O,O-dibutyl hydrogen thiophosphate, compound with 1-octylamine (1:1)

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date 22 June 2016 Experimental completion date 24 June 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Other

Version / remarks:

• Method B.40bis of Commission Regulation (EC) No 440/2008, of 30 May 2008, laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

O,O-dibutyl hydrogen thiophosphate, compound with 1-octylamine (1:1)

EC Number:

300-947-2

EC Name:

O,O-dibutyl hydrogen thiophosphate, compound with 1-octylamine (1:1)

Cas Number:

93964-99-9

Molecular formula:

C8H19N.C8H19O3PS

IUPAC Name:

octan-1-amine dibutyl sulfanylphosphonate

impurity 1

Reference substance name:

Octylamine

EC Number:

203-916-0

EC Name:

Octylamine

Cas Number:

111-86-4

Molecular formula:

C8H19N

IUPAC Name:

octan-1-amine

Test material form:

liquid

Specific details on test material used for the study:

Identification: X-19575 Phosphorothioic acid, O,O-dibutyl ester, compd. with 1-octanamine, CASRN 93964-99-9
Physical state/Appearance: amber colored viscous liquid
Batch: X-019575-00-00
Purity: >95% (treated as 100%)
Expiry Date: 01 June 2017
Storage Conditions: room temperature in the dark

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: Epithelial

Cell source:

other: derived from human skin

Source strain:

not specified

Details on animal used as source of test system:

not applicable

Justification for test system used:

This model incorporates several features, which make it advantageous in the study of potential dermal corrosivity. The target cells are epithelial, derived from human skin, and formed into a stratified, cornified epithelium. Test items are applied to the culture surface, at the air interface, so that undiluted and/or end use dilutions can be tested directly.

Vehicle:

unchanged (no vehicle)

Details on test system:

EpiDerm™ Reconstructed Human Epidermis Model Kit
- Supplier: MatTek
- Date received: June 2016
- EpiDermTM Tissues (0.63cm2) lot number: 23342
- Assay Medium lot number : 061616TMA
Upon receipt of the EpidermTM tissues, the sealed 24 well plate was stored in a refrigerator until use.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C, 5% CO2
- Temperature of post-treatment incubation (if applicable):37°C, 5% CO2

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: Rinsing was achieved by filling and emptying each tissue under a constant soft stream of DPBS to gently remove any residual test item.

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 562 nm (OD 562)

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION:
1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the relative mean tissue viability (% of negative control) is < 50 after the 3 minute exposure OR>/= 50 after 3 minutes exposure and < 15 after 1 hour exposure
- The test substance is considered to be non-corrosive to skin if the relative mean tissue viability (% of negative control) is >/= 50 after a 3 minute exposure and >/= 15 after 1 hour exposure

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 50 µL
- Concentration (if solution): as supplied

NEGATIVE CONTROL (sterile distilled water)
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): as supplied

POSITIVE CONTROL (8.0N potassium hydroxide)
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): 7.92N (specification 7.92 – 8.08N)

Duration of treatment / exposure:

3 minutes
60 minutes

Duration of post-treatment incubation (if applicable):

3 hours MTT incubation

Number of replicates:

2

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute exposure

Value:

104.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The test item was considered to be non corrosive to the skin

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minute exposure

Value:

23.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

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

Other effects / acceptance of results:

Direct MTT Reduction
An assessment found the test item was able to directly reduce MTT. Therefore, an additional procedure using freeze-killed tissues was performed during the determination of skin corrosion potential. However, the results obtained showed a negligible degree of interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the freeze-killed tissues for quantitative correction of results or for reporting purposes.

Assessment of Color Interference with the MTT endpoint
The solution containing the test item was a pale amber color. This color was considered not to have the potential to cause color interference.

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

Any other information on results incl. tables

Test Item, Positive Control Item and Negative Control Item

Mean OD₅₆₂values and viabilities for the negative control, positive control and test item are given in theappendix.

The relative mean viabilities for each treatment group were as follows:

Exposure Period	Percentage Viability
	Negative Control	Positive Control	Test Item
3 minute	100*	3.5	104.4
60 minute	100*	3.8	23.3

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

Quality Criteria

The mean OD₅₆₂for the negative control treated tissues was 1.946 for the 3‑Minute exposure period and 2.041 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 3.8% relative to the negative control following the 60‑Minute exposure period. The positive control acceptance criterion was therefore satisfied.

Mean OD₅₆₂Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Tissue	Exposure Period	MeanOD562of individual tissues	Mean OD562of duplicate tissues	Standard Deviation	Coefficient of Variation (%)	Relative Mean Viability (%)
Negative ControlÅ	3 Minutes	1.918	1.946	0.039	2.0	100*
		1.973
	60 Minutes	2.052	2.041	0.016	0.8
		2.029
Positive Control^Å	3 Minutes	0.062	0.069	0.010	na	3.5
		0.076
	60 Minutes	0.075	0.077	0.003	na	3.8
		0.079
Test Item	3 Minutes	2.074	2.033	0.059	2.9	104.4
		1.991
	60 Minutes	0.464	0.477	0.018	3.7	23.3
		0.489

 

OD= Optical density

Å=   The control groups were shared withEnvigo Research Ltd. study number FY73XC

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

na=  Not applicable

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.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

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

Executive summary:

Introduction

The purpose of this test is to evaluate the corrosivity potential of the test item using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes.

Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test item.

Methods

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. The test item was found to directly reduce MTT and therefore additional non-viable tissues were incorporated into the testing for correction purposes. 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 mL samples were transferred to the appropriate wells of a pre-labeled 96‑well plate. The optical density (OD) was measured at 562 nm (OD₅₆₂).

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

Results

The relative mean viabilities for each treatment group were as follows:

Exposure Period		Percentage Viability
		Negative Control		Positive Control		Test Item
3 minute		100*		3.5		104.4
60 minute		100*		3.8		23.3

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

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

Conclusion

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