Reaction products of 2-(chloromethyl)oxirane and glycerol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: well-documented publication, which meets basic scientific principles, performed similar to OECD guideline 482 i.a. on the read-across substances glycidol und butylglycidyl ether

Data source

Reference

Reference Type:

publication

Title:

Mutagenicity of alkyl glycidyl ethers in three short-term assays

Author:

Thompson, E.D., Coppinger, W.J., Piper, C.E., McCarroll, N., Oberly, T.J., Robinson, D.

Year:

1981

Bibliographic source:

Mutation Research, 90 (1981) 213-231

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)

Deviations:

not applicable

GLP compliance:

no

Type of assay:

DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro

Test material

Test material information

Constituent 1

Reference substance name:

glycidol

IUPAC Name:

glycidol

Constituent 2

Reference substance name:

2,3-epoxypropan-1-ol

EC Number:

209-128-3

EC Name:

2,3-epoxypropan-1-ol

Cas Number:

556-52-5

Molecular formula:

C3H6O2

IUPAC Name:

oxiran-2-ylmethanol

Constituent 3

Reference substance name:

n-butylglycidyl ether

IUPAC Name:

n-butylglycidyl ether

Constituent 4

Reference substance name:

Butyl 2,3-epoxypropyl ether

EC Number:

219-376-4

EC Name:

Butyl 2,3-epoxypropyl ether

Cas Number:

2426-08-6

Molecular formula:

C7H14O2

IUPAC Name:

2-(butoxymethyl)oxirane

Test material form:

not specified

Details on test material:

- Name of test material (as cited in study report): glycidol; C-4 (butyl-GE), butyl glycidyl ether
- Substance type: pure active
- Analytical purity:
97.7% (glycidol)
99.1% (butyl glycidyl ether)
- Impurities (identity and concentrations):
0.4% C4 alcohol (butyl glycidyl ether)
- Other:
Glycidol and the butyl glycidyl ether were obtained in high purity by the following procedure: Glycidol, and butyl glycidyl ether were purchased and purified by fractional vacuum distillation.
The substances were characterized by gas liquid chromatography on a Hewlett Packard instrument equipped with a 2-m stainless steel column packed with SP2100 (Supelco). Oxirane levels were determined as described by Walker (Walker, R.O. (Ed.) (1978) Oxirane Oxygen, Official and Tentative Methods of the American Oil Chemists Society, Tentative Method Cd 9-57, 508 South Sixth Street, Champaign, IL 61820.). Free epichlorohydrin was determined by gas liquid chromatography of the head space.

Method

Target gene:

not applicable

Species / strain

Species / strain / cell type:

mammalian cell line, other: WI38

Details on mammalian cell type (if applicable):

WI38 cells were obtained from the American Type Culture Collection.

Metabolic activation:

with and without

Metabolic activation system:

S9 of liver homogenate (250 mg of liver per ml) from adult male Swiss-Webster mice. The following cofactors were added to the S9: nicotinamide (3.05 mg/ml), glucose-6-phosphate (16.1 mg/ml), MgCI2 * 6 H20 (5.08 mg/ml), and NADP (0.765 mg/ml).

Test concentrations with justification for top dose:

The maximal testable level was set just below the level which produced cytotoxicity as demonstrated by a decrease in the amount of [3H]thymidine incorporated into the DNA. Test concentrations:
0, 0.375, 0.75, 1.5, 3.0, 6.0 µg/ml (glycidol without S9); 0, 0.037, 0.111, 0.333, 1.0, 3.0 µg/mL (glycidol with S9)
0, 0.24, 0.36, 0.53, 0.8, 1.2 µg/ml (butyl glycidyl ether without S9); 0, 0.5, 1.0, 2.0, 4.0, 8.0 µg/mL (butyl glycidyl ether with S9)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

N-dimethylnitrosamine

Remarks:

The positive controls were 4-nitroquinoline-N-oxide (4NQO), a compound that induces UDS in the absence of metabolic activation, and dimethylnitrosamine (DMN), a compound that induces UDS in vitro only with metabolic activation.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: The cells were grown to confluency and maintained in medium containing 0.5% serum for 5-6 days preceding the UDS assays. The cultures were incubated for 1 h with 10 exp -2 M hydroxyurea (HU) before each assay.
- Exposure duration: For testing in the absence of metabolic activation, the cells were exposed simultaneously to the compounds and to 3H-TdR for 3 h. For testing with metabolic activation, the cells were incubated with the compound, 3H-TdR, and the metabolic activation preparation for 1 h and then with only 3H-TdR in culture medium for an additional 3 h.

NUMBER OF REPLICATIONS: 6

DETERMINATION OF CYTOTOXICITY
- Method: other: decrease in the amount of [3H]thymidine incorporated into the DNA

Statistics:

Results are the average of 6 replicate samples at each dose level.

Results and discussion

Test results

Test results 1

Species / strain:

mammalian cell line, other: WI38

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

glycidol

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Test results 2

Species / strain:

mammalian cell line, other: WI38

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

glycidol

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Test results 3

Species / strain:

mammalian cell line, other: WI38

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

butyl glycidyl ether; demonstrable, although not considered positive responses were obtained for butyl glycidyl ether in the presence of S9.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Any other information on results incl. tables

The results of the unscheduled DNA synthesis assays are shown in Table 1. Preliminary tests were performed to define the maximal testable levels of the chemicals. The response index was calculated by dividing the amount of thymidine incorporation in the test results by the thymidine incorporation in the solvent control.

A compound was considered positive in this assay if a dose-related increased in the amount of [3H]thymidine incorporated into DNA over at least 3 concentrations with the highest response equal to at least twice the solvent control was attained. Positive responses were obtained for glycidol in the presence of the S9 activating system. Demonstrable, although not considered positive responses were obtained for butyl glycidyl ether in the presence of S9. The remainder of the assays were negative.

Table 1: Levels of the UDS in WI38 cells following exposure to graded doses of glycidol and butyl glycidyl ether

Compound	Without S9			With S9
	Concentration (µl/ml)	Thymidine incorporation^a	Response index^b	Concentration (µl/ml)	Thymidine incorporation^a	Response index^b
Glycidol	6.0	94	<1	3.0	207	3.4
	3.0	94	<1	1.0	171	2.8
	1.5	89	<1	0.333	120	2.0
	0.75	92	<1	0.111	86	1.4
	0.375	82	<1	0.037	62	1
	Solvent control	111	1	Solvent control	61	1
	Positive control^c	1087	9.8	Positive control^c	346	5.7
Butyl glycidyl ether	1.2	39	<1	8.0^d	68	1
	0.8	46	<1	4.0^d	128	1.9
	0.53	65	<1	2.0	75	1.1
	0.36	75	<1	1.0	69	1
	0.24	46	<1	0.5	67	1
	Solvent control	70	1	Solvent control	67	1
	Positive control^c	1054	15.0	Positive control^c	314	4.7

a DPM / µg DNA.

b Response index = ratio of test / control.

c With metabolic activation - dimethylnitrosamine (5 x 10 exp -2 M). Without metabolic activation - 4-nitroquinoline oxide (10 exp 5 M).

d Oil droplets formed on the surface of the medium.

