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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Data of a whole battery of robust high quality in vitro studies as well as an in vivo test show that the test item does not possess any mutagenic or genotoxic properties.
Diethylene glycol was not mutagenic in the bacterial reverse mutation test in the absence and the presence of metabolic activation. In addition, a SCE and HGPRT assay were negative with and without metabolic activation. In addition, an in vivo micronucleus test with mouse according to the OECD TG 474 under GLP conditions was also negative.

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

Genetic toxicity in vivo

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

Additional information

Genetic toxicity in vitro

Bacterial reverse mutation tests

A test for bacterial gene mutagenicity was conducted with diethylene glycol according to the OECD TG 471 under GLP conditions with the following bacterial strains: Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1537 and E. coli WP2 uvrA (BASF, 2013). The test concentrations were 0, 33, 100, 333, 1000, 2500, and 5000 µg/plate for the standard plate test with and without S9-mix, and for the preincubation test with and without S9-mix, respectively. Negative (sterility and solvent) and positive controls were considered. Under the experimental conditions chosen, the test item diethylene glycol was not mutagenic in the bacterial reverse mutation test in the absence and the presence of metabolic activation. Neither precipitation nor cytotoxicity were noticed. All negative and positive controls were as expected and confirmed the validity, suitability and sensitivity of the test method and system used.

This result is supported by a an older study provided by Union Carbide (UCC, 1984), which also reported a negative result for diethylene glycol tested according to the EU Method B10, which is similar to the OECD TG 471, using the following strains of Salmonella typhimurium: TA 98, TA 100, TA 1535, TA 1537 and TA 1538. In this study, the tested concentrations ranged between 1000 and 11800 µg/plate and testing was done with and without S9 mix.

In a less reliable and poor reported study of Krug et al.(1986) the mutagenic potential of diethylene glycol in Salmonella tester strains TA 98, TA 100, TA 102, and TA 104 in the presence and absence of liver homogenate was investigated. The S9-mix was prepared from Aroclor 1254 pretreated female rats. A weak mutagenic effect was detected in strain TA 104 in the presence of "S9-Mix" (maximum: 2,2 fold increase over the spontaneous reversion frequency at 315 µmol DEG/plate). Due the limited available documentation of these results they were not taken into account for classification and labeling.  

Sister chromatid exchange assay  

A sister chromatid exchange assay with CHO cells tested in absence and presence of S9 mix was conducted with the test item equivalent or similar to OECD TG 479 (Union Carbide, 1984). Diethylene glycol at concentrations of 30 - 50 mg/mL did not produce dose-related, statistically significant increases in the incidence of chromosome aberrations in CHO cells in tests conducted with and without the addition of a rat-liver homogenate, S9 metabolic activation system. A single statistical indication of an increase above control values was obtained for the lowest dose sampled at 12 h after test initiation in the test with S9 activation. This result was not repeated at higher doses and the level of the increase was essentially the same as the variation in the concurrent negative control values. For these reasons, the statistical indication was not considered biologically relevant. The positive control agents, cyclophosphamide and triethylenemelamine, both produced significant increases in chromosome aberrations indicative of the appropriate, reliability of the test system. The control cultures employing only the culture growth medium had low and acceptable levels of chromosome aberrations typical for these cultured cells. Diethylene glycol was concluded to lack significant genotoxic activity under the conditions employed for this in vitro test system.  

HGPRT

A HGPRT assay with CHO cells tested in absence and presence of S9 mix was conducted with the test item equivalent or similar to OECD TG 473 (Union Carbide, 1984). The relative cytotoxicity of the various concentrations, tested both in the presence and absence of an S9 metabolic activation system, was determined by measuring the relative growth of treated and control cells incubated overnight following removal of the test chemical. It was observed that diethylene glycol was not highly cytotoxic when tested either with or without S9 metabolic activation. A concentration of 50 mg/mL produced 16% inhibition of growth without S9 and 27% inhibition with S9. For the definitive tests, a concentration range between 30 to 50 mg/mL was tested in the mutagenicity tests with and without S9. In the main test, diethylene glycol was neither genotoxic nor cytotoxic to CHO cells under the conditions of this in vitro test system.

Genetic toxicity in vivo

An in vivo micronucleus test with mouse was conducted with diethylene glycol according to the OECD TG 474, under GLP conditions (BASF, 2009). The male animals received a single intraperitoneal administration of the test item at following dose levels: 0, 500, 1000 and 2000 mg/kg bw. The single intraperitoneal administration of the test item in mice did not lead to a relevant increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was close to the range of the concurrent vehicle control in all dose groups and at all sacrifice intervals. Also, it was within the range of the historical vehicle control data. Both positive control substances, cyclophosphamide for clastogenicity and vincristine sulfate for spindle poison effects, led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei. Thus no indication for mutagenic potential was noted even after administration of very high doses and for a non-relevant worst case administration route (i.p.).

In less reliable studies (Barilyak, 1985 and Krug, 1986) DEG was further tested for in vivo genotoxicity:

An increase in chromosome damage in the bone marrow cells was reported after administration of 1/5 of the LD50 of DEG by gavage in hamster (Barilyak, 1985). In rats, DEG administration (dose not reported) caused dominant lethal mutations (Barilyak, 1985). In a micronucleus test (species not reported), a single intraperitoneal injection of 60% of LD50 of DEG caused chromosomal fragments. This induction was suppressed when the animals were pretreated during 7 days with a low daily dose of DEG (4 % of the LD50) (Krug et al., 1986). Due the limited available documentation of these results they were not taken into account for classification and labeling.  

SUMMARY  

In summary, the data of a whole battery of robust high-quality in vitro studies as well as results from an in vivo micronucleus test show that the test item does not possess any mutagenic or genotoxic properties. In addition, DEG and other ethylene glycols have no structural alerts that are known to lead to DNA reactivity and the overall evidence points to negative genotoxicity potential. Even though some old and very poorly documented abstracts show some positive results, new state of the art studies are available clearly documenting the absence of any genotoxic effects. The questionable results were thus judged as disregarded studies.

In a recently conducted literature search (August 2014), no further relevant information on genetic toxicity were identified (please refer to IUCLID section 12).

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is not considered to be classified for mutagenicity under Regulation (EC) No 1272/2008.