Reaction mass of N,N,N',N'-tetrabutylmethylenediamine and dibutylamine

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 March - 21 August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EC) No 761/2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N,N,N',N'-tetrabutylmethylenediamine

EC Number:

243-678-5

EC Name:

N,N,N',N'-tetrabutylmethylenediamine

Cas Number:

20280-10-8

Molecular formula:

C17H38N2

IUPAC Name:

dibutyl[(dibutylamino)methyl]amine

Constituent 2

Reference substance name:

Dibutylamine

EC Number:

203-921-8

EC Name:

Dibutylamine

Cas Number:

111-92-2

Molecular formula:

C8H19N

IUPAC Name:

1-Butanamine, N-butyl-

Test material form:

liquid

Details on test material:

Clear, light yellow liquid
Storage: 2 to 8 °C, in the dark
Expiry date: 01-Jan-19

Specific details on test material used for the study:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 2-8 ºC, in darkness
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: N/A
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No, investigations of MTT and colour interference concluded that the test item did not interfere with the test system.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: N/A
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material): Applied as supplied

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable): N/A

OTHER SPECIFICS: N/A

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiDermTM SIT (EPI-200) three-dimensional human skin model, (lot 25800) comprising a reconstructed epidermis with a functional stratum corneum, supplied by MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

Source strain:

other: N/A

Details on animal used as source of test system:

N/A

Justification for test system used:

Guideline specific test system

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm SIT (EPI-200) 3D human skin model (supplied by MatTek In Vitro Life Science, Slovakia)
- Tissue batch number(s): 25803
- Production date: 05 April 2017
- Shipping date: 05 April 2017
- Delivery date: 05 April 2017
- Date of initiation of testing: 05 April 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 ºC
- Temperature of post-treatment incubation (if applicable): 37 ºC

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Rinsed with PBS (minimum of 3 rinses)
- Observable damage in the tissue due to washing: No
- Modifications to validated SOP: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: not reported
- Wavelength: 570 nm
- Filter: Not reported
- Filter bandwidth: Not reported
- Linear OD range of spectrophotometer: Not reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: acceptable (provided in CoA; OD540-570 between 1.0-3.0)
- Barrier function: acceptable (provided in CoA; ET-50 between 4.77 and 8.72 h)
- Contamination: acceptable (provided in CoA; no contamination - screened for HIV-1-virus; Hep B and Hep C; bacteria, yeast and other fungi)

NUMBER OF REPLICATE TISSUES: 3 per test group

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- n/a

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after exposure is ≤ 50 %, realtive to the negative control.
- The test substance is considered to be non-irritant to skin if the viability after exposure is > 50 %, relative to the negative control.
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439: N/A, as per OECD 439 guidance

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 µL
- Concentration (if solution): Undiluted

VEHICLE
- Amount(s) applied (volume or weight with unit): N/A
- Concentration (if solution): N/A
- Lot/batch no. (if required): N/A
- Purity: N/A

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): phosphate buffered saline (PBS) - concentration not reported

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5 % w/v

Duration of treatment / exposure:

60 mins exposure

Duration of post-treatment incubation (if applicable):

Post exposure and rinsing the tissues were placed on the appropriate medium and incubated for 41 hours and 20 minutes.

Upon completion of the recovery period, each tissue was rinsed with PBS before being placed on 0.3 mL of 1 mg/mL MTT in PBS and incubated for three hours at 37 ºC. Following MTT immersion, the samples were extracted in isopropanol for 2 hours.

Number of replicates:

3 per test group

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of three test item replicates

Value:

3.5

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:

- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: No
- Colour interference with MTT: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: Not reported

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The OD values for the negative controls were between ≥0.8 and ≤2.8 (1.117).
- Acceptance criteria met for positive control: Viability of tissues was ≤ 20 % (4.9 %).
- Acceptance criteria met for variability between replicate measurements: Yes, ≤18 % (ca. 6 and 7 % for the negative and positive control groups, respectively).
- Range of historical values if different from the ones specified in the test guideline: Both negative and positive control values fall within the current historical control ranges.

Any other information on results incl. tables

Table 2      Mean OD₅₇₀ Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

 

Item	OD570 of tissues (corrected mean)	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)	% COV Relative mean viability (%)
Negative Control Item	1.191	106.6	100	5.848	5.848
	1.091	97.7
	1.068	95.7
Positive Control Item	0.059	5.3	4.9	0.329	6.713
	0.052	4.6
	0.054	4.8
Test Item	0.038	3.4	3.5	0.557	15.907
	0.034	3.0
	0.046	4.1

OD= Optical Density

SD= Standard deviation

 

*= The mean viability of the negative control tissues is set at 100 %

 

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Under the conditions of this study the test substance is considered to be irritant to the skin and is classified as Category 2 in accordance with UN GHS and EU CLP regulation.

Executive summary:

OECD 439 (2017) - The skin irritation potential of Reaction mass of N,N,N',N'-tetrabutylmethylenediamine and dibutylamine was assessed using an EpiSkin Reconstructed Human Epidermis (RHE) Model Kit in accordance with OECD Guidance 439 and GLP.

 

Triplicate tissues were exposed to the test item for 60 minutes. At the end of the exposure period the test item was rinsed and the tissues incubated for a further 40 h in the presence of maintenance solution which would be used for possible inflammatory mediator determination. Each tissue was then loaded with MTT for 3 h and any resultant colour extracted. After incubation and extraction with isopropanol, the solutions were aliquoted for absorbance measurements. Absorbency at 570 nm of each well was measured using a spectrophotometer.

 

Mean viability of tissues exposed to the test substance after 60 minutes were 3.5 %. It was considered unnecessary to perform IL-1a analysis as the results of the MTT test were unequivocal. The quality criteria required for acceptance of the results was met.

 

Under the conditions of this study the test substance is considered to be irritant to the skin and is classified as Category 2 in accordance with UN GHS and EU CLP regulation.