Ethylene oxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Agurell et al. (1991) conducted a non-guideline, non-GLP bacterial reverse mutation assay using two S.typhimurium strains (TA 100 and TA 1535) without metabolic activation. Test concentrations ranged from 0 – 12 mM. The result of the experiment was positive for genotoxicity.

Agurell et al. (1991) performed a non-guideline sister chromatid exchange assay in human peripheral lymphocytes without metabolic activation using concentrations between 0 – 120 mM. GLP compliance was not specified. The result of the experiment was positive for genotoxicity. 

Kolman et al. (1989) conducted a non-guideline mammalian cell transformation assay in mouse embryo fibroblasts C3H/10T1/2 without metabolic activation. Tested concentrations were 2.5, 7, 7.5 mM. GLP compliance was not specified. A positive result was found.

Hatch et al. (1986) performed a non-guideline mammalian cell gene mutation assay in Chinese hamster lung fibroblasts (V79) without metabolic activation using concentrations between 1.125 – 13.5 mg/l (625 - 7500 ppm). GLP compliance was not specified. A positive result was found.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

according to Maron, D.M. and Ames, B.N.: Mut. Res. 113, 173-215

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

S. typhimurium strains

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain / cell type:

S. typhimurium TA 100

Metabolic activation:

without

Metabolic activation system:

no data

Test concentrations with justification for top dose:

0 - 12 mMh

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

EO gave a dose-dependent increase in the number of revertants on both tester strains.

Conclusions:

In conclusions, under the conditions chosen the test substance induced mutations in the salmonella typhimurium strains TA 100 and TA 1535 and thus, is considered to be mutagenic.

Reference 2

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

according to P. Perry and S. Wolff; Nature 252, 156 - 158, 1974

GLP compliance:

not specified

Type of assay:

sister chromatid exchange assay in mammalian cells

Target gene:

human peripheral lymphocytes

Species / strain / cell type:

lymphocytes: human peripheral

Metabolic activation:

without

Metabolic activation system:

no data

Test concentrations with justification for top dose:

0 - 120 mMh

Species / strain:

hepatocytes: human peripheral

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Conclusions:

In conclusion, the test substance was tested positive in the sister chromatid exchange assay in human peripheral lymphocytes.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

Detailed methods have been previously described [Casto et al, 1973; Hatch and Anderson, 1985; Hatch et al, 1985).

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

- Supplier: Matheson Gas Co., East Rutherford
- Physical appearance: gas
- Purity: 10% in nitrogen

Target gene:

V79 cells

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Metabolic activation system:

no data

Test concentrations with justification for top dose:

1.125 - 13.5 mg/l (original value: 625 - 7500 ppm)

Statistics:

Statistical significance was determined using a table of ratios (Casto et al, 1973 derived from the Lorenz table (Lorenz, 1962) that is based on the poisson distribution. The increased transformation frequency (TF) was considered statistically significant at the 5% or 1 % confidence level if the enhancement ratio exceeded the appropriate value obtained from the Lorenz table.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

The cytotoxicity measurements for the calculation of the mutation frequencies were determined after 24 hr or 5 days of recovery after treatment and probably do not reflect the absolute cytotoxicity induced by EO. The data from experiment 1 indicate that the mutation frequency increased from six Oua(r) mutants per 10(6) survivors at 625 ppm EO to 128 Oua(r) mutants per 10 survivors at 7,500 ppm EO with a relative survival of 13% at the highest concentration. In experiment 2, cytotoxicity was evident at lower concentrations compared to experiment 1. However, the absolute mutation frequency for ouabain resistance was higher in experiment 2 at 1,250 ppm EO. In experiment 1, the mutation frequency for resistance to 6-TG increased from nine mutants per 10(6) survivors at 625 ppm E0 to 283 mutants per 10(6) survivors at 7,500 ppm EO. The absolute mutation frequency for 6-TG resistance was increased in experiment 2 compared to experiment 1 to 478 TGr mutants per 10(6) survivors at 5,000 ppm. In both experiments for both genetic markers clear concentration- response relationships were evident. The spontaneous mutation frequencies were 1-4 Oua(r) and 3-15 TG(r) mutants per 106 survivors determined in non-chemically treated V79 cells exposed to 80% nitrogen/20% oxygen in the chambers. MNNG, which has been reported previously to be mutagenic to V79 cells, was used as a positive control to characterize the sensitivity of the cells to a strong mutagen. The MNNG-treated cells were not treated in exposure chambers but were concurrently treated with the EO-exposed cells. MNNG induced 82-103 Oua(r) mutants and 401-608 TGr mutants per 106 survivors at a concentration of 0.5 pg/ml. The use of HEPES buffer and the rocking procedure probably reduced the plating efficiency of V79 cells in these studies compared to studies performed in plastic Petri dishes using Williams' E medium.

