Ethylene oxide

Carcinogenicity

Description of key information

Snellings et al. (1984) performed a chronic study in 120 Fischer 344 rats per sex per dose which was equivalent or similar to OECD 453. GLP compliance was not specified. Animals were exposed for 6 – 24 months, 6 hours per day 5 days per week to 10, 33, and 100 ppm ethylene oxide vapor. Mortality was observed and was statistically significant and numerically greater in the 100 and 33 ppm group, respectively. Clear evidence of carcinogenicity was found. Brain tumours were elevated at 33 and 100 ppm in a dose-related response. Also peritoneal mesothelioma at a male rat-specific site and leukemias of a Fischer rat-specific site (spleen mononuclear cell leukemia) were observed at increased rates but considered to be of low relevance to man. Female rats experienced a statistically significant increase in the percentage of rats with two or more neoplasms compared to the control at 10 ppm. One or more biologically significant adverse effects have been documented in rats exposed to 10 ppm and thus, a NOAEC could not be established.

 

Garman et al. (1985) performed a non-guideline two-year inhalation study in Fischer 344 rats both male and female. GLP compliance was not specified. Animals were exposed for two years 6 hours per day 5 days per week to 33 and 100 ppm ethylene oxide vapor. Clear evidence of carcinogenicity was found. At the 100 and 33 ppm dose groups an increased number of brain tumours were seen in males and females. Thus, a NOAEC could not be established.

Further investigations on the dose-response relationship and of different types of genotoxicity are presently undertaken in order to possibly identify thresholds of carcinogenicity of non-linear elements for a low dose extrapolation.

 

Lynch et al. (1984) conducted a non-guideline study using 80 male Fischer 344 rats per dose. GLP compliance was not specified. Animals were exposed for two years 7 hours per day 5 days per week to 50 and 100 ppm ethylene oxide vapor. Clear evidence of carcinogenicity were reported. A NOAEC was not established.

 

NTP (1987) performed a sub-chronic chronic non-guideline study using 50 B6C3F1 mice per sex per dose. GLP compliance was not specified. Animals were exposed for 102 weeks 6 hours per day 5 days per week to 50 and 100 ppm of ethylene oxide vapor. No significant differences in survival were observed between any groups of either sex. Evidence of carcinogenic activity for mice was found. Lung tumours were clearly increased at 100 ppm in both sexes and marginally increased at 50 ppm (close to the historical range). Thus, a NOAEC could not be established. The human relevance of these tumours is far from clear since EO exposure has never been associated with lung tumours in man in many epidemiological studies.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via inhalation route

Link to relevant study records

Reference

Reference 1

Endpoint:

carcinogenicity: inhalation

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

GLP compliance:

not specified

Specific details on test material used for the study:

- Supplier: Union Carbide Corporation
- Physical appearance: liquid
- Purity: > 99.9%

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

Source: Microbiological Associates, Walkersville, Maryland, USA
Age of animals: 6 weeks
General health status was determined during a 2-week quarantine period. Rats accepted were those for which no abnormal clinical signs were observed and whose body weights were within plus or minus two standard deviations of the mean weight of all rats of the same sex.
All animals were housed and exposed, five per cage, in wire-mesh stainless steel cages. Light/dark cycle: 12 h. Animals were exposed during the light period at approx. the same time each day. The temperature and humidity controlling devices of the room were set to maintain the environment between 20 and 24°C and 40 - 60% rel. humidity. Feed and water were removed during the exposure period but were available ad libitum at all other times.

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on exposure:

Exposure and analytical systems. A stainles-ssteel cylinder containing liquid ethylene oxide was heated to approximately 35°C. The vapor generated was metered into 4400-liter stainless-steel and glass inhalation chambers. Chamber temperature and relative humidity were controlled, and the values for these parameters were recorded during each exposure.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chamber atmospheric concentrations in both the test and control chambers were monitored by a gas chromatograph equipped with a fame ionization detector. The column was packed with Tergitol-TMN surfactant (30%) and sodium methylate (3%) on Chromosorb W-NAW support material (Klein et al., 1983). Approximately eight analyses were obtained from each chamber within each 6-hr exposure period.

