Reaction mass of [[(2-hydroxyethyl)imino]bis(methylene)]bisphosphonic acid and Phosphonic acid, P-[(tetrahydro-2-hydroxy-2-oxido-4H-1,4,2-oxazaphosphorin-4-yl)methyl]-

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-08-19 to 2014-08-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted to the appropriate OECD test guideline and in compliance with GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

2008

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Version / remarks:

2008

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction Mass of [[(2-hydroxyethyl)imino]dimethylene]bisphosphonic acid and [(2-hydroxy-2-oxo-1,4,2-oxazaphosphinan-4-yl)methyl]phosphonic acid

EC Number:

911-811-2

Molecular formula:

C4H13NO7P2 and C4H11NO6P2

IUPAC Name:

Reaction Mass of [[(2-hydroxyethyl)imino]dimethylene]bisphosphonic acid and [(2-hydroxy-2-oxo-1,4,2-oxazaphosphinan-4-yl)methyl]phosphonic acid

Test material form:

other: liquid at room temperature

Test animals

Species:

human

Strain:

other: not relevant

Details on test animals or test system and environmental conditions:

- Source: Episkin, Lyon, France

Test system

Type of coverage:

other: not relevant

Preparation of test site:

other: not relevant

Vehicle:

unchanged (no vehicle)

Controls:

other: not relevant

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL

Duration of treatment / exposure:

not relevant

Observation period:

not relevant

Number of animals:

not relevant

Details on study design:

Preliminary tests:
1. Test for direct MTT reduction with the test item:
To identify any test item interference, 50 µl of test item was added to 2 ml of a 0.3 mg/ml freshly prepared MTT solution. The mixture was incubated in darkness at ±37°C for 3 hours (±5 minutes) under continuous stirring.
A negative control was tested concurrently by adding 50 µl of water in place of the test solution, in the same manner.
The colour of both solutions was observed.
2. Test for the detection of the colouring potential of the test item:
10 µl of test item was added to 90 µl of water for injection in a transparent recipient. After 1 hour of mixing, the colouration was checked.

Main test:
On 12-well plate was used for each of the 3 exposure times (3 minutes, 1 hour and 4 hours) for test item-treated tissues.
Positive (glacial acetic acid) and negative (0.9% NaCl) controls were placed on separate plates.
Test item, and negative and positive controls were applied on duplicate tissues.
1. Pre-incubation of the tissues on day 0:
2 ml of 37°C maintenance medium was added to 2 wells per plate as follows:
- duplicate wells for each test item and negative control exposure time and for the 4-hour positive control exposure time.
One Episkin tissue was transferred into each maintenance medium pre-filled well.
All 12-well plates were incubated at 37°C, 5% CO2, >95% humidity for 1-48 h pre-incubation.
2. Treatment of tissues:
2 ml of 37°C assay medium was added to 2 wells per 12-well plate as follows:
- duplicate wells for each test item and negative control exposure time and for the 4-hour positive control exposure time.
The tissues were removed from the incubator and one tissue was transferred in to each assay medium pre-filled well.
The test item and controls were applied on each designated tissue.
The lids were replaced on each plate before incubation at room temperature as follows:
- positive control for 4 hours (±10 minutes)
-test item and negative control for 3 minutes (±5 seconds), 1 hours (±5 minutes) and 4 hours (±10 minutes)
3. Rinsing of tissues:
All treated tissues were rinsed with D-PBS at the end of the designated incubation period
4. MTT viability assay:
Two empty wells were filled with 2 ml MTT solution (0.3 mg/ml), and the corresponding tissues were placed in these wells. Each plate was protected from light and incubated for 3 hours ±15 minutes at 37°C, 5% CO2 in a humidified incubator.
After incubation, the underside of each tissue culture insert was blotted. The tissues were removed using a biopsy punch. Any tissue discolouration was evaluated with the naked eye.
For each tissue, the epidermis was separated from the collagen matrix. Both parts were put in a stoppered plastic tube and 0.5ml acidified isopropanol were added. After vortexing, each tube was protected from light and left at room temperature overnight to extract the formazan (reduced MTT).
5. Optical density measurements:
After the extraction period, each tube was vortexed, and centrifuged if needed. Each tube was used to fill 2 consecutive wells of a 96-well plate with 200 µl if extract per well. A separate 96-well plate was used for the test item dose formulation. For each plate, the OD value of 4 wells containing 200 µl of acidified isoproponal only was used as the blank.
The OD was measured at a wavelength of 570 nm.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

4 hours exposure

Value:

82

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour exposure

Value:

84

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes exposure

Value:

104

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

The mixture of 50 µL test item per 2 mL MTT solution showed no reduction of MTT compared to the negative control (0.9% NaCl). The mixture did not turn blue/purple.

The mixture of 10 µL of the test item per 90 µL water showed no colouring and the test item was presumed to not stain the tissue.

Following the 3 minute exposure period, a blue discolouration of the test item-treated tissues was noted. The discolouration was representative of viable tissues. Following the 1 and 4 hour exposure periods, a blue discolouration was noted on one tissue and a blue/white discolouration on the second tissue. This discolouration was representative of viable or semi-viable tissues, respectively. The controls confirmed the validity of the study.

The relative mean viabilities of the test item-treated tissues were: - 104% for the 3 minutes exposure, - 84 % for the 1 hour exposure, - 82% for the 4 hour exposure. All mean viabilities were ≥35%. Table 1: Mean tissue viability, standard deviations and difference (%) between replicate tissues for the test item, the negative and positive controls

Group	Exposure duration	cOD570 nm		Viability (%)
		Mean	SD	Mean	SD	Difference (%)
Negative control	3 min	1.068	0.023	100	2	3
	1 h	1.057	0.015	100	1	2
	4 h	1.138	0.051	100	4	6
Positive control	4 h	0.027	0.006	2	1	n.c.
Test item	3 min	1.112	0.011	104	1	1
	1 h	0.884	0.293	84	28	39
	4 h	0.935	0.129	82	11	16

cOD = blank corrected optical density

n.c. = not calculated

SD = standard deviation

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In an in vitro Human Skin Model Test (EPISKIN-SM™) conducted to OECD TG 431 and in compliance with GLP, the mean relative tissue viability (% negative control) was ≥35% after 4-hour exposure for [[(2-hydroxyethyl)imino]bis(methylene)]bisphosphonic acid. This result is negative for skin corrosivity potential.