Ethane-1,2-diol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Monoethylene glycol was not mutagenic in the bacterial reverse mutation test and in an in vitro mammalian chromosomal aberration test in the absence and the presence of metabolic activation.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 Oct - 30 Nov 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- lot/bacht: Tank 25 20.08.2012

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature; temperature below 40 °C, light exclusion, protected against humidity
- Stability under test conditions: The stability of the test substance under storage conditions throughout the study period was guaranteed by the sponsor until 31 Jul 2013; the sponsor holds this responsibility

Target gene:

his operon for S. typhimurium strains
trp operon for the E. coli strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

other: TA 98: rfa-, uvrB-, R-factor; TA 100: rfa-, uvrB-, R-factor; TA 1535: rfa-, uvrB-; TA 1537: rfa-, uvrB; WP2: trp-; uvr A-

Metabolic activation:

with and without

Metabolic activation system:

S9 liver mix prepared from Wistar rats treated with 80 mg/kg bw phenobarbital i.p. and β-naphthoflavone orally, each on three consecutive days.

Test concentrations with justification for top dose:

First experiment (standard plate test, with and without metabolic activation, 3 plates/dose or control): 0, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Second experiment (preincubation test with and without metabolic activation, 3 plates/dose or control): 0, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Third experiment (preincubation test with and without metabolic activation, 3 plates/dose or control): 0, 33, 100, 333, 1000, 2500 and 5000 µg/plate

Remark: due to contamination affecting the whole second experimental series, a third experiment was added.

Vehicle / solvent:

- Vehicle/solvent used: ultrapure water
- Justification for choice of solvent/vehicle: good solubility of the test item in the vehicle

Untreated negative controls:

yes

Remarks:

sterility control

Negative solvent / vehicle controls:

yes

Remarks:

ultrapure water

True negative controls:

no

Positive controls:

yes

Remarks:

with S9-mix

Positive control substance:

other: 2-aminoanthracene (2-AA)

Remarks:

2.5 µg/plate in DMSO for TA 1535, TA 1537, TA 100, TA 98; 60 µg/plate in DMSO for E. coli WP2 uvrA

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

without S9-mix

Positive control substance:

other: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)

Remarks:

5 µg/plate in DMSO for TA 1535 and TA 100

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

without S9-mix

Positive control substance:

9-aminoacridine

Remarks:

100 µg/plate in DMSO for TA 1537 Migrated to IUCLID6: (AAC)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

without S9-mix

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

5 µg/plate in DMSO for E. coli WP2 uvrA Migrated to IUCLID6: (4-NQO)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

(without S9-mix)

Positive control substance:

other: 4-nitro-o-phenylendiamine (NOPD)

Remarks:

10 µg/plate in DMSO for TA 98

Details on test system and experimental conditions:

STANDARD PLATE TEST (SPT)
According to Ames et al., Mut Res 31: 347-364 (1975) and Maron & Ames, Mut Res 113: 173-215 (1983)

In the standard plate test, tubes were filled with 2mL portions of soft agar and kept in a water bath at 42 to 45°C. This soft agar consisted of 100 mL agar and 10 mL amino acid solution. As amino acid solution for the soft agar was used 0.5 mM histidine and 0.5 mM biotin for TA strains and 0.5 mM tryptophan for the E. coli strain.
Then following components are added:
0.1 mL test solution or vehicle
0.1 mL fresh bacterial culture
0.5 mL S9 -mix or phosphate buffer
After mixing samples were poured onto Vogel-Bonner (minimal glucose agar plates) plate and incubated for 48 - 72 hrs in the dark at 37°C.

PREINCUBATION TEST (PIT)
According to Yahagi et al. Mut Res 48: 121-129 (1977) and Matsushima et al., In: Norpoth, K.H. and R.C. Garner, Short-Term Test Systems for Detecting Carcinogens, Springer Verlag Berlin, Heidelberg, New York (1980)

For the preincubation test 0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL of either S9 mix or phosphate buffer were incubated at 37°C for 20 minutes. After addition of 2 mL soft agar, samples were poured onto agar plates and incubated again at 37°C for 48 to 72 hrs.
For the E. coli strain, plate test differed again in mixture of amino acid solution of the soft agar, the histidine component used for the TA strains being replaced by tryptophan.

Evaluation criteria:

An assay is accepted when the following criteria are met:
1.) number of colonies in the negative control is in the historical control range
2.) no indication of bacterial contamination (checked by sterility control)
3.) number of colonies in the positive controls are in the range of historical control data
4.) titer of viable bacteria is ≥ 10 E+8/mL

Toxicity is detected by:
1.) decrease in the number of revertants
2.) titer reduction
3.) clearing or diminution of the background lawn

Precipitation:
As long as no interference between precipitation and colony counting occurs is 5 mg/plate set as maximum dose even for relatively insoluble compounds.

A test chemical is to be considered as mutagenic when:
1.) increase of number of revertant colonies is reproducible and dose-related.
2.) in at least 1 tester strain doubling of colony counts with or without S-9 mix or after adding a metabolizing system is seen.

