Disodium piperazine-1,4-diethanesulphonate

Endpoint summary

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 Aug 2017 to the 01 Sep 2017

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Specific details on test material used for the study:

Identification: 1,4 piperazinediethanesulfonic acid disodium
salt
Appearance: White powder
Purity/Composition: No correction factor required
Test item storage: At room temperature
Stable under storage conditions until: 30 November 2018 (retest date)
CAS number: 76836-02-7
EC number: 278-562-3

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis.

Details on animal used as source of test system:

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

Justification for test system used:

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

Vehicle:

unchanged (no vehicle)

Details on test system:

Tissues
On the day of receipt the tissues were kept on agarose and stored in the refrigerator. On the next day, at least one hour before starting the assay the tissues were transferred to 6-well plates with 0.9 ml DMEM.

DMEM (Dulbecco’s Modified Eagle’s Medium)
Supplemented DMEM, serum-free supplied by MatTek Corporation.

MTT medium
MTT concentrate (5 mg/ml) diluted (1:5) with MTT diluent (supplemented DMEM). Both supplied by MatTek Corporation.

Environmental conditions
All incubations, with the exception of the test item incubation of 3 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 56 - 84%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.3 - 37.1°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 and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data
these deviations are considered not to affect the study integrity.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Test item was applied directly on top of the skin tissue and was spread to match the size of the tissue

Duration of treatment / exposure:

Two tissues were used for a 3-minute exposure to the test item and two for a 1-hour exposure.

Number of replicates:

The test was performed on a total of 4 tissues per test item together with a negative control and positive control.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes

Value:

96

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no indication of corrosion

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour

Value:

94

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

12% after the 1-hour exposure

A test item is considered corrosive in the in vitro skin corrosion test if: a) The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%. b) In addition, a test item considered non-corrosive (viability > 50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test item is decreased below 15%. A test item is considered non corrosive in the in vitro skin corrosion test if: a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%. b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.

The test item was checked for color interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because the solutions did not turn blue / purple nor a blue / purple precipitate was observed it was concluded that the test item did not interfere with the MTT endpoint.

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, 1,4 piperazinediethanesulfonic acid disodium salt is not corrosive in the in vitro skin corrosion test under the experimental conditions described.

Executive summary:

The objective of this study was to evaluate 1,4 piperazinediethanesulfonic acid disodium salt for its ability to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200)). The possible corrosive potential of the test item was tested through topical application for 3 minutes and 1 hour. The study procedures described in this report were based on the most recent OECD and EC guidelines. Batch 29040-44484 of the test item was a white powder. Skin tissue was moistened with 25 µl of Milli-Q water and at least 25 mg of the test item was applied directly on top of the skin tissue. The positive control had a mean relative tissue viability of 12% after the 1-hour exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit <2.8) and the laboratory historical control data range. In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was < 17%, indicating that the test system functioned properly. Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 96% and 94%, respectively. Because the mean relative tissue viability for the test item was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment the test item is considered to be not corrosive.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15th - 25th August 2017

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Specific details on test material used for the study:

Identification: 1,4 piperazinediethanesulfonic acid disodium
salt acid disodium salt
Appearance: White powder
Purity/Composition: No correction factor required
Test item storage: At room temperature
Stable under storage conditions until: 30 November 2018 (retest date)
CAS number: 76836-02-7
EC number: 278-562-3

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on animal used as source of test system:

This model is a three-dimensional human epidermis model, which consists of adult humanderived 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.

Vehicle:

unchanged (no vehicle)

Details on test system:

On the day of receipt the tissues were transferred to 12-well plates and pre-incubated with pre-warmed Maintenance Medium for approximately 22.5 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 item 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 83 - 99%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.4 - 37.1°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

No correction was made for the purity/composition of the test compound.
The solid test item was applied directly on top of the skin tissue. 1,4 piperazinediethanesulfonic acid disodium salt was spread to match the size of the tissue.

