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Description of key information

Terephtalic acid, compound with hexane-1,6-diamine (1:1) was not-irritating for the skin in an OECD 439 test.
Terephtalic acid, compound with hexane-1,6-diamine (1:1) was not corrosive and borderline not irritating in an OECD 437 test.
Based on these results, the substance is considered as not irritating for both the skin and the eye.

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:
from 30 April may to 09 July 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study, OECD 439 compliant
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
06 May 2013
Species:
human
Strain:
other: skin model
Details on test animals or test system and environmental conditions:
Test system
EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 14-EKIN-018).
This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

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).

Source
SkinEthic Laboratories, Lyon, France.

Preparation and preincubation
Tissues
On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for 26 hours at 37°C. Maintenance medium and Assay medium were supplied by Skinethic Laboratories, Lyon, France.

MTT medium
MTT concentrate (Sigma Aldrich, Zwijndrecht, The Netherlands; 3 mg/ml in PBS) diluted (10x) in Assay medium (final concentration 0.3 mg/ml).

Environmental conditions
All incubations, with the exception of the test substance incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 72 - 89%), containing 5.0 ± 0.5% CO2 in air in the dark at 37 ± 1°C
(actual range 36.0 - 36.9°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the humidity occurred that were caused by opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Type of coverage:
other: not applicable
Preparation of test site:
other: not applicable
Vehicle:
unchanged (no vehicle)
Controls:
other: not applicable
Duration of treatment / exposure:
15 minutes
Number of animals:
Not applicable
Details on study design:
Test for reduction of MTT by the test substance:
Terephthalic acid, hexane-1,6-diamine (1:1) was checked for possible direct MTT reduction before the study was started. To assess the ability of the test substance to reduce MTT, 11.0 mg of the test substance was added to 2 ml MTT solution (0.3 mg/ml in PBS). The mixture was incubated for 3 hours at 37°C. A negative control, sterile Milli-Q water was tested concurrently.

Application/Treatment of the test substance:
The test was performed on a total of 3 tissues per test substance together with negative and positive controls. The skin was moistened with 5 µL Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test substance to the tissue and the solid test substance (11.06 to 12.06 mg; with a small glass weight boat) was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 µL PBS (negative control) and 3 tissues with 25 µL 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test substance. After rinsing the cell culture inserts were each dried carefully and moved to a new well on 2 ml pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.

Cell viability measurement:
After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 ml MTT-medium (0.3 mg/ml). The tissues were incubated for 3 h at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in prelabeled microtubes and extracted with 500 µl isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for 70 hours. 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. Skin irritation potential of the test substance was classified according to remaining cell viability following exposure of the test substance.

Electronic data capture:
Observations/measurements in the study were recorded electronically using the following programme(s):
REES Centron Environmental Monitoring system version SQL 2.0 (REES Scientific, Trenton, NJ, USA): Temperature and humidity.
Magellan Tracker 7.0 (TECAN, Austria) for optical density measurement.
Irritation / corrosion parameter:
other: other: Tissue viability
Value:
118
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 15 minutes. (migrated information)
Irritant / corrosive response data:
Terephthalic acid, hexane-1,6-diamine (1:1) was checked for possible direct MTT reduction by adding the test substance to MTT medium. Because no colour change was observed it was concluded that Terephthalic acid, hexane-1,6-diamine (1:1) did not interact with MTT.
Results shows the mean tissue viability obtained after 15 minutes treatment with Terephthalic acid, hexane-1,6-diamine (1:1) compared to the negative control tissues. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with Terephthalic acid, hexane-1,6-diamine (1:1) compared to the negative control tissues was 118%. Since the mean relative tissue viability for Terephthalic acid, hexane-1,6-diamine (1:1) was above 50% Terephthalic acid, hexane-1,6-diamine (1:1) is considered to be non-irritant.
The positive control had a mean cell viability after 15 minutes exposure of 10%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 9%, indicating that the test system functioned properly.

Table 1: Mean absorption in thein vitroskin irritation test with Terephthalic acid, hexane-1,6-diamine (1:1)

 

 

A

(OD570)

B

(OD570)

C

(OD570)

Mean

(OD570)

 

SD

Negative control

0.966

1.118

1.135

1.073

±

0.093

Test substance

1.322

1.214

1.253

1.263

±

0.055

Positive control

0.084

0.108

0.135

0.109

±

0.025

OD = optical density

SD = Standard deviation

Triplicate exposures are indicated by A, B and C.

Test substance = Terephthalic acid, hexane-1,6-diamine (1:1)

 

In this table the values are corrected for background absorption (0.041). Isopropanol was used to measure the background absorption.

