1,2-dihydro-3H-1,2,4-triazol-3-one

Endpoint summary

Description of key information

In a in vitro EPIDERM skin Corrosion Test performed according to the OECD guideline 431 and in compliance with GLP requirement, the corrosivity potential of the test item using the EpiDerm™ Human Skin Model was evaluated after treatment periods of 3 and 60 minutes.

The optical density (OD) was measured at 570 nm (OD570).

The mean OD570 for negative treated tissues was 2.095 and 2.057, respectively, for the 3 and 60 - minute exposure period. The relative mean tissue viability for the positive control treated tissues was 2.8% relative to the negative control following the 60-minute exposure period. The control acceptance criteria were therefore satisfied.

In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied and the test item is considered to be non-corrosive to the skin.

In a Bovine Corneal Opacity and Permeabilty (BCOP) Assay, performed according to the OECD guideline 437 and in compliance with GLP requirement, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability.

The positive control In Vitro Irritancy Score was within the range of 65.1 to 123.3. The positive control acceptance criterion was therefore satisfied.The negative control gave opacity of ≤2.4 and permeability ≤0.072. The negative control acceptance criteria were therefore satisfied.

With a IVIS of 87.8, the test item is classified according to the prediction model as Category 1. UN GHS or EU CLP Causes serious eye damage

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:

19/07/2017-21/07/2017

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)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: hemring/Batch 0727/16
- Expiration date of the lot/batch: 28 April 2019
- Purity test date: 99%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
- Stability under test conditions: The test item was used as supplied.
- Solubility and stability of the test substance in the solvent/vehicle:
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was used as supplied.
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material)

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)

OTHER SPECIFICS:

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Not specified

Source strain:

other: Epidermm Reconstructed Human Epidermis Model Kit

Details on animal used as source of test system:

NA

Vehicle:

unchanged (no vehicle)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Duration of treatment / exposure:

3 and 60 minutes

Number of replicates:

2

Species:

other: NA

Strain:

other: NA

Details on test animals or test system and environmental conditions:

NA

Type of coverage:

other: NA

Preparation of test site:

other: NA

Vehicle:

other: NA

Controls:

other: NA

Amount / concentration applied:

NA

Duration of treatment / exposure:

NA

Observation period:

NA

Number of animals:

NA

Details on study design:

NA

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

at 3 min

Value:

>= 87.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not valid

Remarks on result:

other: NA

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

at 60 min

Value:

>= 78

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: NA

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: The MTT solution containing the test item did not turn blue/purple. This was taken to indicate the test item did not reduce MTT
- Direct-MTT reduction: The MTT solution containing the test item did not turn blue/purple. This was taken to indicate the test item did not reduce MTT
- Colour interference with MTT: The solution containing the test item did not become colored. This was taken to indicate the test item did not have the potential to cause color interference.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
yes


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline:

REMINDER ON QUALITY CRITERIA
The results of the assay are considered acceptable if the following assay acceptance criteria are achieved:
Negative Control
The absolute OD570 of the negative control treated tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. The mean OD570 of the two negative control tissues should be ≥ 0.8 and ≤ 2.8 for each exposure time, which ensures that the tissue viability meets the acceptance criteria.
Positive Control
Potassium Hydroxide 8.0N solution is used as a positive control. An assay meets the acceptance criterion if mean relative tissue viability of the 60 minute positive control is < 15%.
Coefficient of Variation
In the range 20 and 100% viability, the Coefficient of Variation between tissue replicates should be ≤ 30%.

Mean OD570 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Tissue	Exposure period	Mean OD570 of individual tissues	Mean OD570 of duplicate tissues	Standard deviation	Coefficent of Variation (%)	Relative Mean Viability
Negative control	3 minutes	2.000	2.098	0.134	6.4	100
		2.190
	60 minutes	2.122	2.057	0.092	4.5
		1.992
Positive control	3 minutes	0.063	0.069	0.008	na	3.3
		0.074
	60 minutes	0.058	0.057	0.002	na	2.8
		0.055
Test item	3 minutes	1.889	1.827	0.088	4.8	87.2
		1.765
	60 minutes	1.638	1.604	0.048	3.0	78.0
		1.570

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was considered to be non-corrosive to the skin.
Classification of corrosivity potential is based on relative viabilities for both exposure times according to the EU CLP Regulation (EC) No 1272/2008 UN GHS (cf table in the study report).

Executive summary:

In a in vitro EPIDERM skin Corrosion Test (Lacey F.E., 2018), performed according to the OECD guideline 431 and in compliance with GLP requirement, the corrosivity potential of the test item using the EpiDerm™ Human Skin Model was evaluated after treatment periods of 3 and 60 minutes.