Conclusions:

In conclusion, under the conditions tested, the test substance induced a positive effect in the gene mutation assay and thus, was considered to be mutagenic.

Reference 4

Endpoint:

in vitro transformation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

according to Meyer, A. et al.: In vitro cell transformation assay. UKEMS Sub-Committee on Guidelines for Mutagenicity Testing (Part II A),Ch. 6,123-135

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell transformation assay

Target gene:

mouse embryo fibroblasts C3H/10T1/2

Species / strain / cell type:

mammalian cell line, other: mouse embryo fibroblasts C3H/10T1/2

Metabolic activation:

without

Metabolic activation system:

no data

Test concentrations with justification for top dose:

2.5, 7, 7.5 mMh

Species / strain:

mammalian cell line, other: mouse embryo fibroblasts C3H/10/T1/2

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

Cell transformation in vitro of C3H/10T1/2 cells using gamma-radiation and ethylene oxide (EtO), in both the absence and presence of the cancer promoter, 12-O-tetradecanoylphorbol-1 3-acetate (TPA), was studied. TPA promotes transformation of the cells to the same extent. In the dose ranges studied the average enhancement of the transformation frequency was 2.4 and 2.5 for EtO and gamma-radiation, respectively. The rad-equivalence of EtO in the presence of TPA was calculated to be 75 ± 52 rad/mMh (95% confidence interval) which is consistent with the value 78 + 14 rad/mMh (95% confidence interval) obtained withaut TPA treatment.

Conclusions:

In conclusion, in the in vitro cell transformation assay presented and under the conditions chosen, the test substance was positive in mutagenic activity and thus, considered to be a mutagenic.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Manjanatha et al. (2017) conducted a non-guideline cII Mutation Assay and ACB-PCR using 10 male B6C3F1 mice per dose. GLP compliance was not specified. Animals were exposed for 6 hours per day, 5 days per week for 4, 8, and 12 weeks to concentrations of 10, 50, 100, and 200 ppm. Results confirmed a genotoxic effect.

 

Parsons et al. (2013) performed a non-guideline ACB-PCR using 10 male B6C3F1 mice per dose that were exposed for 6 hours per day, 5 days per week for 4, 8, and 12 weeks to 10, 50, 100, and 200 ppm. GLP compliance was not specified. Genotoxicity of ethylene oxide was confirmed.

Embree et al. (1977) performed a non-guideline modified dominant lethal assay using Long-Evans rats of both sexes. 10 rats per sex were exposed to a concentration of 1000 ppm for 4 hours. 30 animals were used as control animals. GLP compliance was not specified. A positive reaction was found.

Lynch et al. (1984) conducted a non-guideline sister chromatid exchange assay using 12 male monkeys per dose that were exposed for two years. GLP compliance was not specified. Chosen concentrations were 50 and 100 ppm and animals were exposed for 7 hours per day and 5 days per week. Results concerning genotoxicity were ambiguous. 

 

Interestingly, the data of Generoso (1986, 1990), Tates (1995) and Donner (2009) indicate that many genotoxic effects appear to require dose levels of more than 20 ppm. Moreover, doses consisting of high concentrations and shorter exposure times appear to be more effective than lower concentrations and longer exposure times (though AUC in both cases being equal). Further investigations are being initiated in order to corroborate such findings.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

The question addressed in this study was whether EO can induce point mutatuins. Therefore, the neutral cII gene was investigated which detects only point and very small scale deletion mutations.

