Reaction mass of 2,2'-(Ethylenedioxy)diethanol and 3,6,9-Trioxaundecane-1,11-diol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance was not considered to be genotoxic based on data of structural analogues.

Triethylene glycol was tested for its mutagenic potential in Salmonella typhimurium and Escherichia coli, in the standard plate test (SPT) and the preincubation test (PIT), according to the OECD TG 471 (BASF, 2012). Under the experimental conditions chosen, the test item is not mutagenic in the bacterial reverse mutation assay in the absence and presence of metabolic activation.
Further in vitro data on triethylene glycol (BRRC, 1986) and diethylene glycol (BRRC 1984). These data refer to a chromosome aberration assay, a sister chromatid exchange assay and an HGPRT gene mutation assay.
All available in vitro data revealed that the test item is not a mutagenic/genotoxic substance.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Additional information is available in the endpoint summaries and the read-across justification (see section 13).

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

other: Chinese hamster epithelial liver cell line (CHEL)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Result from read across (CAS 112-27-6)

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Additional information is available in the endpoint summaries and the read-across justification (see section 13).

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

S. typhimurium, other: TA1535, TA1537, TA98, TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

slight decrease in the number of his+ revertants reported for TA 1537 without S9 mix at 2500 and 5000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

other: S. typhimurium TA1535, TA1537, TA98, TA100 and E.coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: SPT Experiment 1

Remarks:

Result read-across source CAS No. 112-27-6

Reference 3

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Additional information is available in the endpoint summaries and the read-across justification (see section 13).

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Result from read across (CAS 112-27-6)

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The test substance was not considered to be genotoxic based on data of structural analogues.

In vivo MN studies with tetraethylene glycol produced no clastogenic effects. Tetraethylene glycol was negative in the dominant lethal test.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

Additional information is available in the endpoint summaries and the read-across justification (see section 13).

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

male/female

Genotoxicity:

negative

Remarks:

CA test

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Result from read across (CAS 112-60-7)

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

DLA

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Result from read across (CAS 112-60-7)

Key result

Sex:

male/female

Genotoxicity:

positive

Remarks:

MNT

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Result from read across (CAS 112-60-7)

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

There is are no studies available on the genetic toxicity of the substance itself (target, EC 907-131-0). Therefore studies from structural analogues (DEG, TEG, TetraEG) have been used to cover this endpoint.

 

 

 

 

 

Genetic toxicity in vitro

Bacterial reverse mutation tests

TEG was tested for its mutagenic potential in Salmonella typhimurium and Escherichia coli, in the standard plate test (SPT) and the preincubation test (PIT), according to the OECD TG 471 (BASFSE, 2012) under GLP conditions. The tester strains were: TA1535, TA100, TA1537, TA98 and E. coli WP2 uvrA. Testing was done in absence and presence of S9 mix. Negative and positive controls were included. The test concentrations were as follows:

Experiment 1 (all strains, standard plate test with and without S-9 mix; 3 plates/dose): 0; 33; 100; 333; 1000; 2500 and 5000 µg/plate

Experiment 2 (E.coli, standard test repeated with S-9 mix, due to inconclusive results in the first experiment; 3 plates/dose): 0; 33; 100; 333; 1000; 2500 and 5000 µg/plate

Experiment 3 (all strains, preincubation test with and without S-9 mix; 3 plates/dose): 0; 33; 100; 333; 1000; 2500; 5000 µg/plate

When tested in all S. typhimurium strains mentioned above under SPT and PIT conditions, with and without S9 mix, the test item did not induce an increase in his+ revertant colonies at concentrations up to 5000 µg/plate. A cytotoxic effect was observed in the SPT in absence of S9 mix for TA 1537 only, at 2500 and 5000 μg/plate. For E. coli tested in the SPT in absence of S9 mix, no increase in trp+ revertant colonies at concentrations up to 5000 µg/plate was noticed. In contrast, when tested in presence of S9 mix, a slight increase in revertants (factor 1.6) was observed at the top dose of 5000 μg/plate, which however was not reproducible in a second standard plate test (Experiment 2) and in the preincubation test (Experiment 3). Thus, the finding was regarded as not relevant. No cytotoxic effect was noticed for E.coli. No test item precipitation was observed within the concentration range tested, with and without S9-mix. Referring to controls, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study. Thus, under the experimental conditions chosen, TEG is not mutagenic in the bacterial reverse mutation assay in the absence and presence of metabolic activation.

