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Description of key information

An in vitro Human Skin Model Test (EPISKIN-SM™) conducted to OECD TG 431 and GLP, is presented as key data for irritation (CitoxLAB, 2015). A mean relative tissue viability (of ≥35%  following a 4 hour exposure tor [[(2-hydroxyethyl)imino]bis(methylene)]bisphosphonic acid is reported. The result is negative for skin corrosivity potential. 
The known pH of the commercial product is <2. Based on the negative result from the in vitro skin corrosivity study and the known pH of the test material, a classification on a precautionary principle is proposed for skin and eye irritation for HEBMP-H.

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:
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
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
2008
Qualifier:
according to
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Version / remarks:
2008
GLP compliance:
yes (incl. certificate)
Species:
human
Strain:
other: not relevant
Details on test animals and environmental conditions:
- Source: Episkin, Lyon, France
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 period4. 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.
Irritation / corrosion parameter:
% tissue viability
Value:
>= 35
Negative controls validity:
valid
Positive controls validity:
valid

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

Interpretation of results:
not classified
Conclusions:
In an in vitro Human Skin Model Test (EPISKIN-SM™) conducted to OECD TG 431 and 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.
Executive summary:

In an in vitro Human Skin Model Test (EPISKIN-SM™) conducted to OECD TG 431 and 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.

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The key study for skin irritation is an in vitro Human Skin Model Test (EPISKIN-SM™) which was conducted according to OECD TG 431 and GLP (CitoxLAB, 2015). The reported mean relative tissue viability (expressed as % negative control) was ≥35% after a 4 hour exposure to HEBMP-H. The result is negative for skin corrosivity potential. The top-down, tiered approach to assessing the irritation/corrosion potential of a test material is generally followed up with the OECD TG 439 which discerns between non-irritant and Category 2 irritant substances. However, taking into consideration the known pH of the commercial product which is <2 and the negative result for corrosion, the test material is expected to cause irritancy and classification for skin irritation is proposed based on a precautionary principle.

The endpoint for eye irritation is waived because the substance is expected to cause damage to the eye and a precautionary classification is proposed.


Justification for selection of skin irritation / corrosion endpoint:
Not corrosive; predicted irritant based on pH. The study was conducted according to current OECD guideline and in compliance with GLP.

Justification for selection of eye irritation endpoint:
The irritation endpoint for HEBMP-H is waived, on the grounds of the expected corrosive effects based on the known commercial product pH of the acid form (<2).

Justification for classification or non-classification

HEBMP-H is classified as Skin Irritant Category 2; H315: Causes skin irritation; and for Irreversible effects on the Eye Category 1; H318: Causes serious eye damage, based on the available data and the known pH of the commercial product of <2 , in accordance with the CLP Regulation (EC 1272/2008).