2-Propanol, 1,3-bis[(3-methyl-2-buten-1-yl)oxy]-

Endpoint summary

Description of key information

Based on the results of two skin in vitro tests it was concluded that DPNG fulfilles the CLP classification criteria as skin irritant category 2, H315

Based on the results of two eye in vitro tests it was concluded that DPNG fulfilles the CLP classification criteria as eye irritant category 2, H319

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-07-29 to 2019-08-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted : 2015-07-28

Deviations:

no

GLP compliance:

yes

Test system:

human skin model

Remarks:

EpiDerm Sit (EP-200)

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Not specified

Source strain:

not specified

Justification for test system used:

EPI-200 SIT kit is recommended in the test method.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPI-200 SIT
- Tissue lot number: 30926
- Production date: 2019-07-25
- Date of initiation of testing: 2019-07-29

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After the exposure, each tissue insert was rinsed fifteen times with PBS(-). Furthermore, tissue inserts were completely submerged three times in 150 mL of PBS(-). Finally, tissue inserts were rinsed once inside and outside with PBS(-). Remaining PBS(-) was removed from inside and outside of tissue insert. The tissue inserts were transferred into the upper wells of new 6-well pates filled with 0.9 mL/well of fresh medium.
- Observable damage in the tissue due to washing: Not reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
PRE-TEST
- MTT concentration: 30µL / mL MTT medium
- Incubation time: 60 minutes
NYLON MESH
- 30µl of test substance was dropped onto a nylon mesh on a glass slide. After 60 minutes at room temp., the corrosion of the nylon mesh was evaluated microscopically. Corrosion was not observed.
MAIN TST
-MTT concentration: 0.3 mL/well of MTT medium
-Incubation time: 180+- 5 minutes
- Spectrophotometer: Multimode Microplate Reader (FlUOstar Omega, BMG LABTECH) at 570 nm


FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability positive control: Mean ± SD: 2.0 ± 0.3 %; Minimum: 1.3 %; Maximum: 2.6 %
- OD negative control: Mean ± SD: 2.071 ± 0.196; Minimum: 1.787; Maximum: 2.397
- OD positive control: Mean ± SD: 0.042 ± 0.007; Minimum: 0.024; Maximum: 0.062

NUMBER OF REPLICATE TISSUES: 3; 2 to for tissue-binding test

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues
- N. of replicates : 3
- Method of calculation used: The mean of blank was subtracted from ODs of each tissue inserts and the mean value was calculated in each tissue insert to obtain PD of each tissue insert. The cell viability of each tissue was calculated by the following formula:
Cell viability (%) = (OD of each tissue insert of each treatment group : Mean OD of the negative control substance group) × 100
The mean and standard deviation (SD) of cell viabilities in each treatment group were calculated by the cell viability of each tissue insert.

NUMBER OF INDEPENDENT TEST EXPERIMENTS TO DERIVE FINAL PREDICTION: 2

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the viability greater or equal than 50% (UN GHS Category: 1or 2)
- The test substance is considered to be non-irritant to skin if the viability is less than 50% (UN GHS Category: not classified)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL/CONTROLS:
- Amount applied: 30 µL

Duration of treatment / exposure:

-test substance applied onto each tissue surface at 1 minutes interval

Duration of post-treatment incubation (if applicable):

After the last tissue insert was exposed, all plates were incubated for 35 ± 1 minutes.
All plates were taken out of the incubator and placed at room temperature until 60 ± 1 minutes was completed for the first exposed tissue insert.

Number of replicates:

3

Species:

other: EpiDerm tissue

Strain:

not specified

Details on test animals or test system and environmental conditions:

TEST KIT
- Source: MatTek Corporation
- Receipt date: 2019-07-29

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 37
- Humidity (%): humid conditions

Type of coverage:

not specified

Preparation of test site:

not specified

Vehicle:

not specified

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL/NEGATIVE CONTROL/POSITIVE CONTROL
- Amount(s) applied: 30 µL

Duration of treatment / exposure:

1 minute

Observation period:

After the last tissue insert was exposed, all plates were incubated for 35 ± 1 minutes.
All plates were taken out of the incubator and placed at room temperature until 60 ± 1 minutes was completed for the first exposed tissue insert.

