[No public or meaningful name is available]

Endpoint summary

Description of key information

The substance was tested for skin corrosion and eye irritation according to OECD TG 431 and OECD TG 437. Both studies were carried out under GLP. Skin corrosion potential of the substance was identified. Additionally, the substance turned out as an eye irritant and is classified into UN GHS Category 1.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14.06.-05.08.2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

18 June 2019

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: The EpiDerm™ tissues were provided as kits (e.g. EPI-200-SIT, MatTek)

Source strain:

other: not applicable

Details on animal used as source of test system:

not applicable

Justification for test system used:

The EpiDerm™ Skin Model is a well-established organotypic, three-dimensional model of the human epidermis and is used for in vitro experiments since many years. It is known for its similarity to human skin.

Vehicle:

unchanged (no vehicle)

Remarks:

The test item was applied directly atop the EpiDerm™ tissue using an application spoon avoiding compression of the test item. To ensure good contact with the skin the test item was moistened with 25 μL H2O.

Details on test system:

Upon receipt of the EpiDerm™, the tissues were inspected visually and transferred into 6-well plates containing 0.9 mL pre-warmed assay medium per well. The 6-well plates were pre-incubated in a humidified incubator at 37 +/- 1 °C, 5.0% CO2 / 95% air for at least 1 h. Then the medium was replaced by 0.9 mL fresh assay medium and the surface was dried using a sterile cotton tip. The 6-well plate used for the 3 min experiment was placed back into the incubator. The other plate was used for the 60 min treatment. About 1 h before the end of the first treatment period the MTT solution was prepared and pre-warmed in the incubator.

60 min experiment: the tissues were treated with each dose group in duplicate, starting with the
negative control. Start time was recorded with dosing of the first tissue. A constant time interval of e.g. 20 sec. was kept between dosing. Then the 6-well plate was incubated at 37 +/- 1 °C, 5.0% CO2 / 95% air.

3 min experiment: the tissues were treated with each dose group in duplicate, starting with the
negative control. Start time was recorded with dosing of the first tissue. A constant time interval of 20 sec. was kept between dosing.
After 3 min of application, the first insert was removed from the 6-well plate with forceps. Using a wash bottle, the tissue was gently rinsed about 20 times with PBS (phosphate buffered saline) to remove any residual test item. Excess PBS was removed by gently shaking the insert and blotting bottom with blotting paper. The insert was placed in a prepared 24-well “holding plate“ containing 300 μL prewarmed assay medium per well. All inserts were treated in the same manner.
Then the inserts were dried again and transferred into a prepared 24-well “MTT assay plate“ containing 300 μL pre-warmed MTT solution. The plate was incubated for 3 h at 37 +/- 1 °C, 5.0% CO2 / 95% air.

60 min experiment: after 60 min application, the first insert was removed from the 6-well plate with forceps. Using a wash bottle, the tissue was gently rinsed about 20 times with PBS to remove any residual test item. Excess PBS was removed by gently shaking the insert and blotting bottom with blotting paper. The insert was placed in a prepared 24-well “holding plate“ containing 300 μL prewarmed assay medium per well. All inserts were treated in the same manner. Then the inserts were dried again and transferred into a prepared 24-well “MTT assay plate“ containing 300 μL pre-warmed MTT solution. The plate was incubated for 3 h at 37 +/- 1 °C, 5.0% CO2 / 95% air.

3 min and 60 min experiment: after the 3 h MTT incubation period the MTT solution was aspirated. The wells were refilled with PBS and the PBS was aspirated. The rinsing was repeated twice and the tissues were dried. Then the inserts were transferred into 12-well “extraction plates“. 2 mL of isopropanol were pipetted into each insert: thus, the insert was covered from both sides.
The extraction plates were sealed in zip-bags to inhibit isopropanol evaporation. Extraction was carried out either over night without shaking at room temperature or, alternatively, at least 2 h with shaking at room temperature.
After the extraction period the inserts were pierced with an injection needle to allow the extracts to run through the tissues into the corresponding wells. Then the inserts were discarded and the extraction plates were placed on a shaker for 15 min.
Per each tissue 3 x 200 μL aliquots of the extract were transferred into a 96-well plate and OD was
measured at 570 nm without reference wavelength in a plate spectrophotometer using isopropanol as
a blank.


MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 min, 60 min
- Spectrophotometer: plate spectrophotometer
- Wavelength: 570 nm

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 mg + 25 μL H2O

Duration of treatment / exposure:

3 min, 60 min

Number of replicates:

2 tissues per time time point

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes exposure

Value:

ca. 13

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes exposure

Value:

ca. 102.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Test results are attached as pdf file

Conclusions:

The test item showed corrosive effects. The mean relative tissue viability (% negative control) was reduced below 15% after 60 min treatment but not below 50% after 3 min treatment. The test item is therefore classified as “corrosive“ in accordance with a combination of optional UN GHS sub-categories 1B and 1C.

Executive summary:

The potential of the test item to induce skin corrosion was analysed by using the three-dimensional
human skin model EpiDerm™, comprising a reconstructed epidermis with a functional stratum
corneum.
In the present study Dibuthyltetraethylguanidinium chloride was applied topically to the EpiDerm™ tissue for 3 min and 60 min followed by immediate determination of cytotoxic effects via MTT reduction
assay.
Corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained
after both treatment periods had been compared to the corresponding negative control tissues.
The test item has no non-specific MTT-reducing or colouring potential, therefore no additional controls
were necessary.
The test item showed corrosive effects. The mean relative tissue viability (% negative control) was
reduced below 15% (13.0%) after 60 min treatment but not below 50% (102.3%) after 3 min treatment.
The controls confirmed the validity of the study. The mean OD570nm of the two negative control tissues
was ≥ 0.8 and ≤ 2.8 for each exposure period (1.760, 1.747). The mean relative tissue viability
(% negative control) of the positive control was ≤ 15% (6.1%) after 60 min treatment. The coefficient
of variation (CV) (in the range of 20 – 100% viability) of replicate tissues of all dose groups was ≤ 30%
(0.6% - 4.0%).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (corrosive)

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:

2021-07-26 to 2021-07-28

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 i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

26 June 2020 (adopted)

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

The assay uses isolated corneas obtained as a by-product from animals freshly slaughtered.
On the test day, fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories. Immediately after arrival of the eyes, cornea preparation was initiated.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

750 µL of the test item preparation or the control substance was introduced into the anterior chamber

Duration of treatment / exposure:

4 hours ± 5 minutes

Number of animals or in vitro replicates:

3 corneas for the test item
3 corneas as negative controls
3 corneas as positive control

Details on study design:

The eyes were carefully examined for defects and any defective eyes were discarded.
The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing HBSS. Before the corneas were mounted in corneal holders (Duratec GmbH) with the endothelial side against the O-ring of the posterior chamber, they had been visually examined for defects and any defective cornea had been discarded. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with RPMI 1640 medium (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI 1640 medium). The posterior chamber was always filled first. The corneas were incubated for one hour at 32 +/- 1 °C.

After the equilibration period, the medium was removed from both chambers and replaced with fresh complete RPMI 1640 medium. An initial measurement was performed on each of the corneas using the opacitometer. Three corneas with illuminance readings approximately equivalent to the median illuminance of all corneas were selected as negative-control corneas. The illuminance of each cornea was read and recorded. Only corneas that had an initial illuminance reading I > I0/1.1651 lux were used for the assay. The medium was removed from the anterior chamber and replaced with the test item or control.
750 µL of the test item preparation or the control substance was introduced into the anterior chamber (closed-chamber method). After 4 hours ± 5 minutes incubation at 32 +/- 1 °C either the test substance or the control substance was removed and the epithelium washed at least three times with MEM (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI 1640 medium (without phenol red). The anterior chamber was refilled with complete RPMI 1640 medium and an illuminance measurement was performed. Also, each cornea was observed visually and pertinent observations were recorded.
After the illuminance measurement was performed, the medium was removed from both chambers of the holder. The posterior chamber was refilled with fresh complete RPMI 1640 medium. 1 mL of a 5 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes at 32 +/- 1 °C. Then the medium from the posterior chamber was removed and its optical density at 490 nm (OD490) was determined, using a spectrophotometer (Jenway 6405 UV/VIS).

Irritation parameter:

in vitro irritation score

Value:

ca. 131.57

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Test results are attached as pdf file

Conclusions:

According to the evaluation criteria the test item Dibutyltetraethylguanidinium chloride is classified into UN GHS Category 1.

Executive summary:

The eye irritancy potential of Dibutyltetraethylguanidinium chloride was investigated in the bovine corneal opacity and permeability assay.
Preparation of the test item: The test item was suspended with physiological saline 0.9% NaCl to give a 20% concentration.
Visual observation after treatment: All 3 corneas treated with Dibutyltetraethylguanidinium chloride showed complete opacity of the tissue.
Mean in vitro irritation score: 131.57
Classification: UN GHS Category 1
The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid.
The negative control responses resulted in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)