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Toxicological information

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Description of key information

In the key in vitro Human Skin Model Test (EPISKIN-SM™), conducted according to OECD Test Guideline 431 and in compliance with GLP, an aqueous solution of HEBMP-H containing 50.6% w/w active acid was tested for skin irritation (CitoxLAB, 2015). A mean relative tissue viability (of ≥35%  following a 4-hour exposure for HEBMP-H, [[(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, classification as Skin Irritant Category 2 is applied based on the precautionary principle. Based on the known pH, classification as Eye Damage Category 1 is applied.

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
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
2008
Qualifier:
according to guideline
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Version / remarks:
2008
GLP compliance:
yes (incl. QA statement)
Species:
human
Strain:
other: not relevant
Details on test animals or test system 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 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.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
4 hours exposure
Value:
82
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 hour exposure
Value:
84
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minutes exposure
Value:
104
Vehicle controls validity:
valid
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:
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.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irreversible damage)

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 Test Guideline 431 and in compliance with GLP (CitoxLAB, 2015). In the study, an aqueous solution of HEBMP-H containing 50.6% w/w active acid was tested for skin irritation. 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 Test Guideline 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 skin irritation and classification as Skin Irritant Category 2 is applied based on the precautionary principle.

The endpoint for eye irritation is waived because the substance is expected to cause severe damage to the eye and a precautionary classification as Eye Damage Category 1 is applied.

Justification for classification or non-classification

Discussion on classification conclusion for skin irritation

The skin irritation classification decision for HEBMP-H is based on pH level and information on the known impurities, phosphoric acid, phosphonic acid and hydrogen chloride, which are present at concentrations above 1% and have classifications listed in Annex VI of Regulation (EC) No 1272/2008.

The registered substance is typically manufactured as aqueous solutions containing about 50% w/w solids hence about 50% w/w water. However, in accordance with the definition in REACH of a substance, water (as a solvent that may be separated) is not considered to be a constituent. The concentration ranges specified in the boundary composition (see Section 1.2) are based on the substance as registered (without water); therefore, concentration ranges in the product as sold are approximately half of the values quoted here.

Phosphoric acid (CAS 7664-38-2; EC 231-633-2) is present at the concentration range of 0-≤2 % w/w in the registered substance (equivalent to 0-1% of a 50% aqueous solution; no solids products are on the market). It is classified for skin corrosion Cat 1B according to Annex VI of CLP Regulation (EC) No 1272/2008 and has a specific concentration cut-off limit of C ≥ 25 % for triggering classification for skin corrosion Cat 1. Since phosphoric acid is present at <25 %, it does not affect the classification conclusion of HEBMP-H.

Phosphonic acid (CAS 13598-36-2, EC 237-066-7) is present at the concentration range of 0-≤5 % w/w in the registered substance HEBMP-H (equivalent to 0-2.5% of a 50% aqueous solution; no solids products are on the market). It is classified for skin corrosion Cat 1A according to Annex VI of CLP Regulation (EC) No 1272/2008. If phosphonic acid is present at concentration ≥5 % w/w it triggers classification for skin corrosion Cat 1, while if it is present at concentration <5 % w/w, it triggers classification for skin irritation Cat 2. Based on the available key skin irritation data for the registered substance, HEBMP-H, no skin irritation potential was observed and there were no triggers for skin irritation/corrosion classification. Therefore, it is considered that the presence of phosphonic acid at the concentration range of 0-≤5 % w/w together with the known pH of HEBMP-H <2, supports the classification conclusion of skin irritation Category 2.

Hydrogen chloride (CAS 7647-01-0, EC 231-595-7) is present at the concentration range of 0-≤10 % w/w (equivalent to 0-5% of a 50% aqueous solution; no solids products are on the market). It is classified for skin corrosion Cat 1A according to Annex VI of CLP Regulation (EC) No 1272/2008. If hydrogen chloride is present at concentration ≥5 % w/w it triggers classification for skin corrosion Cat 1, while if it is present at concentration <5 % w/w, it triggers classification for skin irritation Cat 2. Based on the available key skin irritation data for the registered substance, HEBMP-H (including HCl as an impurity), no skin irritation potential was observed and there were no triggers for skin irritation/corrosion classification. Therefore, available data, the presence of HCl at 0-5% in commercial solutions of the substance and the known pH of HEBMP-H <2, support the classification conclusion of skin irritation Category 2.

HEBMP-H is classified for skin irritation Category 2 (H315: Causes skin irritation) and for irreversible eye damage Category 1 (H318: Causes serious eye damage), based on the available data and the known pH of the commercial product of <2 , according to Regulation (EC) No 1272/2008.