Number of animals:

n/a

Irritation / corrosion parameter:

% tissue viability

Value:

5.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

As a result of skin irritation test, the cell viability treated by DPNG was 5.3%, falling below 50% which the judgement criteria of skin irritation.

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative/positive control: The mean OD in the negative control substance was 1.823. The mean cell viability in the positive control substance was 2.0%.
- Acceptance criteria met for variability between replicate measurements: The SDs of cell viabilities in the negative and the positive control substances, and the test substance were 6.7%, 0.2% and 2.7%, respectively.
- Range of historical values: Cell viability positive control [%]: 2.0 ± 0.3

As a result of tissue-binding test, the staining ratio was 0.2%, it was < 5%. Therefore, correction of OD was not conducted.

The mean OD in the negative control substance was 1.823. The mean cell viability in the positive control substance was 2.0%. The SDs of cell viabilities in the negative and the positive control substances, and the test substance were 6.7%, 0.2% and 2.7%, respectively. These results indicated that the present study was appropriately performed.

As a result of skin irritation test, the cell viability treated by DPNG was 5.3%, falling below 50% which the judgement criteria of skin irritation.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Remarks:

As a result of skin irritation test, the cell viability treated by DPNG was 5.3%, falling below 50% which the judgement criteria of skin irritation.

Conclusions:

It was concluded that DPNG was "Irritant" (UN GHS Category 1 or 2) under the present test conditions.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

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:

Adopted: 2019-06-18

Deviations:

no

GLP compliance:

yes

Test system:

human skin model

Remarks:

EpiDerm SCT kit

Cell type:

non-transformed keratinocytes

Cell source:

other: donor - All cells used to produce EpiDermTM are purchased or derived from tissue obtained by MatTek Corporation from accredited lnstitutions, ln all cases, consent was obtained by these Institutions from the donor or the donor's legal next of kin, for use

Source strain:

not specified

Justification for test system used:

EpiDerm SCT kit is recommended in the test method.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm SCT kit
- Tissue batch number: 32116
- Production date: 2019-11-07
- Delivery date: 2019-11-11
- Date of initiation of testing: 2019-11-08

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After the exposure, each tissue twenty times with PBS(-). Inside and outside the tissue inserts were wiped with a sterile cotton swab. The tissue inserts were placed into a 24-well plate (Corning) filled with 300 µL of fresh medium.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
PRE-TEST
- MTT concentration: 1mg MTT/ml MTT medium
- 50 µl test substance + 1 ml MTT medium
- Incubation time: 60 minutes

NYLON MESH
- 30µl of test substance was dropped onto a nylon mesh on a glass slide. After 60 minutes at room temp., the corrosion of the nylon mesh was evaluated microscopically. Corrosion was not observed.

MAIN TEST
- MTT concentration: 1mg MTT/ml MTT medium
- 50µl test substance or 50 µl control substance per vial
- Incubation time: 180+- 5 minutes
- Spectrophotometer: Multimode Microplate Reader (FlUOstar Omega, BMG LABTECH) at 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- OD:
3 min exposure:
Negative control: 2.004 ± 0.166
Positive control: 0.157 ± 0.049
60 min exposure:
Negative control: 1.951 ± 0.152
Positive control: 0.053 ± 0.020

NUMBER OF REPLICATE TISSUES: 2

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the mean cell 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:

TEST MATERIAL/NEGATIVE CONTROL/POSITIVE CONTROL
- Amount applied: 50 µL

Duration of treatment / exposure:

3 minutes and 60 minutes

Duration of post-treatment incubation (if applicable):

180 minutes

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60-minutes

Value:

89.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: It is concluded that DPNG was "Non-corrosive" (UN GHS Category 2 or not classified) under the present test conditions.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3-minutes

Value:

103.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: It is concluded that DPNG was "Non-corrosive" (UN GHS Category 2 or not classified) under the present test conditions.