Applicant's summary and conclusion

Conclusions:

negative without metabolic activation both glycidol and butyl glycidyl ether
negative without metabolic activation butyl glycidyl ether
positive with metabolic activation glycidol
negative '1,2,3-propanetriol, glycidyl ethers' , derived from read-across

Unscheduled DNA synthesis was induced by glycidol in the presence of an uninduced mouse liver S9 fraction, whereas butyl glycidyl ether was clearly negative for UDS induction. The effect of S9 in this assay was just the opposite of that seen in the mouse lymphoma assay (Thompson, 1981), i.e. S9 appears to increase the amount of UDS whereas it reduced the mutagenic response in the mouse lymphoma assay: There, the mutagenic potential of the ethers increases in the presence of the S9 fraction, which reduces the mutagenic potential of glycidol. The epoxide hydrase present in induced S9 fractions has been reported to lower the mutagenic potential of an epoxide by converting it to the diol (Ortiz De Montellano, P.R., and A.S. Boparai (1978) Aliphatic 3,4-epoxyalcohols, Metabolism by epoxide hydrase and mutagenic activity, Biochim. Biophys. Acta 544, 504-513.).
The decrease in mutagenic potency in the presence of the metabolic activation system in the mouse lymphoma assay was probably due to inactivation of the epoxide group by epoxide hydrase in the induced rat liver S9 or by endogenous metabolism by the cells. It has been shown that rat liver S9 contains higher levels of epoxide hydrase than S9 from mice and that induction with Aroclor further increases the epoxide hydrase levels (Oesch, F. (1972) Mammalian epoxide hydrases; Inducible enzymes catalysing the inactivation of carcinogenic and cytotoxic metabolites derived from aromatic and olefinic compounds, Xenobiotica, 3,305-340.). However, these effects are consequently less relevant for humans and the results gained in the UDS assay by uninduced mouse S9 mix is more relevant for human risk assessment.
Ohtani et al. showed in a bacterial mutation assay that epoxide compounds which have higher molecular weight and lower solubility showed neither any killing effect nor mutagenic effect in the DNA repair test as well as the reversion test. The reason seems to be that their molecular sizes and solubilities are not small and high enough to pass through the cellular membrane and to reach DNA (Ohtani H., Nishioka H. (1981), Mutagenic activity of epoxide compounds as constituents of resins in bacterial test system, Science and engineering review of Doshisha University).
Similar conclusion can be drawn in this assay: Since '1,2,3-propanetriol, glycidyl ethers' has a higher molecular weight than the tested compounds, it can be reasonably concluded that '1,2,3-propanetriol, glycidyl ethers' will not induce any positive results in the UDS assay, and can be hence considered as negative with and without metabolic activation.

Executive summary:

In an unscheduled DNA synthesis assay similar to OECD guideline 482, WI38 cells were exposed to the read-across substances for '1,2,3-propanetriol, glycidyl ethers', glycidol and butyl glycidyl ether at concentrations of 0, 0.375, 0.75, 1.5, 3.0, 6.0 µg/ml (glycidol without S9); 0, 0.037, 0.111, 0.333, 1.0, 3.0 µg/ml (glycidol with S9); 0, 0.24, 0.36, 0.53, 0.8, 1.2 µg/ml (butyl glycidyl ether without S9); 0, 0.5, 1.0, 2.0, 4.0, 8.0 µg/ml (butyl glycidyl ether with S9).

Glycidol and butyl glycidyl ether were tested just below the level which produced cytotoxicity. The positive controls (4-nitroquinoline-N-oxide (4NQO), dimethylnitrosamine (DMN)), induced the appropriate response.

There was no evidence or a dose related positive response for both compounds without metabolic activation that unscheduled DNA synthesis, as determined by radioactive tracer procedures, was induced. Glycidol induced a dose-related positive response with metabolic activation, butyl glycidyl ether induced demonstrable, although not considered positive responses with metabolic activation. Based on the extrapolation from glycidol over butyl glycidyl ether to '1,2,3-propanetriol, glycidyl ethers', taking into account molecule size, side chain length(s) and lipophilicity, it can be concluded that '1,2,3-propanetriol, glycidyl ethers' would reveal negative results both with and without S9 mix in the UDS assay.

The study was classified as reliable with restrictions (Klimisch 2) and satisfies the requirements for OECD guideline 482 for other genotoxicity data.