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative (sterile distilled water) and positive (8.0N Potassiumm hydroxyde) control groups were treated for each exposure period. At the end of the exposure period, the test item was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT loading each tissue was placed in 2 mL Isopropanol for MTT extraction.

At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density (OD) was measured at 570 nm (OD570).

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

The mean OD570 for the negative control treated tissues was 2.095 for the 3-Minute exposure period and 2.057 for the 60-Minute exposure period. The negative control acceptance criteria were therefore satisfied.

The relative mean tissue viability for the positive control treated tissues was 2.8% relative to the negative control following the 60-Minute exposure period. The positive control acceptance criterion was therefore satisfied.

In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.

The quality criteria required for acceptance of results in the test were satisfied.

The test item was considered to be non-corrosive to the skin.

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:

26/09/2017-26/09/2017

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)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Chemring/Batch 0727/16
- Expiration date of the lot/batch: 28 April 2019
- Purity test date: 99%
- Physical state/Appearance: off white powder

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark
- Stability under test conditions: For the purpose of this study the test item was used as supplied.
- Solubility and stability of the test substance in the solvent/vehicle:
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: For the purpose of this study the test item was used as supplied.
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material)

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)

OTHER SPECIFICS:

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: adult cattle, from a local abattoir as a by-product from freshly slaughtered animals
- Age at study initiation: 12-60 months old

The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Approximately 0.725 g of the solid test item was found to adequately cover the corneal surface.

Duration of treatment / exposure:

240 minutes (4 hours)

Observation period (in vivo):

NA

Number of animals or in vitro replicates:

Three corneas were randomly allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.

Details on study design:

At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed three times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post-treatment opacity reading was taken and each cornea was visually observed.

Irritation parameter:

in vitro irritation score

Run / experiment:

None

Value:

>= 87.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not valid

Remarks on result:

other: NA

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system:

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes


Opacity Measurement
The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.

Permeability Measurement
The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.

In Vitro Irritancy Score
The following formula was used to determine the In Vitro Irritancy Score:
In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value)
Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced a response through only one of the two endpoints.

Visual Observation
The condition of the cornea was visually assessed post treatment.

CRITERIA FOR AN ACCEPTABLE TEST
For an acceptable test the following positive control criterion should be achieved:
20% w/v Imidazole was used for positive control purposes.
The test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the historical mean during 2016 for this testing facility. Therefore the In Vitro Irritancy Score should fall within the range of 65.1 to 123.3.
For an acceptable test the following negative control criteria should be achieved:
Sodium chloride 0.9% w/v was used for negative control purposes. The test was acceptable if the negative control produced an In Vitro Irritancy Score which is less than or equal to the upper limit for background opacity and permeability values during 2016 for bovine corneas treated with the respective negative control. When testing solids the negative control limit for opacity should be ≤2.4 and for permeability ≤0.072.

Corneal Opacity and Permeability Measurement

Individual and mean corneal opacity measurements and individual and mean corneal permeability measurements are given in Appendix 1 of this sutdy report.

Corneal Epithelium Condition

The condition of each cornea is given in Appendix 2 of the study report.

The corneas treated with the test item were cloudy post treatment. The corneas treated with the negative control item were clear post treatment. The corneas treated with the positive control item were cloudy post treatment.

In Vitro Irritancy Score

The In Vitro irritancy scores are summarized as follows:

Treatment	In vitro Irritancy Score
Test item	87,8
Negative control	0,6
Positive control	109,7

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

With a IVIS of 87,8, the test item is classified according to the prediction model as Category 1. UN GHS or EU CLP Causes serious eye damage.

IVIS Classification
> or=3 No category. Not requiring classification to UN GHS or EU CLP
>3; < or= 55 No prediction of eye irritation can be made
>55 Category 1. UN GHS or EU CLP Causes serious eye damage

Executive summary:

In a Bovine Corneal Opacity and Permeabilty (BCOP) Assay (Henzell G., 2018), performed according to the OECD guideline 437 and in compliance with GLP requirement, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability.

The undiluted test item was applied for 240 minutes. Negative and positive control items were tested concurrently.The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

The positive control In Vitro Irritancy Score was within the range of 65.1 to 123.3. The positive control acceptance criterion was therefore satisfied.The negative control gave opacity of ≤2.4 and permeability ≤0.072. The negative control acceptance criteria were therefore satisfied.

With a IVIS of 87.8, the test item is classified according to the prediction model as Category 1. UN GHS or EU CLP Causes serious eye damage

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Triazolone is not classified for skin irritation but is classified as a Category 1 eye irritant