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes: The ODs in the negative control substance group in the 3-minute and 60-minute exposures were 1.848 and 1.689, respectively.
- Acceptance criteria met for positive control: Yes: The mean cell viability in the positive control substance group in the 60-minute exposure was 2.8%.
- Acceptance criteria met for variability between replicate measurements: The CVs between the two tissue inserts were = 30% for all substances which the cell viability was from 20% to 100%.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

As a result of skin corrosion test in the 3-minute and 60-minute exposures, the cell viabilities treated by the test substance were 103.6% and 89.4%, respectively. It is concluded that DPNG was "Non-corrosive" (UN GHS Category 2 or not classified) under the present test conditions.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation, other

Type of information:

calculation (if not (Q)SAR)

Adequacy of study:

weight of evidence

Study period:

2021

Reliability:

1 (reliable without restriction)

Justification for type of information:

WoE to derive the classification based on in vitro testing

Qualifier:

according to guideline

Guideline:

other: weight of evidence according to the OECD Integrated Approaches to Testing and Assessment (IATA) for serious eye daman and eye irritation (OECD Guidance Document 263)

Irritation parameter:

mean percent tissue viability 

Run / experiment:

RhCE assay

Value:

27.4

Irritation parameter:

in vitro irritation score

Remarks:

BCOP-Test

Value:

8.66

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

The combination of results from these two assays and the potential of the substance to cause skin irritation leads to the conclusion that DPNG is an eye irritant but does not qualify as a Category 1 irritant. By default, the appropriate designation is Category 2.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-03-31 to 2021-03-31

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Abattoir Vion Beef B.V., Buchloe, Germany (isolated corneas obtained as by-product from animals freshly slaughered)
- Storage, temperature and transport conditions of ocular tissue: transported in HBSS containing Pen/Strep on ice to the laboratory.
- Time interval prior to initiating testing: Immediately after arrival of the eyes, cornea preparation was initiated.
- Selection and preparation of corneas: 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. The corneas were incubated for one hour at 32 ± 1 °C.

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

750 µg/L of the test substance or the control substance was introduced into the anterior chamber.

Duration of treatment / exposure:

After 10 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 after 2 hours incubation at 32 +- 1 °C. Also, each cornea was observed visually and pertinent observations were recorded.

Duration of post- treatment incubation (in vitro):

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 4 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes at 32 ± 1 °C.

Number of animals or in vitro replicates:

3 corneas for the test item
3 corneas as negative controls treated with physiological saline 0.9% NaCl
3 corneas as positive control treated with ethanol 100%

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
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. 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 (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI). The posterior chamber was always filled first. The corneas were incubated for one hour at 32 ± 1 °C.

QUALITY CHECK OF THE ISOLATED CORNEAS:
Only corneas that had an initial illuminance reading I > I0/1.1651 lux were used for the assay. Eyes that were noted to have defects were discarded and not used on the study.

NUMBER OF REPLICATES: 3
NEGATIVE CONTROL USED: yes
POSITIVE CONTROL USED: yes
APPLICATION DOSE AND EXPOSURE TIME: 750 µg/L and 10 minutes
REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: The epithelium was washed at least three times with MEM (containing phenol red). Once the medium was free of test item, the cornea was finally rinsed with complete RPMI (without phenol red).
- POST-EXPOSURE INCUBATION: 2 hours at 32 ± 1 °C

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacimeter (BASF-OP3.0, Duratec GmbH)
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry
- Others: Pertinent observations
SCORING SYSTEM: In Vitro Irritancy Score (IVIS).
This was detailed in the study report as follows:
= 3 = no category
> 3; = 55 = no stand-alone prediction can be made
>55 = Category 1.
An identification of test substances that should be classified as irritating to eyes (UN GHS Category 2 or Category 2A) or test substances that should be classified as mildly irritating to eyes (UN GHS Category 2B) cannot be made.

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used: Yes.

