Di(µ-2,2´,2´´-nitrilotris(ethanol)-diperchlorato)dinatrium

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

non mutagenic (Ames)

non clastogenic (Chromosome aberration)

non mutagenic (UDS and SCE, triethanolamine)

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Non mutagenic (SLRL, Mammalian Erythrocyte Micronucleus, triethanolamine, perchlorate)

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genetic Toxicity in vitro

The substance was tested in two in vitro experiments for its mutagenic potential and for its potential to induce chromosomal aberrations. Furthermore, available data in literature on one of the dissociation substance (triethanolamine) are also presented. Justification of Read Across is given in Section 13 of IUCLID.

The substance was tested for its mutagenic effects to Salmonella typhimunum strains (TA97a, TA 98, TA 100, TA 102, TA 1535) in a test by the plate incorporation and a verification test by using the pre-incubation method, according to the OECD Guideline 471 and EU Method B.13/14. The test was performed with and without the addition of rat-liver homogenate metabolising system. The compound was tested at concentrations in the range of 50 - 5005 μg/ml and 1250 - 4998 μg/ml in the first and second test respectively. Negative (plates containing no compound but only the vehicles used) and positive controls (plates containing a known mutagen) were used in parallel with the test material. No significant increase in the numbers of revertant colonies was recorded for any of the bacterial strains in any of the dose level tested, with and without metabolic activation. The substance was found to be non-mutagenic under the conditions of this test.

The potential of the substance to induce structural chromosome aberrations was assessed in the in-vitro test according to the OECD Guideline 473 and EU Method B.10. Evaluation of cytogenetic damage induced in V79 cells (cell line from the lung of the Chinese Hamster) in the absence and the presence of metabolic activation was performed in two independent experiments. In Experiment I, the exposure period was 4 hrs with and without metabolic activation. In Experiment II the exposure period was 4 hrs with S9 mix and 18 hrs and 28 hrs without S9 mix. The chromosomes were prepared 18 hrs (Exp. I and II) and 28 hrs (Exp. II) after the start of treatment with the test item. Per culture, at least 100 metaphase plates were scored for structural chromosome aberrations. In both experiments, both in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item were within the range of the solvent control values and the laboratory’s historical control data. Under the experimental conditions reported, the test item did not induce structural chromosome aberrations in V79 cells.

Triethanolamine was assessed for its potential to induce sister chromatid exchanges and chromosome aberrations to Chinese Hamster Ovary cells after the exposure to the substance with and without metabolic activation. The cells were incubated with the test material in three different doses in two different tests (SCE and ABS). Each test consisted of concurrent solvent and positive controls and three doses of test material. In the SCE test without S9, the first of two trials was negative. In the second trial, a significant increase in SCEs was observed at the highest dose tested (2520 µg/ml), but the trend test was negative (P≥0.025), and the trial was concluded to be equivocal. Severe cytotoxicity limited the number of cells that could be scored at this dose. Overall, the SCE test was considered to be negative. Cytotoxicity was also noted at the highest dose tested (4030 µg/ml) in the Abs test without S9. No induction of SCEs or Abs was observed with or without S9. Triethanolamine was also evaluated for its potential to cause DNA damage by measuring UDS in primary rat hepatocytes in vitro. The substance was tested at concentrations ranging from 5.0 to 1000.0 nl/ml in Williams Medium E with 10% fetal calf serum. The negative control (medium only) and positive control (2 -acetylaminofluorene) groups run in parallel. The treatment caused an extreme vacuolation at 1000 nl/ml and reduced the cell survival to app. 65 %; concentrations of 100 nl/ml and less were not cytotoxic to the cell cultures. None of the criteria used to indicated UDS were even approached and a dose-related response was not observed.The treatment had no effect on the rate of DNA repair, indicating a lack of genotoxicity of triethanolamine in this cell culture assay.

Genetic toxicity in vivo

No data were available on the genetoxicity potential of the substance. Data available on the dissociation substances were used for the assessment of the in vivo genotoxicity of the substance. Justification on Read Across is given in Section 13 of IUCLID.

The results obtained by two studies on triethanolamine and one study on perchlorate suggest that the substances are negative for genotoxicity.

The potential effects of perchlorate on bone marrow micronucleus formation in rats were investigated. 6 male and 6 female Sprague-Dawley rats were orally exposed to five concentrations of the substance (0.01 to 10 mg/kg bw/day) via the drinking water for 90 days. After 90 days bone marrow slides obtained from the vehicle control, positive control and high-dose (10 mg/kg bw) groups were stained and evaluated. 1000 polychromatic erythrocytes (PCEs) per slide were analysed for the frequency of micronucleated cells while the ratio of PCEs/NCEs (normochromatic erythrocytes) was also determined to assess potential cytotoxicity. No test article-related changes in bone marrow micronucleus formation or PCE/NCE ratios were observed in males or female after 90 days of treatment.