Further, a test for bacterial gene mutagenicity was conducted with DEG 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.

HGPRT

A HGPRT assay with CHO cells conducted with DEG in absence and presence of S9 mix equivalent or similar to OECD TG 476 (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.

Chromosome aberration assay

A GLP-compliant in vitro chromosome aberration assay with CHO cells tested in absence and presence of S9 mix was conducted with TEG equivalent or similar to OECD TG 473 (BRRC, 1986). TEG at concentrations of 35, 42 and 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. No cytotoxicity was observed. All controls were valid. TEG was concluded to lack significant genotoxic activity under the conditions employed for this in vitro test system.  

Further, DEG was tested in a chromosome aberration assay with CHO cells in absence and presence of S9 mix equivalent or similar to OECD TG 473 (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.  

Sister chromatid exchange assay

A sister chromatid exchange assay with CHO cells tested in absence and presence of S9 mix was conducted with TEG equivalent or similar to OECD TG 479 (BRRC, 1986). TEG at concentrations of 24 - 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. No dose-related effects of exposure on the incidence of SCEs were evident and the test agent was inactive in the present in vitro assay. No cytotoxicity was observed. All controls were valid. TEG was concluded to lack significant genotoxic activity under the conditions employed for this in vitro test system.

 

Chromosome aberration assay

In a publication by Biondi (2002) an in vitro chromosome aberration assay with CHEL and CHO cells tested in absence and presence of S9 mix was conducted with TetraEG equivalent or similar to OECD TG 473. CHEL cells were exposed to 1.8, 2.5, 3.5, 5.0, and 7.0 mM TetraEG and CHO cells were exposed to 0.6, 1.1, 1.7, 13.7, 21.6 and 30.2 mM TetraEG. TetraEG induced marked and dose-related increases in aberration-bearing CHEL cells compared with the relevant solvent control values. Statistical significance was achieved for TetraEG at the three higher dose levels. The same trend was also observed at later sampling times (24 h), although the incidence of aberrant cells was slightly lower in absolute terms. The aberrations found were mainly chromatid exchanges and breaks. The authors also assessed TetraEG in CHO cells in the presence and absence of rat liver S9 mix. The results obtained indicate that TetraEG induced marked and dose-related increases in aberration-bearing cells compared with the relevant control values both in the presence and absence of S9. Statistical significance was only achieved at the two higher dose levels employed, which were substantially different in the presence (1.1 and 1.7 mM) and absence of S9 (21.6 and 30.2 mM). It should be noted here that while in the presence of S9 mix TetraEG showed a very strong clastogenic response at relatively low dose levels, in its absence TetraEG induced chromosomal aberrations only at dose levels consistently exceeding the limit of 10 mM (21.6 and 30.2 mM), likely determined by 'secondary' effects generated by the non-physiological conditions. These findings corroborate the evidence that TetraEG requires metabolic activation to exert its clastogenic effects. The large increase in mitotic index observed in the CHO experiment with TetraEG without S9 was apparently due to cell synchronization caused by, most probably, non-specific subtoxic effects of the test compound. No cytotoxicity was observed. All controls were valid. Based on the results, TetraEG was concluded to induce significant genotoxic activity under the conditions employed for this in vitro test system.

 

Genetic toxicity in vivo

Chromosome aberration assay

A GLP-compliant in vivo mammalian bone marrow chromosome aberration test with male and female Sprague-Dawley rats was conducted with TetraEG equivalent or similar to the OECD TG 475 (BRRC 1988). Three concentrations of TetraEG were evaluated for induction of chromosome aberrations using dosage levels of 1250, 2500 and 5000 mg/kg. 5 rats/sex/group were administered the appropriate concentrations of the test material or the vehicle control by oral gavage. Volumes of the delivered test or control solutions were kept at a constant volume of 10 mL/kg. An injection volume of 1 mL/kg of the positive control agent was administered as a single i.p. injection. Animals were sacrificed at time intervals of 12, 24 or 48 h after administration of the test material. Fifty metaphase cells were evaluated for incidence and type of chromosome damage for each animal/dose/sample time. Each cell was evaluated for chromosome number, specific type of chromosome- or chromatid-type aberrations and further classified for deletions and exchanges. No mortality or adverse clinical signs were observed. No statistically significant or dose-related increases in chromosome aberrations above vehicle control values were observed with either male or female rats sacrificed at any of the 3 time intervals after dosing. The control samples were all valid. It was concluded that TetraEG was not considered to be clastogenic under the conditions of this in vivo test system.