GLP compliance:

not specified

Type of assay:

other: cII Mutation Assay and ACB-PCR

Specific details on test material used for the study:

- Physical appearance: gas

Species:

mouse

Strain:

B6C3F1

Details on species / strain selection:

Male mice were used because the incidence of EO-exposure related lung tumors was higher in males than females.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Strategene Products Division of Agilent Technologies (supplied through Taconic Farms, Germantown, New York, USA)
- Age at study initiation: 8 week-old
- Acclimation period: at least 1 week

Route of administration:

inhalation: vapour

Vehicle:

no data

Duration of treatment / exposure:

4 weeks, 8 weeks or 12 weeks

Frequency of treatment:

6 h/d, 5 d/w

Post exposure period:

Four days

Dose / conc.:

10 ppm

Remarks:

10.1 ppm (analytical). 4 weeks exposure.

Dose / conc.:

50 ppm

Remarks:

50.0 ppm (analytical). 4 weeks exposure

Dose / conc.:

100 ppm

Remarks:

101.8 ppm (analytical). 4, 8 and 12 weeks exposure

Dose / conc.:

200 ppm

Remarks:

199.2 ppm (analytical). 4, 8 and 12 weeks exposure

No. of animals per sex per dose:

10

Control animals:

yes

Tissues and cell types examined:

right lobe of the lung was used for cII mutation analysis and the left lobe was used for ACB-PCR analysis.

Details of tissue and slide preparation:

Four days after the last exposure, mice were sacrificed and lungs were removed and snap-frozen in liquid nitrogen and shipped to the National Center for Toxicological Research on dry ice for subsequent DNA isolation and mutation analyses. DNA was extracted from the right mouse lung using the RecoverEase DNA Isolation Kit (conducted according to instruction manual). cII mutants were measured by growing infected G1250 bacteria on TB1 plates at 24°C for approximately 42 h.

Evaluation criteria:

cII MFs were determined by the number of mutant plaques divided by the total number of plaques screened.

Statistics:

Mutant and mutation frequencies for the cII gene were analyzed as a function of dose-3-time by Proc GLM and the Tukey-Kramer (for unbalanced data) test to evaluate the differences in mutant and mutation frequencies among groups. A value of P < 0.05 was considered significant.
Chi square and the statistical test described by Cariello et al. (1994) were used to analyze and compare cII mutational profiles. The mutation frequency was calculated by multiplying the mutation frequency by the percentage of independent mutations. Mutation frequency was only determined for the control and the 200 ppm groups. A vlaue of P < 0.05 was considered significant.

Sex:

male

Genotoxicity:

positive

Toxicity:

no effects

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not applicable

Additional information on results:

cII mutation frequencies after 4 weeks of exposure were not statistically significant in all concentrations. At 8 weeks and 12 weeks of exposure, only the highest concnetration (200 ppm) induced a significant response ( p < 0.05).

Table 1: The average cII mutant, and mutant frequencies +/- SEM, and induced frequenices in the lung of BB mice exposed to EO for up to 12 weeks

EO (ppm)		4 week- mutantfrequency (mutation frequency)^ax10^-6		Fold-increase mutant frequency over control		4 week-induced mutant frequency^b x10^-6		8 week- mutant frequency (mutation frequency)^ax10^-6		Fold-increase in mutant frequency over control		8 week-induced Mutant frequency^b x10^-6	12 week- mutant frequency  (mutationfrequency)^a 310^26		Fold-increase in mutant frequency over control		12-week induced mutant frequency^b x10^-6
0		17.462.2		—		—		20.063.4		—		—	29.664.1		—		—
		(16.762.1)						(1863.1)					(27.863.7)
10		28.567.1		1.6		11.1		NT		—		—	NT		—		—
50		33.865.8		1.9		16.4		NT		—		—	NT		—		—
100		25.965.1		1.5		8.5		26.162.8		1.3		6.1	30.062.9		1		0.4
200		47.9616.2		2.7		30.5		49.668.5*		2.5		29.6	41.962.8*		1.4		12.3
		(40.5613.4)						(45.367.8)*					(40.862.6)*

