Bis[C5-(linear and branched)-alkyl] benzene-1,4-dicarboxylate

Endpoint summary

Description of key information

One in vitro skin irritation study and two eye irritation/corrosion studies were conducted. In all studies no classification of skin and eye irritation were needed.  

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:

09 July 2014 - 14 July 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Remarks:

Epi-200 SIT

Source species:

human

Cell type:

other: epidermal keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi-200- SIT
- Tissue batch number(s): 19652 Kit G
- Date of initiation of testing: 09 July 2014

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C
- Temperature of post-treatment incubation (if applicable): 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
After the end of the treatment interval the inserts were removed immediately from the 6-well plate and tissues were gently rinsed with DPBS at least 15 times in order to remove any residual test material. After the rinsing the inserts were submerged in DPBS at least three times. Afterwards the inserts were once again rinsed with sterile DPBS from the inside and the outside. Excess DPBS was removed by gently shaking the inserts and blotting the bottom with sterile blotting paper. The tissues were carefully dried using sterile cotton tipped swap.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
On the day of testing the MTT concentrate was diluted with the MTT diluent. The 24-well plates were prepared before the end of the tissue pre-warming period. A volume of 300 µL of the MTT solution was added to each well and the plates were kept in an incubator (37 ± 1 °C, 5 ± 0.5 % CO2) until further use.
After the 42-hours incubation period was completed for all tissues and exposure groups, culture inserts were transferred from the holding plates to the MTT-plates. After a 3-hour incubation period (37 ± 1 °C, 5 ± 0.5 % CO2), the MTT solution was aspirated from the wells, and the wells were rinsed three times with DPBS. Inserts were transferred onto new 24-well plates. The inserts were immersed into extractant solution by gently pipetting 2 mL extractant solution (isopropanol) in each insert. The level rose above the upper edge of the insert, thus tissues were completely covered from both sides. The 24-well plate was sealed to inhibit the isopropanol evaporation. The formazan salt was extracted for 69 hours at room temperature.
After the extraction period was completed, the inserts were pierced with an injection needle to allow the extract to run into the well from which the insert was taken. Afterwards the insert was discarded. The 24-well plates were placed on a shaker for 15 minutes until the solution was homogeneous in colour.
Per each tissue, 3 x 200 µL aliquots of the blue formazan solution were transferred into a 96-well flat bottom microtiter plate from the 15 minutes exposure. OD was read in a microplate reader (Versamax® Molecular Devices, Softmax Pro, version 4.7.1) with a 570 ± 1 nm filter. Mean values were calculated from the 3 wells per tissue.


PREDICTION MODEL / DECISION CRITERIA
For the current test, an irritation potential of a test item according to EU classification R38 (according to directive 67/548/EEC), H315 (according to regulation (EC) 1272/2008), and GHS category 2 according to UN GHS (published 2003, last (3rd) revision 2009) is recommended if the mean relative tissue viability of three individual tissues is reduced below 50% of the negative control.

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):30 µL of the undiluted test item

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL DPBS was used as negative control.

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL of 5% SLS solution in deionised water was used as positive control.

Duration of treatment / exposure:

After pre-incubation of EpiDerm™ tissues was completed, medium was replaced by 0.9 mL of fresh medium per well. The negative and positive control and the test item were added into the insert atop the corresponding EpiDerm™ triplicate tissues. The 6-well plates were placed into the incubator for 35 minutes at 37 ± 1.5 °C, 5 ± 0.5 % CO2, 95 ± 5% RH. Thereafter, plates were removed from the incubator and placed into the sterile hood until the period of 60 minutes was completed.

Number of replicates:

3

Species:

other: human keratinocytes

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

30 µl of the undiluted test item was dispensed directly atop the EpiDerm™ tissue.

Duration of treatment / exposure:

60 minutes

Details on study design:

This in vitro study was performed to assess the irritation potential of Di-(iso)-pentyl terephthalate (DPT) by means of the Human Skin Model Test.
Three tissues of the human skin model EpiDerm™ SIT (EPI-200) (from MatTek Corporation, Bratislava, Slovakia) were treated with the test item, the negative or the positive control for 60 minutes.Each 30 µL of the test item, of the negative control (DPBS), and of the positive control (5% SLS) were applied to each of triplicate tissue and spread to match the tissue size.
The test is followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazoliumbromide], in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison of untreated negative controls is used to predict skin irritation potential and is used for the purpose of classification as irritating or non-irritating.