Duration of treatment / exposure:

6 - 24 months

Frequency of treatment:

6 h/d, 5 d/w

Post exposure period:

none

Dose / conc.:

10 ppm (nominal)

Remarks:

18 mg/m³

Dose / conc.:

33 ppm (nominal)

Remarks:

60 mg/m³

Dose / conc.:

100 ppm (nominal)

Remarks:

183 mg/m³

No. of animals per sex per dose:

120

Control animals:

yes, concurrent no treatment

Details on study design:

Two air control groups were exposed under similar conditions.
Whole body exposures were conducted in a dynamic exposure system where the vapor concentration levels were determined by gas chromatography.

Observations and examinations performed and frequency:

After initiation of exposure, animals were routinely examined for signs of toxic effects including body weight determination and palpation for abnormal tissue masses. After 6 and 12 months of exposure, 10 rats per sex per dose were necropsied. After 18 months, 20 rats per sex per dose were necropsied, and after 24 months (females) or 25 months (males), all remaining rats were necropsied.

Sacrifice and pathology:

A complete necrospy was performed on each animal killed at a scheduled necropsy interval ans on all animals which were found dead or moribund. At the 6-month and final intervals, approx. 50 tissues from each of the rats in the 100-ppm and two air control groups were histopathologically examined (details not mentioned). The same tissues were examined from all rats that died or were killed in a moribund condition from any group. At the 12- and 18-month intervals, app. 15 major organs and tissues were microscopically examined from the rats in the 100-ppm and both control groups. Only tissues with gross lesions were examined from the rats in the 33- and 10-ppm groups at the 6-12-, and 18-month intervals. At the final interval, approx. 20 major organs and tissues were examined from the rats in the 33- and 10-ppm groups. Tissues saved were fixed in 10% neutral buffered Formalin. Tissues submitted for histopatholigic evaluation were stained with hematoxylin and eosin.

Statistics:

Tumor incidence data for each exposure group were compared statistically to the combined data of the two air control groups. For all statistical comparisons, 2-tailed probabilities are reported. For all life table statistical comparisons, the critical ratios 2 were calculated using standard errors. The probabilities associated with the critical ratios and with the results of the Fisher's exact comparisons for tumor incidences were adjusted by the Bonferroni correction to account for the multiplicity of comparisons. Additional statistical tests for positive trends were also used. Time-adjusted trend test analyses were performed to compensate for bias associated with differential mortality in the various treatment groups. The adjusted analyses are sensitive not only to differences in relative tumor frequencies among groups but also to the time of observation of the tumors. Thus, the trend tests indicate not only whether the treatment results in more tumors but also if tumors develop earlier. An additional statistical analysis was also performed. In this test, early deaths are eliminated from the procedure as described by Gart et al.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

effects observed, treatment-related

Details on results:

Exposure concentrations
The actual daily ethylene oxide concentration within the chambers were very close to the target concentration of 100, 33 and 10 ppm. The overall 2-year means (with the coefficient of variation) of the daily measured chamber concentration were 101 (5%), 33 (3%) and 10 (5%) ppm for the high, intermediate, and low exposure concentration.
A viral sialodacryoadenitis (SDA) occurred during the 15th exposure month and exposures to ethylene oxide were temporarily stopped for 2 weeks. During this time, body weight gain and almost all clinical signs returned to preinfection status.

Body weights
A statistically significant depression in body weight gain was noted in the 100-ppm group for both male and female rats beginning about the end of the first exposure month. Following 10 weeks of exposures to 33 ppm of ethylene oxide, an effect on the body weight of only the female rats was observed. These treatment-related effects were generally observed throughout most of the remainder of the study. Furthermore, for both sexes, the body weight curves of the two control groups and the 10-ppm groups were similar throughout the study.