Executive summary:

Ethylene oxide (EO) is a direct acting alkylating agent; in vitro and in vivo studies indicate that it is both a mutagen and a carcinogen. However, it remains unclear whether the mode of action (MOA) for cancer

for EO is a mutagenic MOA, specifically via point mutation. To investigate the MOA for EO-induced mouse lung tumors, male Big Blue (BB) B6C3F1 mice (10/group) were exposed to EO by inhalation, 6 hr/day, 5 days/week for 4 (0, 10, 50, 100, or 200 ppm EO), 8, or 12 weeks (0, 100, or 200 ppm EO).

Lung DNA samples were analyzed for cII mutant frequency (MF) at 4, 8 and 12 weeks of exposure; the mutation spectrum was analyzed for mutants from control and 200 ppm EO treatments. Although EO induced cII MFs were 1.5- to 2.7-fold higher than the concurrent controls at 4 weeks, statistically significant increases in the cII MF were found only after 8 and 12 weeks of exposure and only at 200 ppm EO

(P0.05), which is twice the highest concentration used in the cancer bioassay. Consistent with the positive response, DNA sequencing of cII mutants showed a significant shift in the mutational spectra between

control and 200 ppm EO following 8 and 12 week exposures (P0.035), but not at 4 weeks. Thus, EO mutagenic activity in vivo was relatively weak and required higher than tumorigenic concentrations and

longer than 4 weeks exposure durations. These data do not follow the classical patterns for a MOA mediated by point mutations.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

no guideline followed

Principles of method if other than guideline:

ACB-PCR

GLP compliance:

not specified

Type of assay:

other: ACB-PCR

Specific details on test material used for the study:

- Physical appearance: gas

Species:

mouse

Strain:

B6C3F1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Farms (Germantown, New York, USA)
- Age at study initiation: 8 week-old mice
- Acclimation period: at least one week

Route of administration:

inhalation

Duration of treatment / exposure:

6 h/day 5 d/week for 4 weeks, 8 weeks, 12 weeks

Dose / conc.:

10 ppm

Remarks:

4 week exposure

Dose / conc.:

50 ppm

Remarks:

4 week exposure

Dose / conc.:

100 ppm

Remarks:

4, 8, 12 week exposure

Dose / conc.:

200 ppm

Remarks:

4, 8, 12 week exposure

No. of animals per sex per dose:

10

Control animals:

yes

Tissues and cell types examined:

Isolation of lung DNA for ACB-PCR from the entire left lobe of each mouse lung

Statistics:

The geometric mean for mutant fractions was calculated. The statistical significance of treatment-related effects was assessed by comparing the distribution of samples using a 2-sided Fisher's exact test. Pearson's rank correlation coefficient was calculated on log-transformed data, using a 2-tailed test (correlation between mutant frequency at the cll locus and K-ras mutant frequency).

Sex:

male

Genotoxicity:

positive

Toxicity:

no effects

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not applicable

Conclusions:

In summary, ACB-PCR was used to discern the early effects of EO on K-ras mutation in mouse lung. The results indicate that
1. the type and magnitude of induced K-ras mutations were consistent with their spontaneous occurrence in mouse lung tissue,
2. levels of K-ras codon 12 GGT->GTT and GGT->GAT mutations within individual mouse lung DNA samples were significanrly correlated,
3. EO had a biphasic effect on K-ras mutation in lung tissue, and
4. K-ras mutation and cll mutation were inversely correlated in lung tissues exposed to relatively high EO doses.

These data are consistent with the interpretation that EO inhalation initially alters the fitness of preexisting K-ras mutant cells rather than inducing de novo K-ras mutations in lung, although it cannot be concluded from these results that none of EO's genotoxic effects lead to K-ras mutations.

Executive summary:

Ethylene oxide (EO) is a genotoxicant and a mouse lung carcinogen, but whether EO is carcinogenic through a mutagenic mode of action remains unclear. To investigate this question, 8-week-old male Big Blue B6C3F1 mice (10 mice/group) were exposed to EO by inhalation - 6h/day, 5 days/week for 4 weeks (0, 10, 50, 100, or 200 ppm EO) and 8 or 12 weeks (0, 100, or 200 ppm EO). Lung DNA samples were analyzed for levels of 3 K-ras codon 12 mutations

(GGT->GAT, GGT->GTT, and GGT->TGT) using ACB-PCR.

