Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A bacterial reverse mutation assay with the test item conducted according to OECD 471 and GLP revealed no mutagenic potential of the test item. A micronucleus test in human lymphocytes in vitro was negative. The test item did not induce gene mutations at the HPRT locus in mammalian cells in vitro.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In vitro bacterial reverse mutation assay, Ames

The test substance, waterfree was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay.

STRAINS: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA

DOSE RANGE: 33 µg - 5600 µg/plate (SPT); 33 µg - 5600 µg/plate (PIT)

TEST CONDITIONS: Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats).

SOLUBILITY: No precipitation of the test substance was found with and without S9 mix.

TOXICITY: A weak bacteriotoxic effect was observed in the standard plate test with tester strain TA 1537 with S9 mix at concentrations of 2800 and 5600 µg/plate, only.

MUTAGENICITY:

A biologically relevant increase in the number of his+ or trp+ revertants (factor > 2: TA 100, TA 98 and E.coli WP2 uvrA or factor > 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test without S9 mix or after the addition of a metabolizing system.

CONCLUSION:

Under the experimental conditions of this study, the test substance , waterfree is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

Micronucleus test in human lymphocytes in vitro

The test item, dissolved in deionised water, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in three independent experiments. The following study design was performed:

 

 

Without S9 mix

With S9 mix

 

Exp. I

Exp. IIA

Exp. I & IIB

Stimulation period

 48 hrs

48 hrs

 48 hrs

Exposure period

 4 hrs

20 hrs

 4 hrs

Recovery

16 hrs

¾

16 hrs

Cytochalasin B exposure

20 hrs

20 hrs

20 hrs

Total culture period

88 hrs

88 hrs

88 hrs

 

In each experimental group two parallel cultures were analysed. Per culture at least 1000 binucleated cells were evaluated for cytogenetic damage.

The highest applied concentration in this study (4000 µg/mL of the test item) was chosen with regard to the water content (49.9%) of the test item and with respect to the current OECD Guideline 487.

Dose selection of the cytogenetic experiment was performed considering the toxicity data in accordance with OECD Guideline 487.

In all experimental parts in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied and evaluated concentration.

In Experiment I and IIA in the absence of S9 mix and in Experiment IIB in the presence of S9 mix, no relevant increase in the number of micronucleated cells was observed after treatment with the test item. In Experiment I in the presence of S9 mix, one statistically significant increase in micronucleated cells (1.38 %), above the historical control data range (95 % control limit: 0.16 – 1.08 %) was observed after treatment with 1306 µg/mL. Since no dose-dependency was observed, the finding can be regarded as biologically irrelevant.

Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.

Therefore, the test item is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest required concentration.

Gene mutation in mammalian cells in vitro

The potential of the test item to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster was investigated. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with a treatment period of 4 hours with and without microsomal activation. The second main experiment was performed with a 24 hours treatment period without microsomal activation and a 4 hours treatment period with microsomal activation.The maximum test item concentration of the pre-experiment (4000 µg/mL) was chosen with respect to the current OECD guideline 476 regarding the water content of the test item. The test item was dissolved in deionised water. No substantial and reproducible dose dependent increase of the mutation frequency was observed in the main experiments. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

 

Read-Across Approach

Supporting information is available on two read-across substances 2 -aminoethanol (MEA) and etidronic acid, the single components of the composition. Furthermore, a QSAR prediction was performed. Both Ames assays, on 2 -aminoethanol (MEA) and etidronic acid, yielded negative results. The QSAR prediction with OASIS TIMES was also negative, for the parent substance as well as for potential metabolites. The substance is therefore not considered to be mutagenic in a bacterial reverse mutation assay.

REPORTING FORMAT FOR THE ANALOGUE AND WEIGHT OF EVIDENCE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH

The test item is a multi-constituent substance: etidronic acid composed with 2-ethanolamine. Reliable experimental data are available for both components, etidronic acid and 2-aminoethanol, which are considered to be suitable read across substances.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)

2-aminoethanol CAS 141-43-5 is already registered under REACH. The substance purity (analytical grade) for the Ames test performed was 100%.

Etidronic acid (Phosphonic acid, P,P'-[1-hydroxyethylidene]bis-, CAS 2809-21-4), the second component is not yet registered under REACH. An Ames assay (pre-GLP, pre-OECD) was performed without documentation of the substance purity.

The target substance CAS 42220-47-3 is a composition of etidronic acid and 2-ethanolamine 1:1.

3. ANALOGUE APPROACH JUSTIFICATION (Read across and weight of evidence)

Experimental data i.e. Ames assay are available for 2-aminoethanol and etidronic acid. The Ames test on 2-aminoethanol was performed by the Japanes Ministry of Health and Environment according to OECD guideline 471. The assay is considered to be reliable and well documented. The Ames test on etidronic acid, performed in 1979, is a pre-GLP and pre-OECD study and only briefly documented. Both compounds, etidronic acid as well as 2-aminoethanol, are not considered to be mutagenic. The information given on the two single components is considered to be sufficient to cover the required endpoint information for the composition thereof. In addition a QSAR prediction with the target substance was performed (OASIS TIMES, endpoint: Ames test with metabolic activation). The parent as well as potential metabolites are not considered to be mutagenic.

4. CONCLUSION

Read-across to the single components of the composition and the QSAR prediction for the target substance together are supporting the findings of the key studies that the substance has no mutagenic properties.

Taken together, the three key studies on gene mutation in bacterial and mammalian cells as well as chromosome aberration in mammalian cells conducted with the test item did not indicate any genotoxic potential of the test item. These findings are further supported by QSAR predictions and a read-across approach to the single components of the test item.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Available in vitro studies were unanimously negative. As a result the substance is not considered to be classified for genetic toxicity under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.