 

Dominant lethal assay

A GLP-compliant Rodent Dominant Lethal Assay with male and female Fischer 344 rats was conducted with TetraEG according to EPA OTS 798.5450 (Neeper-Bradley 1993). 100 virgin male rats were assigned to three treatment groups, one positive control group and one vehicle control group (20 males per group). 200 females were similarly assigned to each group. Groups were exposed through drinking water to 5000, 25000 and 50000 ppm TetraEG. The females remained in the male's cage until there was evidence of copulation or until seven days elapsed, whichever came first. If there was no evidence of copulation then the sixth day of cohabitation was designated gd 0. After completion of the first week of breeding, two new naive females were added to each male's cage and observed and measured as described above. Two new females were added to each male's cage weekly for a total of ten weeks to encompass the entire spermatogenic cycle. All study males and females were examined daily for any clinical signs. There were no treatment-related clinical signs of toxicity during the 5-day period in the males. The body weights of all males were equivalent. Water consumption was increased at 50000 and 25000 ppm. The increased urine volume observed at 50000 and 25000 ppm is consistent with the increased water consumption in these dose groups. Urine osmolality and pH were reduced at 50000 ppm. No treatment-related lesions were observed. There were no effects of treatment on terminal body weight or on testes weights. No effects on reproduction or gestational parameters was noted for males and females throughout the 10 week observation period. There were no dominant lethal effects of the test chemical on any stage of spermatogenesis evaluated in this assay. The "no observable effect level for general toxicity was 25000 ppm. Under the conditions of this study, dominant lethal effects were not observed.

 

Micronucleus test

A GLP-compliant in vivo micronucleus assay with male Swiss Webster mice was conducted with TetraEG according to an EPA test guideline (BRRC 1987). 5 male mice per dose were injected with a single dose at 2500, 4000 or 5000 mg/kg bw. Triethylenemelamine (TEM) was used as the positive control agent at dose levels of 0.3 mg/kg and 0.5 mg/kg to demonstrate the sensitivity and responsiveness of the animals in the definitive test. Peripheral blood was sampled for micronucleus evaluation at 30 hr, 48 hr and 72 hr after dosing. One thousand polychromatic erythrocytes per animal were scored at each time point. An additional 1000 polychromatic erythrocytes were scored for the males in the vehicle control group and tetraethylene glycol treated groups at the 30 hr time point. The data at the 30 hr time point was analysed as a mean per 1000 cells. No remarkable decreases in the PCE/NCE ratios relative to the control values were observed in this study at any of the three sampling periods. Analysis of variance testing indicated that there was a significant sex-related difference in the micronucleus response between male and female animals sampled 30 hours post-treatment. Therefore, values were analyzed separately using Fisher's exact test for the male and female mice, respectively. A 2-fold, statistically significant increase (0.05 > p> 0 .01) in relative numbers of micronucleated cells was observed only with the male animals tested with the highest dose level (5000 mg/kg) of tetraethylene glycol. Analysis of variance testing indicated that the data for male and female mice sampled at 48 hr and 72 hr after dosing were not statistically different; thus, values were pooled for Fisher's Exact analyses . No statistically significant or treatment-related increases in numbers of micronuclei were observed with any of the treatment groups at these later intervals. TEM, used as a positive control agent for this study, produced highly significant increases in numbers of micronuclei demonstrating the appropriate sensitivity of the test system. Numbers of micronuclei in the vehicle control animals were in a low and acceptable range for this test system at all sampling times.

Because of the low level increases and equivocal nature of the test results for the male animals sampled at the 30 hr sample period, an additional 1000 PCEs were evaluated for incidence of micronuclei for each of the treated and vehicle control animals. The additional scoring showed that the significant effect at the 5000 mg/kg dose was retained and the lowest dose gained significance above control. However, these increases are within the range of

historical controls and no dose-response relationship was demonstrated (i.e., micronuclei were increased in low-dose male mice to a greater degree than in mid-dose mice). No increases were noted for females. Overall, tetraethylene glycol is therefore considered to be negative (non-clastogenic) under the conditions of this test.

Justification for classification or non-classification

Based on the available information classification for genetic toxicity is not warranted in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. (EC) 1272/2008.