 

Table 2: Mean tissue viability in thein vitroskin irritation test with Terephthalic acid, hexane-1,6-diamine (1:1):

 

Mean tissue viability (percentage of control)

Negative control

100

Test substance

118

Positive control

10

Test substance = Terephthalic acid, hexane-1,6-diamine (1:1)

Individual OD measurements at 570 nm: 

 

A

(OD570)

B

(OD570)

C

(OD570)

Negative control

OD570measurement 1

OD570measurement 2

1.021

0.993

1.216

1.101

1.192

1.160

Test substance

OD570measurement 1

OD570measurement 2

1.390

1.336

1.361

1.149

1.310

1.278

Positive control

OD570measurement 1

OD570measurement 2

0.137

0.113

0.142

0.156

0.174

0.178

OD = Optical density

Triplicate exposures are indicated by A, B and C.

 

Test substance = Terephthalic acid, hexane-1,6-diamine (1:1)
Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
It is concluded that this test is valid and that Terephthalic acid, hexane-1,6-diamine (1:1) is non-irritant in the in vitro skin irritation test under the experimental conditions.
Executive summary:

This study (2014) is an in vitro skin irritation test with Terephthalic acid, hexane-1,6-diamine (1:1) using a human skin model, performed according to OECD 439 guideline and under GLP conditions.

Skin tissue was moistened with 5 µL of Milli-Q water and 11.06 to 12.06 mg of Terephthalic acid, hexane-1,6-diamine (1:1) was applied directly on top of the skin tissue for 15 minutes. After a 42-hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

 

Skin irritation is expressed as the remaining cell viability after exposure to the test substance.

The relative mean tissue viability obtained after 15 minutes treatment with Terephthalic acid, hexane-1,6-diamine (1:1) compared to the negative control tissues was 118%. Since the mean relative tissue viability for Terephthalic acid, hexane-1,6-diamine (1:1) was above 50% after 15 minutes treatment Terephthalic acid, hexane-1,6-diamine (1:1) is considered to be non-irritant.

 

The positive control had a mean cell viability of 10% after 15 minutes exposure. The absolute mean OD570(optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 9%, indicating that the test system functioned properly.

Finally, it is concluded that this test is valid and that Terephthalic acid, hexane-1,6-diamine (1:1) is non-irritant in the in vitro skin irritation test under the experimental conditions.

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

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 05 May to 25 June 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study OECD 437 compliant
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
GLP compliance:
yes
Species:
other: bovine
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Test System: Bovine eyes were used as soon as possible after slaughter on the same day.

Rationale: In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing (1-6). As a consequence a validated and accepted in vitro test for eye irritation should be performed before in vivo tests are conducted. One of the proposed validated in vitro eye irritation tests is the Bovine Corneal Opacity and Permeability (BCOP) test.

Source: Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.

Transport: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
Vehicle:
unchanged (no vehicle)
Controls:
other: not applicable
Duration of treatment / exposure:
240 minutes
Number of animals or in vitro replicates:
Not applicable
Details on study design:
Preparation of corneas:
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 (Invitrogen Corporation, Breda, The Netherlands) containing 1% (v/v) L-glutamine (Invitrogen Corporation) and 1% (v/v) Foetal Bovine Serum (Invitrogen Corporation)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of MC2 (Clermont, France) 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.

Cornea selection and Opacity reading:
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (OP-KIT, MC2, Clermont, France). The opacity of each cornea was read against an air filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

Treatment of corneas and opacity measurements:
The medium from the anterior compartment was removed and 750 µL of the negative control and 20% (w/v) Imidazole solution (positive control) were introduced onto the epithelium of the cornea. Terephthalic acid, hexane-1,6-diamine (1:1) was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (302 to 315 mg).The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 240 ± 10 minutes at 32 ± 1°C. After the incubation the solutions and the test compound were removed and the epithelium was washed at least three times with MEM with phenol red (Eagle’s Minimum Essential Medium, Invitrogen Corporation). Possible pH effects of the test substance on the corneas were recorded. The anterior and the posterior compartment were refilled with fresh cMEM and an opacity determination was performed without any further incubation. After the completion of the incubation period each cornea were inspected visually for dissimilar opacity patterns and the opacity determination was performed.

Opacity measurement:
The opacitometer determined the difference in the light transmission between each control or treated cornea and an air filled chamber. The numerical opacity value (arbitrary unit) was displayed and recorded. The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each positive control or test substance treated cornea was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each positive control or test substance treated cornea.
The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.

Application of sodium fluorescein:
Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Merck) was evaluated.
The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 ml of 5 mg Na-fluorescein/ml cMEM solution. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 ± 5 minutes at 32 ± 1°C.

Permeability determinations:
After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 µL of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader). Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation.
The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution was performed, the OD490 of each reading was corrected for the mean negative control OD490 before the dilution factor was applied to the readings.