The mean OD of the three negative control tissues was calculated. This value corresponds to 100% tissue viability in the current test. For each individual tissue treated with the test item or the positive control the individual relative tissue viability is calculated.

For the test item and the positive control the mean relative viability +/- rel. standard deviation of the three individual tissues was calculated and used for classification according to the following prediction model:

in vitro result in vivo prediction
mean tissue viability < 50% irritant (I), H315 (category 2)
mean tissue viability > 50% non-irritant (NI)

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean relative absorbance value of the test item is corresponding to the cell viability.

Run / experiment:

DPT

Value:

80.1

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:

Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.

Results after treatment with Di-(iso)-pentyl terephthalate (DPT) and the controls

Dose Group	Treat-ment Interval	Absor-bance 570 nm Tissue 1*	Absor-bance 570 nm Tissue 2*	Absor-bance 570 nm Tissue 3*	Mean Absor-bance of 3 Tissues	Rel. Absor-bance [%] Tissue 1, 2 + 3**	Relative Standard Deviation [%]	Mean Rel. Absorbance [% of Negative Control]***
Nega-tive Control	60 min	1.950	2.003	2.336	2.096	93.0 95.5 111.4	10.0	100.0
Positive Control	60 min	0.096	0.104	0.112	0.104	4.6 5.0 5.2	7.5	5.0
Test Item	60 min	1.608	1.640	1.790	1.680	76.7 78.2 85.4	5.8	80.1

The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 80.1% (threshold for irritancy:≤50%), consequently the test item was not irritant to skin. 

 

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: OECD GHS

Conclusions:

In conclusion, the test item Di-(iso)-pentyl terephthalate (DPT) is not irritant to skin according to UN GHS and EU CLP regulation in this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014

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 Ocular Corrosives and Severe Irritants)

Deviations:

no

Principles of method if other than guideline:

Additional the test methods are compatible to Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997.

GLP compliance:

yes (incl. QA statement)

Species:

other: bovine eyes

Details on test animals or tissues and environmental conditions:

Freshly isolated bovine cornea (at least 9 month old donor cattle) from Schlachthof Bensheim, Germany

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Test item or negative or positive control is applied undiluted at a volume of 0.75 mL on the surface of the corneae.

Duration of treatment / exposure:

Incubation time: 10 min

Duration of post- treatment incubation (in vitro):

After the test item or control items, respectively, were rinsed off from the application side with saline, the corneae were incubated at 32 ± 1 °C for further two hours in a vertical position, followed by a second opacity reading.

Number of animals or in vitro replicates:

Number of Corneae:
Negative Control: 3
Positive Control: 3
Test Item: 3

Details on study design:

This in vitro study was performed to assess the corneal damage potential of Di-(iso)-pentyl terephthalate (DPT) by means of the BCOP assay using fresh bovine corneae.
The corneae were distributed as follows:

Negative Control (0.9% w/v NaCl): 3 corneae

Positive Control (2-Ethoxyethanol): 3 corneae

Test Item (DPT): 3 corneae

After a first opacity measurement of the fresh bovine corneae (t0), the neat test item Di-(iso)-pentyl terephthalate (DPT), the positive, and the negative controls were applied to corneae and incubated for 10 minutes at 32 ± 1 °C. The posterior chamber contained incubation medium. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae. Further, the corneae were incubated for another 120 minutes at 32 ± 1 °C in incubation medium, and opacity was measured a second time (t130).
After the opacity measurements permeability (OD490 value) of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.

At the end, an In vitro Irritancy Score (IVIS) is calcuted:
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)
The mean IVIS value of each treated group is calculated from the IVIS values.
Depending on the score obtained, the test item is classified into the following category according to OECD guideline 437:

IVIS
≤ 3: No Category (according to GHS)
> 3; < 55: No prediction can be made
≥ 55: Serious eye damaging according to CLP/EPA/GHS (Cat 1)

Irritation parameter:

in vitro irritation score

Run / experiment:

DPT (Test item)

Value:

4.73

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No serious eye damaging

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:

- Acceptance criteria met for negative control: 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.