Mortality
Very low mortality had been observed prior and after SDA outbreak. During viral infection one or more animals of each group died. However, after 2 weeks of no ethylene oxide exposure, the rate of mortality decreased to the level prior to the infection.
Since the 20th exposure month, mortality increased and was significantly higher for both sexes in the 100- ppm group compared to control groups. From the 21st month on, the values for both sexes of the 33-ppm group were numerically greater than those of both control groups.

Mononuclear cell leukemia
Hematologic evidence of an increased number of animals with malignant mononuclear cells in the peripheral blood was found at the final interval (24 months) among rats exposed to ethylene oxide. Histologic evaluation of the spleens confirmed mononuclear cell leukemia (MNCL) in these animals. The mean relative spleen weight values of rats with leukemia were significantly higher than those of rats without leukemia, averaging approximately 5 times higher in the males and 8 times higher in the females.
The prevalence of histologically confirmed MNCL in the ethylene oxide-exposed rats was greater in the females than in the males. At the final interval, the incidence of MNCL was statistically different (p < 0.001) from the control groups only for the female 100-ppm group; however, the frequencies for the female 10-ppm (11/54), 33-ppm (14/48), and 100-ppm (15/26) groups were 2, 3, and 6 times, respectively, that of the control groups (11/115), indicating a dose-response relationship down to the lowest exposure group.
For the male rats, the prevalence of MNCL in the 100- and 33-ppm groups was numerically, but not statistically significantly, elevated in comparison to the controls, and the prevalence in the three different exposure groups was not significantly related to the concentration (regression analysis not significant; r = ±0.78).

Peritoneal mesothelioma
At 24 months, the prevalence of peritoneal mesotheliomas was elevated (not statistically significant) in the males of the 100- and 33-ppm groups when compared to the low frequencies of the control groups. The frequency of the 100-ppm group was 13%, and that of the 33-ppm group was 10%, whereas the frequencies for the control and 10-ppm groups were between 2 and 4%.

Brain tumors
From the time of the 18-month kill until the end of the study, the frequency of animals with primary brain neoplasms increased, particularly in the males of the 100- and 33-ppm exposure groups. When the data for all the animals that died, as well as the ones killed at a scheduled interval, were statistically analyzed by a time-adjusted trend test, significant probabilities indicating a positive trend were obtained for the male (p < 0.01) and female rats (p < 0.05).
When the data from the final interval were statistically evaluated (Fisher’s exact test), there were no statistically significant differences in the prevalence of brain tumors between groups for either males or females. However, there was a numerical increase in the 100-ppm group.

Pituitary adenoma
By the terminal kill, the prevalence of pituitary adenoma was similar in all groups for male and female rats. However, if the assumption is made that this is a nonincidental tumor, the results of the trend analysis (significance level was p < 0.001) indicated that this tumor developed earlier in female rats than in male rats exposed to 100 ppm of ethylene oxide.

Proportion of rats with neoplasms
The frequencies among rats with multiple neoplasms (benign or malignant) at the final interval were increased in the ethylene oxide-treated rats. The mean values for the number of primary tumors per tumor-bearing rat are given in Tables 1 and 2 for male and female rats, respectively. The mean value of the 100-ppm group for the males was approximately 24% greater than for the controls, and the mean for the females was approximately 69% greater. This finding was statistically evaluated by comparing the ratios of rats with one or more neoplasms, two or more neoplasms, etc., and the results are also presented in Tables 1 and 2. Since the prevalences for most tumor types of both control groups were similar, the control data were combined and statistically compared to the ethylene oxide-treated groups. The frequency among male rats with multiple primary neoplasms was significantly greater for the male rats in the 100-ppm group, whereas it was significantly greater (p < 0.05) for all three exposure groups in the female rats with at least one malignant neoplasm was noted in the 100- and 33-ppm exposure groups when statistically compared to the controls (Table 2).