No measureable level of K-ras codon 12 TGT mutation was detectcd (ie, all lung mutant fractions [MFs] ≤ 10^-3). Four weeks of inhalation of 100 ppm EO caused a significant increase in K-ras codon 12 GGT->GTT MF relative to controls, whereas 50, 100, and 200 ppm EO caused significant increases in K-ras codon 12 GGT->GAT MF. In addition, significant inverse correlations were observed between K-ras codon 12 GGT->GTT MF and cll mutant frequency in the lungs of the same mice exposed to 50, 100, or 200 ppm EO for 4 weeks. Surprisingly, 8 weeks of exposure to 100 and 200 ppm EO caused signi:ficant decreases in K-ras MFs relative to controls. Thus, the changes in K-ras MF as a function of cumulative EO dose were nonmonotonic and were consistent with EO causing early amplification of preexisting K-ras mutations, rather

than induction of K-ras mutation through genotoxicity at codon 12. The possibility that these changes reflect K-ras mutant cell selection under varying degrees of oxidative stress is discussed.

Reference 3

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Principles of method if other than guideline:

The current study was designed to investigate the mutagenic potential of EO and PO in monkeys following 2-year exposures under controlled experimental conditions.

GLP compliance:

not specified

Type of assay:

sister chromatid exchange assay

Specific details on test material used for the study:

- Physical appearance: gas
- Purity: 99.7%

Species:

monkey

Strain:

Macaca fascicularis

Sex:

male

Route of administration:

inhalation

Duration of treatment / exposure:

2 years

Frequency of treatment:

7 h/d, 5 d/w

Dose / conc.:

50 ppm (nominal)

Dose / conc.:

100 ppm (nominal)

No. of animals per sex per dose:

12

Control animals:

yes, concurrent no treatment

Sex:

male

Genotoxicity:

ambiguous

Toxicity:

not specified

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

Toxic effects of the EO exposure were encountered during attempts to culture blood samples from EO-exposed monkeys. In 1 out of 11 animals exposed at 50 ppm EO and 3 out of 12 monkeys exposed at 100 ppm EO, there were minimal (if any) mitosis. In one of these three EO 100 ppm monkeys, where only two metaphases could be evaluated, the SCEs/metaphase were 20 and 42, respectively. Further attempts employing different culture times and varying phytohemagglutinin concentrations and sources were also unsuccessful.

Conclusions:

In conclusion, the test substance revealed an ambiguous test result under the conditions chosen in the sister chromatid exchange assay and chromosome aberration assay.

Reference 4

Endpoint:

genetic toxicity in vivo, other

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

GLP compliance:

not specified

Specific details on test material used for the study:

- Physical appearance: gas

Species:

other: human

Strain:

not specified

Sex:

not specified

Route of administration:

inhalation

Details on exposure:

Peripheral blood from four groups of seven workers from a chemical manufacturing plant in The Netherlands was analyzed for hemoglobin adducts in erythrocytes and for hprt mutants, micronuclei and SCEs in lymphocytes. Group I workers were incidentally exposed to acute high doses of ethylene oxide ranging from 52 to 785 mg/m3. Group II and III workers were chronically exposed to low doses of ethylene oxide for < 5 years or > 15 years respectively. Group IV workers served as unexposed controls and came from the Occupational Health Department.

Remarks on result:

other: This finding implies that the incidental exposure or workers to high concentrations of EtO did not cause any measurable permanent mutational/cytogenetic damage in lymphocytes.