- Acceptance criteria met for positive control: the positive control gives an IVIS that falls within two standard deviations of the current historical mean (updated every three months).

- Range of historical values if different from the ones specified in the test guideline:
Positive Control Negative Control
Mean IVIS 72.53 1.131
Standard Deviation 18.62 0.76
Range of IVIS 3.59 - 153.20 0.00 - 2.84

After the opacity measurements permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C. With the negative control (0.9% (w/v) NaCl solution in deionised water) neither an increase of opacity nor permeability of the corneae could be observed. The positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability of the corneae corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)). Relative to the negative control, the test item Di-(iso)-pentyl terephthalate (DPT) caused a slight increase of the corneal opacity, but permeability effects did not occur. The calculated mean in vitro irritancy score was 4.73. According to OECD 437 (see table in chapter 3.10.3) the test item is not classified as serious eye damaging (CLP/EPA/GHS (Cat 1), but the test item’s hazard for eye damaging cannot be predicted.

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, according to the current study and under the experimental conditions reported, Di-(iso)-pentyl terephthalate (DPT) is not serious eye damaging, but a prediction for the damage hazard cannot be made (GHS).

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05.11.2014 to 01.07.2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

Species / Strain: Rabbit / New Zealand White
Breeder: Isoquimen, SL, 08182 Sant Feliu de Codines, Barcelona, Spain
Selection of species: The rabbit is a commonly used non-rodent species for acute eye irritation/corrosion tests
Identification of animals: By tattooed number
Sex: Female rabbits
Number of animals: 3
Body weight At dosing:
Animal no. 1: 5.2 kg
Animal no. 2: 4.0 kg
Animal no. 3: 5.0 kg
Termination of test
Animal no. 1: 5.2 kg
Animal no. 2: 4.0 kg
Animal no. 3: 5.1 kg
Age (at start of administration): Approx. 8 - 23 months
Duration of study: At least 20 adaptation days, 1 test day and a follow-up period of 72 hours

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

0.1 mL of the test item was administered per animal.

Duration of treatment / exposure:

Single treatment without removal of the test item.

Observation period (in vivo):

The eyes were examined ophthalmoscopically with a slit lamp prior to the administration and 1, 24, 48 and 72 hours after the administration. The eye reactions were observed and registered.
One day before and 24 hours after administration, fluorescein4 was applied to the eyes before being examined to aid evaluation of the cornea for possible lesions. The fluorescein test was repeated on each day of observation.

Number of animals or in vitro replicates:

3

Details on study design:

SCORING SYSTEM:

Reactions were scored according to the following scheme:

CORNEA
no ulceration or opacity 0
scattered or diffuse areas of opacity (other than slight dulling of normal 1
lustre), details of iris clearly visible
easily discernible translucent area, details of iris slightly obscured 2
nacreous areas, no details of iris visible, size of pupil barely discernible 3
opaque cornea, iris not discernible through the opacity 4

IRIS
normal 0
markedly deepened rugae, congestion, swelling, moderate circumcorneal 1
hyperaemia, or injection, iris reactive to light (a sluggish reaction is
considered to be an effect)
haemorrhage, gross destruction, or no reaction to light 2

CONJUNCTIVAE
normal 0
some blood vessels hyperaemic (injected) 1
diffuse, crimson colour; individual vessels not easily discernible 2
diffuse beefy red 3

CHEMOSIS
normal 0
some swelling above normal 1
obvious swelling with partial eversion of lid 2
swelling with lids about half-closed 3
swelling with lids more than half-closed 4


Fluorescein-Test:

DEGREE OF STAINING
no staining 0
scattered or diffuse colouration, details of iris clearly visible 1
easily discernible translucent area, details of iris slightly obscured 2
opalescent areas, details of iris not discernible, extent of pupil difficultly 3
determinable
opaque cornea, iris not discernible through the opacity 4