A test chemical is to be considered as non-mutagenic when:
1.) the number of revertants is inside the range of historical negative control data in 2 experiments performed independently from each other.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

occasionally observed depending on strain and test conditions from about 1000 μg/plate onward

Vehicle controls validity:

valid

Untreated negative controls validity:

other: sterility control, yes

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

occasionally observed depending on strain and test conditions from about 1000 μg/plate onward

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Remarks:

sterility control, yes

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

occasionally observed depending on strain and test conditions from about 1000 μg/plate onward

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Remarks:

sterility control, yes

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

occasionally observed depending on strain and test conditions from about 1000 μg/plate onward

Vehicle controls validity:

valid

Untreated negative controls validity:

other: sterility control, yes

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

occasionally observed depending on strain and test conditions from about 1000 μg/plate onward

Vehicle controls validity:

valid

Untreated negative controls validity:

other: sterility control, yes

Positive controls validity:

valid

Additional information on results:

No precipitation was detected.
No cytotoxic effects were seen in the standard plate test (SPT).
The first preincubation test (PIT) with and without metabolic activation was removed due to contamination and needed to be repeated. In the second PIT, a slight decrease in the number of his+ revertants indicating cytotoxicity could occasionally be seen depending on the strain and test conditions from about 1000 μg/plate onward.
Negative and positive controls were as expected and confirmed the validity and sensitivity of the test method and system.

Experiment 1: Standard plate-incorporation test

SPT without S9-Mix  [mean no. of mutations/ plate]
Dosage [µg/plate]	TA 1535	TA 100	TA 1537	TA 98	WP2 uvrA
Solvent control	11	54	7	24	88
33	11	47	10	23	86
100	12	53	7	22	102
333	11	53	7	26	93
1000	11	62	9	21	91
2500	11	55	6	21	101
5000	11	48	8	22	96
Respective positive control	1096	925	365	642	1102
SPT with S9-Mix  [mean no. of mutations/ plate]
Dosage [µg/ plate]	TA 1535	TA 100	TA 1537	TA 98	WP2 uvrA
Solvent control	11	68	12	29	96
33	12	62	13	29	100
100	13	58	14	29	109
333	12	64	10	34	100
1000	11	59	11	33	100
2500	12	59	13	27	100
5000	13	64	12	34	105
Respective positive control	210	1141	340	1190	417
Experiment 3: Preincubation test* PIT without S9-Mix  [mean no. of mutations/ plate]
Dosage [µg/ plate]	TA 1535	TA 100	TA 1537	TA 98	WP2 uvrA
Solvent control	17	48	9	20	109
33	16	45	8	20	109
100	16	50	7	22	105
333	17	48	8	16	105
1000	17	50	7	20	102
2500	10	50	6	19	103
5000	12	51	4	24	107
Respective positive control	1445	958	349	732	1056
PIT with S9-Mix  [mean no. of mutations/ plate]
Dosage [µg/ plate]	TA 1535	TA 100	TA 1537	TA 98	WP2 uvrA
Solvent control	12	57	8	22	118
33	11	53	7	21	118
100	14	60	8	25	121
333	12	57	9	24	116
1000	14	58	8	21	107
2500	11	55	9	22	100
5000	13	59	8	20	96
Respective positive control	446	1188	636	748	252
*Exp 2 not evaluated because of contamination

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

IN VITRO

A test for bacterial gene mutagenicity was conducted with monoethylene 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 SE 40M0008/07M016, 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 monoethylene glycol was not mutagenic in the bacterial reverse mutation test in the absence and the presence of metabolic activation. No precipitation was noticed. No cytotoxic effects were seen in the standard plate test (SPT). A first preincubation test (PIT) with and without metabolic activation was conducted, which needed to be removed due to contamination and was replaced by a second PIT. In this second PIT, a slight decrease in the number of his+ revertants indicating cytotoxicity could occasionally be seen depending on the strain and test conditions from about 1000 μg/plate onward. 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 previous BASF study conducted 1981 (BASF AG 80/507, 1981) which also reported a negative result in the Ames test, with and without metabolic activation, the test concentrations were 20, 100, 500, 2500 and 5000 µg/plate.

Results of in vitro cytogenetic tests for induction of SCEs and chromosomal Abs with ethylene glycol in CHO cells were negative, with and without Aroclor 1254-induced male Sprague-Dawley rat liver S9. In the SCE and the Abs tests, doses up to 5,000 pg/mL ethylene glycol were tested (NTP 1993).

Further, as supporting information, an in vitro mammalian chromosome aberration test using Chinese hamster ovary cells and testing concentrations ranging from 10 to 100 mg/mL is available (BRRC 1985). Results obtained with and without metabolic activation indicated that monoethylene glycol did not produce significant increases in chromosome aberrations in comparison to control cultures.

Regarding in vitro genotoxicity, the following data further are available, which are considered as weight of evidence. In a publication by McGregor et al. (1991), the results of a L5178Y mouse lymphoma cell forward mutation assay were negative regarding monoethylene glycol. In fact, in this experiment, the results obtained in the absence of S9 were inconsistent. Three trials were performed and in each case ethylene glycol was tested up to 5000 µg/mL, a concentration which failed to induce any significant toxicity. Two statistically significant dose levels were obtained in one of these trials, but this observation was not reproducible, there being no indication of a mutagenic response in the other two. Although this experiment showing the significant response was acceptable according to the study criteria, it was noted that the cloning efficiencies were low in all dose groups and in the vehicle controls. In the two trials performed in the presence of S9 mix, no significant mutagenicity was observed in any monoethylene glycol-treated cultures and the conclusion was that ethylene glycol was not mutagenic in the assay.

 

IN VIVO

No recent study is available regarding in vivo genotoxicity. Nevertheless, in a publication by DePass et al. (1986) a dominant lethal mutagenesis study with monoethylene glycol was reported in addition to a 3-generation reproduction study. Male and female rats were given the test item in daily oral doses (feed) 40, 200 and 1000 mg/kg/day. Slight apparent increases in the dominant lethal mutation index were reported for the high-dose group and the low-dose group, which were considered to be random occurrences, not related to treatment. Thus, the test item did not have a genotoxic effect in this dominant lethal study.

 

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. The available test data revealed not genotoxic potential. As a result the substance is not considered to be classified for genetic toxicity under Regulation (EC) No 1272/2008, as amended for the eighth time in Regulation (EU) No 2016/218.