Additional information on results:

Hemoglobin adduct levels in group I workers were very high and ranged from 1461 to 19913 pmol HOEtVal/g Hb approximately 1 month after the accident. HOEtVal values for group II and III workers fluctuated between 0 and 190 pmol/g Hb corresponding with average EtO exposure levels in the range of < 0.01 and 0.06 mg/m3 EtO.
The statistical analysis of the genetic data did not reveal any statistically significant differences between any combination of worker groups. The genetic tests for group I workers were performed on blood samples collected 89-180 days after the incidental exposure. The absence of enhanced frequencies of mutations, micronuclei and SCEs suggests that significant induction of hprt mutations in vivo did not occur and that persistent preclastogenic lesions were not present in significant amounts when the exposed lymphocytes were put in culture to visualize any induced cytogenetic damage. This finding implies that the incidental exposure to high concentrations of EtO did not cause any measurable permanent mutational/cytogenetic damage in lymphocytes.

Conclusions:

In summary, the present study on hemoglobin adducts in erythrocytes and on hprt mutants, sister-chromatid exchanges and micronuclei in lymphocytes has indicated that acute occupational exposure to high concentrations of the test substance or chronic exposures to low doses of the test substance did not lead to a measurable permanent cytogenetic or mutational damage.

Executive summary:

Peripheral blood from four groups of seven workers from a chemical manufacturing plant in The Netherlands was analyzed for hemoglobin adducts in erythrocytes and for hpti mutants, micronuclei and SCEs in lymphocytes. Group I workers were incidentally exposed to acute high doses of ethylene oxide ranging from 52 to 785 mg/m3. Group II and III workers were chronically exposed to low doses of the test substance for < 5 years or > 15 years respectively. Group IV workers served as unexposed controls and came from the Occupational Health Department. Hemoglobin adduct levels in group I workers were very high and ranged from 1461 to 19913 pmol HOEtVal/g Hb approximately 1 month after the accident. HOEtVal values for group II and III workers fluctuated between 0 and 190 pmol/g Hb corresponding with average test substance exposure levels in the range of < 0.01 and 0.06 mg/m3 of the test substance.

The statistical analysis of the genetic data did not reveal any statistically significant differences between any combination of worker groups. The genetic tests for group I workers were performed on blood samples collected 89-180 days after the incidental exposure. The absence of enhanced frequencies of mutations, micronuclei and SCEs suggests that significant induction of hprf mutations in vivo did not occur and that persistent preclastogenic lesions were not present in significant amounts when the exposed lymphocytes were put in culture to visualize any induced cytogenetic damage. This finding implies that the incidental exposure to high concentrations of the test substance did not cause any measurable permanent mutational/cytogenetic damage in exposed lymphocytes.

Reference 5

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

The present study was undertaken to show that EtO produces such heritable effects by a relevant route of human exposure, namely inhalation, and to generate a dose-response curve that could be explored further as a model for estimating human genetic risk.

GLP compliance:

not specified

Type of assay:

rodent dominant lethal assay

Specific details on test material used for the study:

- Physical appearance: gas

Species:

mouse

Strain:

other: (C3Hx101)F1

Sex:

male

Route of administration:

inhalation

Duration of treatment / exposure:

8.5 weeks: 6 hr/d, 5 d/wk, for 6 wk; then 7 d/wk, for 2.5 wk

Frequency of treatment:

8.5 weeks: 6 hr/d, 5 d/wk, for 6 wk; then 7 d/wk, for 2.5 wk

Dose / conc.:

0.3 mg/L air

Remarks:

165 ppm

Dose / conc.:

0.37 mg/L air

Remarks:

204 ppm

Dose / conc.:

0.45 mg/L air

Remarks:

250 ppm

Dose / conc.:

0.54 mg/L air

Remarks:

300 ppm

No. of animals per sex per dose:

96 exposed animals; 24 control animals

Control animals:

yes

Sex:

male

Genotoxicity:

positive

Toxicity:

not specified

Vehicle controls validity:

not applicable

Negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

Transmitted clastogenic effects of these exposures were measured in terms of induction of dominant lethal mutations and heritable translocations. The concentration response curves for both endpoints are not linear but are markedly concave upward. Significant increases in dominant lethals were detected at all concentrations, except the lowest one. In comparison, the incidences of heritable translocations were significantly increased at oll concentrations.

Conclusions:

In conclusion, the test substance was tested positive in the dominant lethal assay undertaken in male mice.