Irritation parameter:

in vitro irritation score

Value:

8.66

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The eye irritancy potential of 1,3-di(3-Methyl-2-butenoxy)-2-hydroxypropane was investigated in the bovine corneal opacity and permeability assay.
The test item was tested as provided by the sponsor.
The tissue of the 3 corneas treated with 1,3-di(3-Methyl-2-butenoxy)-2-hydroxypropane showed little whitish threads (vein type).
The following mean in vitro irritation score was calculated:
8.66
No stand-alone prediction can be made regarding the classification of the test substance 1,3-di(3-Methyl-2-butenoxy)-2-hydroxypropane according to the evaluation criteria. Further testing in another suitable method is required.
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 should result in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

Opacity

Cornea No.	Test Item	Initial Opacity	Final Opacity	Change of Opacity Value	Corrected Opacity Value
1		2.66	4.52	1.86
2	Negative	3.03	5.41	2.38
3	Control	2.88	3.89	1.02
MV		2.85	4.61	1.75
4		5.20	27.15	21.94	20.19
5	Positive	1.42	26.07	24.65	22.90
6	Control	1.53	21.34	19.81	18.06
MV		2.72	24.85	22.13	20.38
7		3.32	16.33	13.01	11.26
8	Test Item	2.77	11.23	8.46	6.71
9		3.14	13.26	10.12	8.37
MV		3.08	13.61	10.53	8.78

MV = mean value

Permeability

Cornea No.	Test Item	OD490	Corrected OD490 Value
1		0.021
2	Negative	0.028
3	Control	0.036
MV		0.028
4		0.696	0.668
5	Positive	1.173	1.145
6	Control	1.292	1.264
MV		1.054	1.025
7		0.013	-0.015
8	Test Item	0.032	0.004
9		0.016	-0.012
MV		0.020	-0.008

MV = mean value

 

In Vitro Irritation Score

Cornea No.	Test Item	Corrected Opacity	Corrected OD490 Value	IVIS
1		1.86	0.021
2	Negative	2.38	0.028
3	Control	1.02	0.036
MV		1.75	0.028	2.18
4		20.19	0.668
5	Positive	22.90	1.145
6	Control	18.06	1.264
MV		20.38	1.025	35.76
7		11.26	-0.015
8	Test Item	6.71	0.004
9		8.37	-0.012
MV		8.78	-0.008	8.66

MV = mean value

 

Historical Mean In Vitro Irritation Score of the Positive Control

	IVIS Positive Control - Ethanol 100 %
Mean Value (MV)	47.24
Standard Deviation (SD)	8.86
MV- 2xSD	29.52
MV+2xSD	64.95
Number of Replicates providing Historical Mean: 74

Positive controls are updated after every single experiment or at least every 3 months

Interpretation of results:

other: No stand-alone prediction can be made regarding the classification of the test substance 1,3-di(3-Methyl-2-butenoxy)-2-hydroxypropane according to the evaluation criteria. Further testing in another suitable method is required.

Conclusions:

No stand-alone prediction can be made regarding the classification of the test substance 1,3-di(3-Methyl-2-butenoxy)-2-hydroxypropane according to the evaluation criteria. Further testing in another suitable method is required.

Reference 3

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-07-16 to 2019-07-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

Adopted: 2018-06-25

Deviations:

no

GLP compliance:

yes

Species:

other: human keratinocytes

Strain:

other: 4F1188

Details on test animals or tissues and environmental conditions:

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live:
- RhCE tissue, including batch number: EpiOcular tissue (tissue insert, Lot number: 28038, manufactured on 2019-07-11)

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 µL mixed

Duration of treatment / exposure:

Each plate was incubated until 30 ± 2 minutes was completed for the first exposed tissue insert in each plate.

Duration of post- treatment incubation (in vitro):

After the incubation, each tissue insert was completely submerged three times per beaker into three beakers filled with about 100 mL/beaker of PBS(-) for rinsing. After rinsing, PBS(-) was removed from the tissue surface.
The tissue inserts were transferred into a 12-well plate (Corning) filled with 5 mL/well of the fresh medium and soaked 12 ± 2 minutes.
After the soak, the medium was removed from the tissue surface. The tissue inserts were transferred into 6-well plates filled with 1 mL/well of the fresh medium and incubated for 120 ± 15 minutes.
All tissue inserts were transferred into a 24-well plate (Coming) filled with 0.3 mL/well of MTT medium and incubated for 180 ± 15 minutes.