INVOLVED AREA OF CORNEA
none 0
up to 1/4 of the surface 1
1/4 to 1/2 of the surface 2
1/2 to 3/4 of the surface 3
3/4 to whole surface. 4


TOOL USED TO ASSESS SCORE: hand-slit lamp (Reactions) and fluorescein (Degree of staining, involved area of cornea)

Irritation parameter:

cornea opacity score

Basis:

animal: 1-3

Time point:

24/48/72 h

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal: 1-3

Time point:

24/48/72 h

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal: 2-3

Time point:

24/48/72 h

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48 h

Max. score:

1

Reversibility:

fully reversible

Remarks:

Within 72 hours after dosing

Remarks on result:

probability of weak irritation

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

72 h

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal: 1-3

Time point:

24/48/72 h

Remarks on result:

no indication of irritation

Other effects:

- Lesions and clinical observations:There were no systemic intolerance reactions.
- Ophthalmoscopic findings: Conjunctival redness (grade 1) was observed in all animals 1 hour after administration, in animal no. 1 until 48 hours after administration.
The corneae and irises were not affected by administration of the test item.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the present test conditions a single administration of 0.1 mL Di-(iso)-pentyl-terephthalate (DPT) per animal into the conjunctival sac of the right eye of three rabbits caused following changes:
Conjunctival redness (grade 1) was observed in all animals 1 hour after administration, in animal no. 1 until 48 hours after administration.
The corneae and irises were not affected by administration of the test item.
There were no systemic intolerance reactions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation study:

This in vitro study was performed to assess the irritation potential of Di-(iso)-pentyl terephthalate (DPT) by means of the Human Skin Model Test according to OECD TG 439.

Three tissues of the human skin model EpiDerm^™were treated with the test item, the negative (DPBS) or the positive control (5% SLS) for 60 minutes.

After treatment with the test item Di-(iso)-pentyl terephthalate (DPT) the mean relative absorbance value decreased to 80.1% compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

In conclusion, it can be stated that in this study and under the experimental conditions reported, Di-(iso)-pentyl terephthalate (DPT) is not irritant to skin.

Acute eye irritation/Corrosion test:

An acute eye irritation/corrosion test in rabbits was conducted according to OECD guideline 405, EC method B.5. and OCSPP (OPPTS) guideline 870.2400.

A single administration of 0.1 mL Di-(iso)-pentyl-terephthalate (DPT) per animal into the conjunctival sac of the right eye of three rabbits caused following changes:

Conjunctival redness (grade 1) was observed in all animals 1 hour after administration, in animal no. 1 until 48 hours after administration.

The corneae and irises were not affected by administration of the test item.

There were no systemic intolerance reactions.

According to the Globally Harmonized System (GHS) and EC-Regulation 1272/2008 and subsequent regulations, the test item is non-irritating to eyes; classification and labelling of the substance is not necessary.

Bovine corneal opacity and permeability assay (BCOP):

This in vitro study was performed to assess the corneal damage potential of Di-(iso)-pentyl terephthalate (DPT) by means of the BCOP assay using fresh bovine corneae according to OECD guideline 437.

After a first opacity measurement of the fresh bovine corneae, the neat test item Di-(iso)-pentyl terephthalate (DPT), the positive, and the negative controls were applied to corneae and incubated for 10 minutes at 32 ± 1 °C. The posterior chamber contained incubation medium. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae. Further, the corneae were incubated for another 120 minutes at 32 ± 1 °C in incubation medium, and opacity was measured a second time.

After the opacity measurements permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.

With the negative control (0.9% (w/v) NaCl solution in deionised water) neither an increase of opacity nor permeability of the corneae could be observed.

The positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability of the corneae corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)).

Relative to the negative control, the test item Di-(iso)-pentyl terephthalate (DPT) caused a slight increase of the corneal opacity, but permeability effects did not occur. The calculated mean in vitro irritancy score was 4.73. According to OECD 437 the test item is not classified as serious eye damaging (CLP/EPA/GHS (Cat 1), but the test item’s hazard for eye damaging cannot be predicted.

Justification for classification or non-classification

Based on the available studies, the test substance is not classified for skin or eye irritation according to EC Regulation No. 1272/2008.