Duration of treatment / exposure:

After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item.

Duration of post-treatment incubation (if applicable):

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-solution (0.3 mg/ml in PBS). 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 pre-labeled microtubes and extracted with 500 μl isopropanol (Merck, Darmstadt, Germany).

Number of replicates:

3 tissues per test item

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean

Value:

104

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

11%

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

other: Mean Absorption OD570

Run / experiment:

Mean

Value:

1.068

Negative controls validity:

valid

Remarks:

1.028

Positive controls validity:

valid

Remarks:

0.118

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, 1,4 piperazinediethanesulfonic acid disodium salt is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report and should not be classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations

Executive summary:

The objective of this study was to evaluate 1,4 piperazinediethanesulfonic acid disodium salt for its ability to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small model (EPISKIN-SMTM)). The possible skin irritation potential of 1,4 piperazinediethanesulfonic acid disodium salt was tested through topical application for 15 minutes. The study procedures described in this report were based on the most recent OECD and EC guidelines. Skin tissue was moistened with 5 μl of Milli-Q water and at least 10 mg of the test item was applied 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. 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 item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 104%. Since the mean relative tissue viability for the test item was above 50% after 15 ± 0.5 minutes treatment the test item is considered to be non-irritant. The positive control had a mean cell viability of 11% after 15 ± 0.5 minutes exposure compared to the negative control. 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 ≤ 11%,

indicating that the test system functioned properly.

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:

25th to the 26th of August 2017

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Specific details on test material used for the study:

Identification: 1,4 piperazinediethanesulfonic acid disodium salt
Appearance: White powder
Test item storage: At room temperature
Stable under storage conditions until: 30 November 2018 (retest date)

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

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. Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

20% (w/v) solution

Duration of treatment / exposure:

240 +/- 10 minutes

Number of animals or in vitro replicates:

3

Details on study design:

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 (Earle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Fetal 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 +/- 1C. 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 were performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea was read against a cMEM 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.
The medium from the anterior compartment was removed and 750 ul of either the negative control, positive control (20% (w/v) Imidazole solution) or 20% (w/v) solution of the test item was introduced onto the epithelium of the cornea. 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 +/- 1C. After the incubation the solutions were removed and the epithelium was washed at least three times with MEM with phenol red (Earle’s Minimum Essential Medium Life Technologies). Possible pH effects of the test item on the corneas were recorded. Each cornea was inspected visually for dissimilar opacity patterns. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM and the opacity determinations were performed.
Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Sigma-Aldrich, Germany) 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 (Sigma-Aldrich Chemie GmbH, Germany). 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 +/-1C.

Irritation parameter:

in vitro irritation score

Value:

1.7

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

fluorescein leakage

Value:

0.01

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Value:

1.9

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

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 143 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. 1,4 piperazinediethanesulfonic acid disodium salt did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 1.7 after 240 minutes of treatment.

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, since 1,4 piperazinediethanesulfonic acid disodium salt induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Executive summary:

The objective of this study was to evaluate the eye hazard potential of 1,4 piperazinediethanesulfonic acid disodium salt as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test). This report describes the potency of chemicals to induce serious eye damage using isolated bovine corneas. The eye damage of 1,4 piperazinediethanesulfonic acid disodium salt was tested through topical application for approximately 240 minutes. The study procedures described in this report were based on the most recent OECD guideline. Batch of 29040-44484 was a white powder. The test item was applied as a 20% (w/v) solution (750 µl) directly on top of the corneas. 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 143 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. 1,4 piperazinediethanesulfonic acid disodium salt did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 1.7 after 4 hours of treatment. In conclusion, since 1,4 piperazinediethanesulfonic acid disodium salt induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In conclusion, 1,4 piperazinediethanesulfonic acid disodium salt is not corrosive in the in vitro skin corrosion test, is a non-irritant in the in vitro skin irritation test and there was no eye irritation noted in a BCOP study. Therefore, there is no classification is required for skin or eye irritation.