Reference 6

Endpoint:

in vivo mammalian somatic and germ cell study: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

This study provides low-level chronic exposure somatic cytogenetic data generated in mice that can be used to support the shape of the tumour-response in rodents and humans.

GLP compliance:

not specified

Type of assay:

mammalian germ cell cytogenetic assay

Species:

mouse

Strain:

B6C3F1

Sex:

male

Route of administration:

inhalation

Details on exposure:

whole-body exposure

Duration of treatment / exposure:

up to 48 weeks

Frequency of treatment:

6 h/d, 5 d/w

Dose / conc.:

25 ppm (nominal)

Dose / conc.:

50 ppm (nominal)

Dose / conc.:

100 ppm (nominal)

Dose / conc.:

200 ppm (nominal)

No. of animals per sex per dose:

44

Control animals:

yes

Tissues and cell types examined:

somatic and germ cells

Sex:

male

Genotoxicity:

positive

Toxicity:

not specified

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not applicable

Additional information on results:

The frequency of the total chromosomal aberrations in the peripheral blood lymphocytes was significantly increased at the 100 and 200 ppm exposure concentrations at the 12-week time point, at 50, 100 and 200 p.p.m. at the 24-week time point and at all EO concentrations at the 48-week time point. The frequency of stable reciprocal translocations, which can be used as biomarkers, was increased (P < 0.05) at 100 and 200 p.p.m. at the 12-week
time point, at 100 and 200 p.p.m. at the 24-week time point and at 50, 100 and 200 p.p.m. at the 48-week time point. No statistically significant increase could be observed in translocation frequencies at the 6-week time point in the peripheral blood lymphocytes. The exposure–response curves were non-linear when the frequencies of translocations were plotted against EO exposure durations or against EO exposure concentrations. There was no effect of exposure concentration rate on reciprocal translocation frequency. Reciprocal translocations induced in spermatogonial stem cells (observed at the sprematocyte stage) showed significant increases in translocation frequencies over controls at all EO concentrations at 48 weeks. However, increases were small and they did not occur in a dose-responsive manner. The statistically significant increase observed at 12 weeks in the spermatocytes was equivocal. This study provides low-level chronic exposure somatic cytogenetic data generated in mice that can be used to support the shape of the tumour dose–response in rodents and humans The germ cell cytogenetic data are discussed in terms of its relevance for a threshold response for genetic effects at low exposures.

Conclusions:

Inhalation exposure to ethylene dioxide for up to 48 weeks induced reciprocal translocations and other observable aberrations in the peripheral blood lymphocytes with increases being proportional to exposure duration.

Reference 7

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

Modified version of the murine dominant-lethal test as described by Epstein (1973).

GLP compliance:

no

Type of assay:

rodent dominant lethal assay

Specific details on test material used for the study:

- Physical appearance: gas

Species:

rat

Strain:

Long-Evans

Sex:

male/female

Route of administration:

inhalation

Vehicle:

olive oil

Duration of treatment / exposure:

4 hours

Dose / conc.:

1 000 ppm (nominal)

No. of animals per sex per dose:

10 (control); 30 (test group)

Control animals:

yes, concurrent no treatment

Positive control(s):

TEM (triethylenemelamine)

Statistics:

Raw data were tabulated and statistically analyzed using a computer program available at the Stanford Research Institute. The mean data for total and dead implants in pregnant females were analyzed using the Student t-test. Preimplantation loss defined as Corpora lutea minus total implants per pregnant females and dead implants per total implants in pregnant females was analyzed using the t-test on the Freeman-Tukey transformed data. The Chi-square technique was used to detect differences in the fertility index which is defined as the number of females pregnant per number mated.
5 one-way analyses of variance were performed on the control group data. The variables were number of pregnant females, preimplantation loss per pregnant female, number of dead implants per pregnant female, and the ratio of dead implants to the total implants per pregnant female.

