
24/48/72 h
Score:
1.7
Max. score:
3
Reversibility:
fully reversible within: 7 days
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #3
Time point:
24/48/72 h
Score:
1.3
Max. score:
3
Reversibility:
fully reversible within: 7 days
Irritation parameter:
conjunctivae score
Remarks:
chemosis
Basis:
animal #2
Time point:
24/48/72 h
Score:
0.3
Max. score:
4
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
conjunctivae score
Remarks:
chemosis
Basis:
animal: 1 and 3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
iris score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritant / corrosive response data:
- Irritation and corrosion: Irritation consisted of redness, chemosis and discharge. The irritation had completely resolved within 7 days in all animals. No iridial irritation or corneal opacity were observed, and treatment of the eyes with 2% fluorescein 24 hours after test item instillation revealed no corneal epithelial damage.
- Coloration: Remnants of the test item were present in the eye of all rabbits on day 1.
- Toxicity: No signs of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Table 1 Individual eye irritation scores

 

 

Cornea

 

Iris

 

Conjunctivae

 

 

Comments

Animal 

Time after dosing

 

Opacity

(0-4)

Area

(0-4)

Fluor area (%)#

 

 

(0-2)

 

Redness

(0-3)

Chemosis

(0-4)

Discharge

(0-3)

 

 

 

 

 

295*

 1 hour

 

0

0

 

 

0

 

2

1

1

 

b

 

24 hours

 

0

0

0

 

0

 

2

0

0

 

-

 

48 hours

 

0

0

 

 

0

 

1

0

0

 

-

 

72 hours

 

0

0

 

 

0

 

1

0

0

 

-

 

 7 days

 

0

0

 

 

0

 

0

0

0

 

-

293

 1 hour

 

0

0

 

 

0

 

2

1

1

 

b

 

24 hours

 

0

0

0

 

0

 

2

1

0

 

-

 

48 hours

 

0

0

 

 

0

 

2

0

0

 

-

 

72 hours

 

0

0

 

 

0

 

1

0

0

 

-

 

 7 days

 

0

0

 

 

0

 

0

0

0

 

-

294

 1 hour

 

0

0

 

 

0

 

2

1

1

 

b

 

24 hours

 

0

0

0

 

0

 

2

0

0

 

-

 

48 hours

 

0

0

 

 

0

 

1

0

0

 

-

 

72 hours

 

0

0

 

 

0

 

1

0

0

 

-

 

 7 days

 

0

0

 

 

0

 

0

0

0

 

-

 

 

 

 

 

 

 

 

 

 

 

 

 

*Sentinel; #Green staining after fluorescein treatment (percentage of total corneal area)

Comments: b Remnants of the test item in the eye.

Interpretation of results:
GHS criteria not met
Remarks:
Not classified according to Regulation (EC) No. 1272/2008
Conclusions:
Based on these results, Ethylene glycol dibenzoate does not have to be classified and has no obligatory labelling requirement for eye irritation according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) and Regulation (EC) No. 1272/2008.
Executive summary:

Based on these results, Ethylene glycol dibenzoate does not have to be classified and has no obligatory labelling requirement for eye irritation according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) and Regulation (EC) No. 1272/2008.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The in vivo testing was required for registartion outside the EU.

Justification for classification or non-classification

Based on the available data thes substance is not classified as corrosive or irritating.