Principal effects of treatment include depression of body weight gain, enhanced mortality, general increase in tumors, and treatment-associated increases in brain tumors, mesotheliomas, and mononuclear cell leukaemia. Only at the final kill were there apparent treatment-related effects seen in the 10-ppm group. This conclusion was based on the findings of numerically increased incidence of MNCL and a statistically significant increase in the number of rats with multiple primary neoplasms. Based on the observation that the incidences of mononuclear cell leukemia, peritoneal mesothelioma and pituitary adenoma in the air control groups were similar to those reported in the literature, a possible contribution of SDA outbreak to the ethylene oxide exposure related tumors is considered to be unlikely.

Dose descriptor:

NOAEC

Effect level:

< 10 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

histopathology: neoplastic

mortality

Remarks on result:

not determinable

Remarks:

no NOAEC identified.

Critical effects observed:

yes

Lowest effective dose / conc.:

10 ppm

System:

haematopoietic

Organ:

leucocyte development

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Critical effects observed:

yes

Lowest effective dose / conc.:

33 ppm

System:

central nervous system

Organ:

brain

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

yes

Critical effects observed:

yes

Lowest effective dose / conc.:

33 ppm

System:

musculoskeletal system

Organ:

other: mesothelium

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Table 1

 

Number of primary benign and malignant neoplasms^aamong male rats killed after 24 months of expsure

 

	Exposure group (ppm)
Parameter	100	33	10	CI 0	CII 0	CI and CII 0
Number of rats examined	30	39	51	48	49	97
Mean number neoplasms^bper neoplasm-bearing rat	4.1	3.3	3.0	3.2	3.3	3.3
Percentage of rats with
1 or more neoplasms^b	100	100	100	100	98	99
2 or more neoplasms	100	97	94	98	98	98
3 or more neoplasms	83	72	61	75	76	75
4 or more neoplasms	63*	41	35	35	31	33
5 or more neoplasms	47**	18	10	10	12	11
6 or more neoplasms	17	5	2	2	8	5
7 neoplasms	7	3	0	0	2	1
Percentage of rats with
1 or more malignancies^c	40	51	33	27	33	30
2 or more malignancies	17*	5	4	2	2	2

 

^a                The presence of the same neoplasm in bilateral organs was counted as two. Exceptions to this were that mononuclear cell leukemia and peritoneal mesothelioma were tabulated as one neoplasm each per rat, and multiple tumors of the same type in the liver were tabulated once. For certain tissues, only gross lesions were examined in the 33- and 10-ppm groups.

^b                Includes benign and malignant.

^c                Liver neoplastic nodule was not tabulated as a malignancy.

*       p < 0.05 for comparison to combined controls (CI and CII)

**      p < 0.05 for comparisons to CI, CII, and combined controls (CI and CII).

 

 

Table 2

 

Number of primary benign and malignant neoplasms^aamong female rats killed after 24 months of expsure

 

	Exposure group (ppm)
Parameter	100	33	10	CI 0	CII 0	CI and CII 0
Number of rats examined	26	48	54	60	56	116
Mean number neoplasms^bPer neoplasm-bearing rat	2.2	1.7	1.8	1.3	1.3	1.3
Percentage of rats with
1 or more neoplasms^b	92	81	81	80	79	79
2 or more neoplasms	62**	42*	44*	20	20	20
3 or more neoplasms	42**	17	13	3	5	4
4 or more neoplasms	12	0	6	0	0	0
5 neoplasms	4	0	0	0	0	0
Percentage of rats with
1 or 2 malignancies^c	58**	35**	31	10	21	16
2 malignancies	19**	6	6	0	0	0

 

^a                The presence of the same neoplasm in bilateral organs was counted as two. Exceptions to this were that mononuclear cell leukemia and peritoneal mesothelioma were tabulated as one neoplasm each per rat, and multiple tumors of the same type in the liver were tabulated once. For certain tissues, only gross lesions were examined in the 33- and 10-ppm groups.