The mutagenicity of triethanolamine in Drosophila melanogaster was tested by using the sex-linked reccesive ethal (SLRL) assay. Canton-S males were first exposed to the substance by feeding in concentrations of 20000 and 30000 ppm for 3 days. A lethal as judged to have occured if no wild-type males were recovered in the 2 among at least Basc males (or Basc/+ females). As there was no evidence of induced mutation after feeding, the substance was retested by injection into adult males (at 10000, 20000 and 30000 ppm). Canton-S males were mated to three consecutive harems of Basc females to collect germ cells treated at primarily postmeiotic stages. Clusters were identified in four dose groups (control group of 20000 and 30000 -ppm feeding experiment, group of 20000-ppm treated feeding experiment, control group of 10000-ppm injection experiment). No increase in the frequency of sex-linked recessive lethal mutations was observed in germ cells of male Drosophila melanogaster administered the test matterial by feeding or injection. Triethanolamine was also evaluated for its potential to induce formation of micronucleated polychromatic erythrocytes in the peripheral blood of mice. 10 male and 10 female B6C3F₁mice were dermally exposed to three concentrations of the substance (at 1000, 2000 and 4000 mg/kg) per day, for 5 days, for 13 weeks. After the exposure period, peripheral blood samples were taken from the ventral tail vessel of the mice. The smears were fixed in absolute methanol, they were stained, fixed and coded. 10000 NCEs (normochromatic erythrocytes) and 2000 PCEs (polychromatic erythrocytes) were scored for micronuclei per animal per dose group. No significant increases in the frequencies of micronucleated NCEs or PCEs were observed at any dose tested. The substance is considered as not mutagenic under the test conditions.

Data available on the dissociation substances (triethanolamine and perchlorate) are also taken in consideration for the assessment of the mutagenic potential of the substance. Triethanolamine was included in the Community rolling action plan (CoRAP) and was subject to evaluation. The substance evaluation conclusion document (dated September 2015) suggested that mutagenicity was not identified as an area of concern for the substance, but all available information on mutagenicity were still evaluated since carcinogenicity was an area of concern. The in vitro genotoxicity of triethanolamine was investigated in four bacterial reverse mutation assays, a chromosome aberration assay in CHO cells, mammalian gene mutation and SCE assay in mammalian cells. The evaluation member state concluded that the available negative results suggest that the substance is not an in vitro mutagen. As part of CoRAP, perchlorate was also evaluated for its mutagenicity due to a concern regarding the potential of perchlorate to cause cancer in humans. The evaluation Member State Competent Authority (substance evaluation document dated August 10th, 2016) concluded that no further action was required for both mutagenicity and carcinogenicty while the concern for carcinogenicity was not substantiated. Available in vivo and in vitro data on mutagenicity (bacterieal reverse mutation, mammalian gene mutation, mammalian chromosome aberration, erythrocyte micronucleus assay) support that the substance is not genotoxic. This is also in line with evaluations of other risk assessment bodies such as EFSA (2014) and ASTDR (2008).

EFSA (2014) CONTAM Panel (EFSA Panel on Contaminants in the Food Chain), Scientific Opinion on the risks to public health related to the presence of perchlorate in food, in particular fruits and vegetables, last revision on 26 May 2015. EFSA Journal 12:117.

ATSDR (2008) Toxicological profile for Perchlorates, U.S. Department of Health and Human Services, Public Health Service, Atlanta, GA.

Justification for classification or non-classification

According to Annex I: 3.5.2.2 of the CLP regulation n. 1272/2008 substances are allocated to one of two categories (Category 1A-1B and Category 2):

- Category 1A: The classification in Category 1A is based on positive evidence from human epidemiological studies. Substances to be regarded as if they induce heritable mutations in the germ cells of humans.

- Category 1B: The classification in Category 1B is based on: positive result(s) from in vivo heritable germ cell mutagenicity tests in mammals; positive result(s) from in vivo somatic cell mutagenicity tests in mammals, in combination with some evidence that the substance has potential to cause mutations to germ cells. It is possible to derive this supporting evidence from mutagenicity/genotoxicity tests in germ cells in vivo, or by demonstrating the ability of the substance or its metabolite(s) to interact with the genetic material of germ cells; positive results from tests showing mutagenic effects in the germ cells of humans, without demonstration of transmission to progeny; for example, an increase in the frequency of aneuploidy in sperm cells of exposed people.

- Category 2: Substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans. The classification in Category 2 is based on: positive evidence obtained from experiments in mammals and/or in some cases from in vitro experiments, obtained from: somatic cell mutagenicity tests in vivo, in mammals; or other in vivo somatic cell genotoxicity tests which are supported by positive results from in vitro mutagenicity assays. Substances which are positive in in-vitro mammalian mutagenicity assays, and which also show chemical structure activity relationship to known germ cell mutagens, shall be considered for classification as Category 2 mutagens.

The substance was assessed for its genetic toxicity potential in one in-vitro bacteria reverse mutation test (Ames) and it was then further assessed in one in-vitro mammalian chromosome aberration test. Only in strain TA1535 without metabolic activation in the first experiment an increase in the number of revertant colonies was observed. This increase was not concentration - related and barely reached the threshold of the induction factor 2.0. Furthermore, in the verification experiment, no increase in revertants was detected for the specific strain with and without metabolic activation, therefore the increase observed in the first experiment was assessed as not significant. Both in vitro-tests gave negative results.

Furthermore, available data on the dissociation substances, triethanolamine and perchlorate were used for the assessment of mutagenicity potential of the substance. Triethanolamine was tested in two in vitro DNA damage/repair tests in CHO cells and hepatocytes cells of rats and one chromosome aberration test in CHO cells. This substance was also tested in vivo in the SLRL test in order to detect potential lethal mutations in germ line of D.melanogaster and in the mammalian somatic erythrocyte micronucleus test. Perchlorate was only tested in-vivo in the Mammalian Erythrocyte Micronucleus assay. All tests in both dissociation substances were negative supporting the non mutagenicity of the substance. According to the CLP Regulation the substance should not be classified in neither of the two categories for mutagenicity since no test on somatic or germ cells gave a positive result both in vitro and in vivo.