Sex:

male/female

Genotoxicity:

positive

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Following the 4 hr 1000-ppm EO exposure, all of the exposed rats showed slight signs of EO toxicity. These included central depression, diarrhea, and occular and respiratory irritation. Within 34 hr, however, all rats appeared normal and no deaths were recorded during the experiment. The TEM and negative control groups showed no signs of toxicity after dosing and also sustained no deaths during the experiment. One male within the control group produced no pregnancies for the full 10 weeks of the experiment.
Before analysis of the results, all data pertaining to this animal and the females caged with him were discarded.
No significant changes between groups were seen in the last 5 weeks of the experiment. In comparing the EO data with the negative control data, significant differences were apparent in both the fertility index and the indices dealing with postimplantational losses.
The fertility index or the number of rats pregnant per number mated shows a significant decrease in the EO test group in weeks 3 and 4. Other significant decreases were seen in the total implants per pregnant female (week 2), dead implants per pregnant female (weeks 2, 3, and 5) and the mutagenic index (dead implants per total implants) for weeks 1, 2, 3, and 5.
The TEM positive control group, when compared to the negative control, also showed significant decreases in total implants per pregnant female (weeks 2, 3 and 4), dead implants per pregnant female (each week up to 5), and in the mutagenic index (each week up to 5). Except for week 4 in the TEM group, neither EO nor TEM treatment resulted in significant changes in average pre-implantation loss per pregnant female. TEM treatment did not significantly change the fertility index.
The results of the analysis of variance on the data of the control group showed no significant differences between or within males over the various weeks of the experiment.

Conclusions:

In conclusion, under the consitions chosen, the test substance generated positive results and thus, is considered to be mutagenic.

Reference 8

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

The present publication was designed to determine dominant-lethal response in male mice as affected by varying the dose and dose-rate.

GLP compliance:

not specified

Type of assay:

rodent dominant lethal assay

Specific details on test material used for the study:

- Physical appearance: gas

Species:

mouse

Strain:

other: C3H x 101F1

Sex:

male

Route of administration:

inhalation

Duration of treatment / exposure:

4 consecutive days

Frequency of treatment:

6 h per day

Dose / conc.:

3.24 mg/L air (nominal)

Remarks:

x hr cumulative daily dose

Dose / conc.:

0.54 mg/L air (nominal)

Remarks:

6 hrs/d

Dose / conc.:

1.08 mg/L air (nominal)

Remarks:

3 hrs/d

Dose / conc.:

2.16 mg/L air (nominal)

Remarks:

1.5 hrs/d

No. of animals per sex per dose:

total: 96

Control animals:

yes

Sex:

male

Genotoxicity:

positive

Additional information on results:

In the dose-response study, male mice were exposed by inhalation to ethylene oxide (EtO) for 4 consecutive days. Mice were exposed for 6 hr per day to 300 ppm, 400 ppm, or 500 ppm EtO for a daily total of 1,800, 2,400, or 3,000 ppm x hr (total exposures of 7,200, 9,600 and 12,000 ppm x hr), respectively. In the dose-rate study, mice were given a total exposure of 1, 800 ppm x hr per day, also for 4 consecutive days, delivered either at 300 ppm in 6 hr, 600 ppm in 3 hr, or 1,200 ppm in 1.5 hr. Quantitation of dominant-lethal responses was made on matings involving sperm exposed as late spermatids and early spermatozoa, the most sensitive stages to EtO. In the dose-respone study, a dose-related increase in dominant-lethal mutations was observed, the dose-response curve proved to be nonlinear. In the dose-rate study, increasing the exposure concentrations resulted in increased dominant-lethal responses.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Mode of Action Analysis / Human Relevance Framework

A non-guideline study conducted by Dow Chemical (2015) in compliance with GLP was intended to reveal the mechanism of action of ethylene oxide using 356 male B6C3F1 mice (total number). Animals were exposed for 4, 8, 12 weeks, 6 hours per day 5 days per week to concentrations of 10, 50, 100, 200 ppm. The exact mechanism of action could not be identified.

Justification for classification or non-classification

Classification, Labeling, 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.

As a result, the substance requires classification as a mutagen of category 1 B, H340 (Index number 603-023-00-X in Annex VI, part 3, Table 3.1 (list of harmonized classification and labelling of hazardous substances) of Reg. (EC) No 1272/2008).