Number of animals or in vitro replicates:

Duplicate tissue inserts were used for the test substance, negative control substance and positive control substance, respectively. Duplicate tissue inserts were used to check the tissue-binding of the test substance (tissue-binding test).

Details on study design:

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable)

Exposure: 30 ± 2 minutes
Post-exposure immersion: 12 ± 2 minutes
Post-exposure incubation: 120 ± 15 minutes

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: Methylthioglycolate, Hydroxyethylacrylate, 2,5-Dimethyl-2,5-hexanediol, Sodium oxalate, 2,4,11,13-Tetraazatetradecane-diimidamide, N,N''-bis(4-chlorophenyl)-3,12-diimino-, di-D-gluconate (20%, aqueous), Sodium benzoate, Dietely, toluamide, 2,2-Dimethyl-3-methylenebicyclo[2.2.1]heptane, 1-Ethyl-3-methylimidazolium ethylsulphate, Dicaprylyl ether, Piperonyl butoxide, Polyethylene glycol (PEG-40) hydrogenated castor oil, 1-(4-Chlorophenyl)-3-(3,4-dichlorophenyl)urea, 2,2'-Methylene-bis-(6-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)-phenol), potassium tetrafluoroborate
- Number of tissue replicates used per test chemical and controls: 2
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): 570 nm

- For hCE cells: data of QC check
Tissue viability: pass (1.208 ± 0.014); acceptance criteria: OD (540-570 nm) [0.839 - 3,000]
Barrier function: pass (34:05 min); acceptance criteria: OD (540-570 nm) [12.67-36.53 min]
Sterility: pass (no contamination)

- Description of evaluation criteria:
Mean cell viability = 60%: Irritant (UN GHS Category: 1 or 2)
Mean cell viability > 60%: Non-irritant (UN GHS Category: not classified)

- Complete supporting information for the specific RhCE tissue construct or hCE cells used: OCL-200 EIT kit (MatTek Corporation)
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals
- Positive and negative control means and acceptance ranges based on historical data:
OD negative control: 1.399 ± 0.335
OD positive control: 0.354 ± 0.088
Cell viability positive control: 25.7 ± 5.2%

Irritation parameter:

percent tissue viability 

Value:

27.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes (mean OD = 1.505)
- Acceptance criteria met for positive control: yes (mean cell viability = 26.7%)

The test results are shown in Tables 1 and 2.

As a result of the tissue-binding test, the staining ratio was 0.3 % that was 60%. However, the cell viability in the test substance was ≤ 60%.   Therefore, correction of the cell viability was not conducted.

The mean OD in the negative control substance group was 1.505. The mean cell viability in the positive control substance group was 26.7%. The differences of two cell viabilities in the negative and the positive control substances, and the test substance were 1.0%, 3.9% and 3.3%, respectively. These results indicated that the present study was appropriately performed.

As a result of the eye irritation test, the cell viability treated by DPNG was 27.4%, falling below 60% which was the judgement criteria of eye irritation.

Interpretation of results:

other: No prediction can be made

Conclusions:

Mean cell viability is >= 60%. No stand-alone prediction can be made regarding the classification of the test substance.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

As a result of an in vitro skin irritation test (OECD guidance 439), the cell viability treated by DPNG was 5.3% falling below 50% which the judgement criteria of skin irritation. As a result of skin corrosion test in the 3-minute and 60-minute exposures, the cell viabilities treated by the test substance were 103.6% and 89.4%, respectively, leading to the conclusion that DPNG is non-corrosive. Therefore DPNG was classified as skin irritant category 2.

The eye irritation assessment of DPNG was based on the results from two separate in vitro studies, the BCOP (OECD 437) and RhCE (OECD test 431).  The results from both tests indicate DPNG has eye irritation potential but does not qualify as a Category 1 irritant.