Electronic data capture:
Observations/measurements in the study were recorded electronically using the following programme: Magellan Tracker 7.0 (TECAN, Austria) for optical density measurement.
Irritation parameter:
other: IVIS score
Basis:
mean
Time point:
other: 240 minutes
Score:
4.5
Irritant / corrosive response data:
Terephthalic acid, hexane-1,6-diamine (1:1) induced ocular irritation through one endpoint (opacity), resulting in a mean in vitro irritancy score of 4.5 after 240 minutes of treatment.
Other effects:
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 (20% (w/v) Imidazole) was 165 and within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.
Interpretation of results:
other: Not corrosive
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
IVIS score is 4.5. The substance is therefore not corrosive for the eye. No conclusion can be made between non irritating or irritating for the eye classifications even if IVIS score is only slightly over the limit for the not irritating classification.
Executive summary:

This study (2014) is a screening for the eye irritancy potential of Terephthalic acid, hexane-1,6-diamine (1:1) using the Bovine Corneal Opacity and Permeability test (BCOP test, OECD 437), under GLP conditions. It was tested through topical application for approximately 240 minutes.

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 (20% (w/v) Imidazole) was 165 and within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Terephthalic acid, hexane-1,6-diamine (1:1) induced ocular irritation through one endpoint (opacity, resulting in a mean in vitro irritancy score of 4.5 after 240 minutes of treatment.

Since Terephthalic acid, hexane-1,6-diamine (1:1) induced an IVIS ≤55, the substance is definitely not corrosive for the eye. As the IVIS is > 3 it is not possible to conclude that the substance is not irritating for the eye and no definitive classification can be made.

However with an IVIS score of 4.5, the substance is very close to the limit for the non irritating classification.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation:

This study (2014) is an in vitro skin irritation test with Terephthalic acid, hexane-1,6-diamine (1:1) using a human skin model, performed according to OECD 439 guideline and under GLP conditions.

Skin tissue was moistened with 5 µL of Milli-Q water and 11.06 to 12.06 mg of Terephthalic acid, hexane-1,6-diamine (1:1) was applied directly on top of the skin tissue for 15 minutes. After a 42-hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

Skin irritation is expressed as the remaining cell viability after exposure to the test substance.

The relative mean tissue viability obtained after 15 minutes treatment with Terephthalic acid, hexane-1,6-diamine (1:1) compared to the negative control tissues was 118%. Since the mean relative tissue viability for Terephthalic acid, hexane-1,6-diamine (1:1) was above 50% after 15 minutes treatment Terephthalic acid, hexane-1,6-diamine (1:1) is considered to be non-irritant.

The positive control had a mean cell viability of 10% after 15 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 9%, indicating that the test system functioned properly.

Finally, it is concluded that this test is valid and that Terephthalic acid, hexane-1,6-diamine (1:1) is non-irritant in the in vitro skin irritation test under the experimental conditions.

Eye irritation:

This study (2014) is a screening for the eye irritancy potential of Terephthalic acid, hexane-1,6-diamine (1:1) using the Bovine Corneal Opacity and Permeability test (BCOP test, OECD 437), under GLP conditions. It was tested through topical application for approximately 240 minutes.

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 (IVIS) of the positive control (20% (w/v) Imidazole) was 165 and within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Terephthalic acid, hexane-1,6-diamine (1:1) induced ocular irritation through one endpoint (opacity), resulting in a mean in vitro irritancy score of 4.5 after 240 minutes of treatment.

Since Terephthalic acid, hexane-1,6-diamine (1:1) induced an IVIS ≤ 55, the substance is definitely not corrosive for the eye. As the IVIS is > 3 it is not possible to conclude that the substance is not irritating for the eye and no definitive classification can be made.

However with an IVIS of 4.5, the substance is very close to the limit for the non irritating classification. In the in-vitro skin irritation OECD 439 study performed on the substance, the results showed a complete absence of toxicity as the treated tissues exhibited a 118 % viability compared to the controls. The OECD 439 test is based on the same principle than in-vitro tests that would have allowed to concluded on the final eye irritation classification (i.e. OECD 492).

Therefore, taking together the borderline almost negative response result obtained for the substance in the BCOP (OECD 437) and the completely negative response obtained in the performed in-vitro skin irritation test we conclude that Terephtalic acid, compound with hexane-1,6-diamine (1:1) is most likely not irritating for the eye and that no classification is required for this endpoint in a weight of evidence approach.


Justification for selection of skin irritation / corrosion endpoint:
Only study available. GLP and OECD 439 compliant.

Justification for selection of eye irritation endpoint:
Only study available. GLP and OECD 437 compliant.

Justification for classification or non-classification

Based on the results obtained in the performed skin and eye irritation tests, the substance terephtalic acid, compound with hexane-1,6-diamine (1:1) is not classified for skin or eye irritation according to the regulation 1272/2008/EC criteria.

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