^b                Includes benign and malignant.

^c                Liver neoplastic nodule was not tabulated as a malignancy.

*       p < 0.05 for comparison to the combined controls (CI and CII)

**      p < 0.05 for comparisons to CI and/or CII, and combined controls (CI and CII).

Conclusions:

In conclusion, biologically significant adverse effects related to ethylene oxide exposure were observed at all concentrations tested.

Executive summary:

Inhalation of EtO resulted in a significant depression of body weight gain in the 100- and 33-ppm exposure groups and a significant increase in mortality in the 1000 ppm group. Through 18 months of exposure to EtO, no statistically significant increases in tumor incidence were observed. After 18 months, the incidence of primary brain tumors was increased for both sexes. Statistical evaluation indicated a treatment-related response, particularly for the male rats, in the 100- and 33-ppm exposure groups. After 24 months ofexposure, histologic findings confirmed hematologic evidence that exposure to EtO resulted in an increased prevalence of mononuclear cell leukemia, which is a neoplasm that is common in aged Fischer 344 rats. This increase was dose related and increased for each of the three exposure concentrations. The percentage of female rats with multiple neoplasms was also greater inall three exposure groups than in controls. Furthermore, in both the 100- and 33-ppm exposure groups, the percentage of female rats with at least one malignant neoplasm was increased. An increased frequency of peritoneal mesothelioma was treatment related in the male rats exposed to 100 or 33 ppm of EtO. This study has shown biologically significant adverse effects at all concentrations tested. The incidences of mononuclear cell leukemia, peritoneal mesothelioma, and primary brain tumors in the air-control rats were similar to those reported in the literature. The possible contribution of a sialodacryoadenitis viral outbreak (which occurred during the 15th exposure month) to the EtO exposure-related tumors is unknown, though unlikely.

Reference 2

Endpoint:

carcinogenicity: inhalation

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:

no data

GLP compliance:

not specified

Specific details on test material used for the study:

- Physical appearance: gas

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Route of administration:

inhalation

Type of inhalation exposure (if applicable):

not specified

Duration of treatment / exposure:

2 years

Frequency of treatment:

6 h/d, 5 d/w

Dose / conc.:

33 ppm

Dose / conc.:

100 ppm

Control animals:

yes, concurrent no treatment

Details on results:

Brain tumours: 3 representative sections of the brain from each rat were evaluated. 23  primary brain tumours were found, 2 in control animals. At the 100 and 33  ppm dose groups an increased number of brain tumours were seen in males and females.

Dose descriptor:

NOAEL

Effect level:

< 33 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: neoplastic

Remarks on result:

not determinable

Remarks:

a NOAEL could not be determined

Critical effects observed:

yes

Lowest effective dose / conc.:

33 ppm

System:

central nervous system

Organ:

brain

Treatment related:

yes

Dose response relationship:

yes

Reference 3

Endpoint:

carcinogenicity: inhalation

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:

Evaluation of the chronic toxicity and carcinogenic potential using an exposure route relevant to workers.

GLP compliance:

not specified

Specific details on test material used for the study:

- Physical appearance: gas
- Purity: 99.7%

Species:

rat

Strain:

Fischer 344

Sex:

male

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

not specified

Duration of treatment / exposure:

2 years

Frequency of treatment:

7 h/d, 5 d/w

Post exposure period:

none

Dose / conc.:

50 ppm (nominal)

Remarks:

91.5 mg/m³

Dose / conc.:

100 ppm (nominal)

Remarks:

183.1 mg/m³

No. of animals per sex per dose:

80

Control animals:

yes, concurrent no treatment

Details on study design:

80 rats per group were exposed at 0 (shared control), 50, or 100 ppm EO for about 7 hr/day, 5 days/week for 2 years. Each chamber housed 80 rats during the 7-hr exposure period. Rats were housed in separate animal rooms during the periods of no oxide exposure, with control animals housed in separate rooms from the exposed animals to preclude any potential for oxide exposure due to offgasing. Forty rats from each shipment were assigned to a shared group of 80 control animals which were exposed in chambers to filtered air.
Animal exposures were conducted in 4.5-m3 stainless-steel and glass inhalation chambers. Chambers were operated under dynamic flow conditions at a pressure of -0.25 cm water with tangential airfeed manifolds maintained at 40 liters/min. Chamber airflows provided 12 to 15 air changes/hr. Temperature and humidity were maintained at 23 + 3°C and 50 + 10% respectively.
Chamber concentrations were monitored two to four times per hr.

Observations and examinations performed and frequency:

Individual animal body weights for test and control groups were recorded weekly for the first 10 weeks, biweekly for the next 20 weeks (Weeks 11-30), monthly for the next 64 weeks (Weeks 31-95), and weekly for the last 10 weeks (Weeks 96-105). Rats were individually observed twice a day, once in the morning and once in the afternoon, for clinical signs, morbidity, and mortality.
Hematology indices, including hematocrit, hemoglobin, RBC and WBC counts, clotting time, and differential counts, were evaluated at the 104 week termination.
The following parameters were also evaluated at termination: alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine phosphokinase (CPK), blood urea nitrogen (BUN), creatinine (CRE), sorbitol dehydrogenase (SDH), and albumin/globulin ratio (AG). Urinalyses conducted at termination included measurements of acetone, albumin, glucose, blood, casts, crystals, WBC, RBC, bacteria, and epithelial cells.

Sacrifice and pathology:

All rats which died or were killed underwent a complete necropsy. The following tissues were weighed: lung, liver, kidneys, adrenals, spleen, testes and brain. Complete gross examinations were performed on all animals, and a standard set of 34 tissues plus all gross lesions were fixed in 12% buffered formalin.

Statistics:

Group body weight comparisons were made using a multivanate analysis of covariance. The body weights from each animal (until the death of the first animal of the groups being compared) served as multiple dependent variables, initial weight as the covariance, and exposure groups as the independent variable. If statistically significant results (p < 0.05) were obtained with the MANCOVA, comparisons of exposure groups at various time periods were assessed by the Duncan multiple range test. Using the number of days survived on study, survival functions were computed and cumulative functions were plotted for each group. The actuarial method was used to compute the survival function. The Lee-Desu statistic was used for comparisons of survival functions. For absolute organ weights, relative organ weights, hematology, and clinical chemistry parameters, a comparison of distributions of values was made for each variable using the Kruskal-Wallis test, followed by multiple comparisons if the initial test were significant at p < 0.05. The incidences of mononuclear cell leukemia and peritoneal mesothelioma were analyzed for group differences by the Mantel-Haenszel Method. The method of
Peto et al. was used to compare glioma incidences. The incidences of other tumors or pathological lesions in the exposure groups were compared to the control groups by a x2 test. If a significant association was found between incidence and exposure, further analyses were completed to see if both exposure concentrations differed from the controls. In all cases, except for RBC and WBC counts where differences in absolute numbers necessitated comparisons of the E0 and PO groups to their individual uncombined control groups, each exposure group was compared to the combined rat control group.

Details on results:

Some significant differences in the percentage of neutrophils and lymphocytes were seen between control and exposed groups; however, these changes occurred in the absence of any statistically significant differences in total WBC counts, so these differences were not interpreted to be of toxicologic significance.
Rats exposed at 50 and 100 ppm had a higher incidence of inflammatory lesions of the lungs, nasal cavities, trachea, and internal ear. The lungs of these rats had an increased incidence of bronchiectasis, bronchial epithelial hyperplasia, and inflammatory changes consistent with the manifestations seen in chronic respiratory disease complex in rodents.
Rats exposed at 50 and 100 ppm had a high incidence of proliferative and degenerative lesions of the adrenal cortex, consisting of vacuolation and hyperplasia or hypertrophy of the cells of the zona fascicularis. In some of these rats the areas of hyperplasia and hypertrophy formed distinct nodules which compressed the adjacent cortical tissue. The skeletal muscle myopathy observed in a higher incidence in the 100 ppm rats consisted of multifocal areas of atrophy and degeneration of skeletal muscle fibers.
Other non-neoplastic alterations seen in exposed and control rats were typical of aged F344 rats.
A statisticaily significant association of EO exposure with increased occurrence of mononuclear cell leukemia was found (p = 0.03) between the control and 50 ppm rats. The mononuclear cell leukemia was typical of the leukemia reported to occur in the F344 rat. Hepatosplenomegaly was a common gross finding and focal to multifocal splenic fibrosis was usually observed.
When comparing the control group with the 100 ppm group, a non-significant association was found (p = 0.15).
An association of EO exposure and increased incidence of peritoneal mesotheliomas was also found. Generally, the tumors were present on the tunica vaginalis surrounding the testes and epididymis and, on occasion, spread to the pentoneal surfaces of the organ within the pentoneal cavity.
During histopathologic evaluation of brain tissue, several exposed rats were identified with mixed cell gliomas. None were found in the control group. The term glioma was used because the tumors contained mixed cell components with areas of both astrocyte and oligodendroglia cells within the same tumor. Two rats with gliomas were found in the 50 ppm EO group, one dying prior to termination. Five rats with gliomas were found in the 100 ppm EO group; four of these rats died prior to scheduled termination.
In addition to the gliomas observed, two additional rats exposed at 50 ppm EO and four additional rats exposed at 100 ppm EO had increased numbers of glial cells, termed gliosis. In these cases no evidence of inflammation nor neuronal degeneration or necrosis were found which would stimulate the glial response. Hence, these gliosis cases may represent incipient gliomas.

Dose descriptor:

NOAEC

Effect level:

< 50 ppm (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: neoplastic

histopathology: non-neoplastic

Remarks on result:

not determinable

Remarks:

no NOAEC identified

Critical effects observed:

yes

Lowest effective dose / conc.:

50 ppm

System:

male reproductive system

Organ:

testes

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

yes

Critical effects observed:

yes

Lowest effective dose / conc.:

50 ppm

System:

central nervous system

Organ:

brain

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Conclusions:

clear evidence of carcinogenicity

Reference 4

Endpoint:

carcinogenicity: inhalation

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 internal NTP standard

GLP compliance:

not specified

Specific details on test material used for the study:

- Physical appearance: liquid

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

50 mice of each sex were exposed to air containing ethylene oxide at target concentrations of 0 (chamber control), 50, or 100 ppm, 6 h/d, 5 d/w for 102 weeks. The mice were to the study laboratory at 5 weeks of age, quarantined for 21 days, and then placed on study at 8 weeks of age. Thereafter, a complete necropsy was performed on 5 animals per sex to assess their health status.
The animals were housed continuously in exposure chambers from approx. 1 wk before exposure started until they were killed at the end of the study. During non-exposure periods, the front doors of the exposure chambers were open while air continued to be exhausted through the chambers. All animals were housed individually. Feed and water were available ad libitum except during exposure periods, when only water was available.

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

not specified

Duration of treatment / exposure:

102 weeks

Frequency of treatment:

6 h/d, 5 d/w

Dose / conc.:

0.09 mg/L air (nominal)

Remarks:

50 ppm (nominal)

Dose / conc.:

0.18 mg/L air (nominal)

Remarks:

100 ppm (nominal)

No. of animals per sex per dose:

50

Control animals:

yes, concurrent no treatment

Observations and examinations performed and frequency:

All animals were observed two times per day, and clinical signs were recorded once per week. Individual body weights were recorded once per week for the first 13 weeks of the study and once per months thereafter. Mean body weights were calculated for each group. Animals found moribund and those surviving to the end of the studies were humanely killed.

Sacrifice and pathology:

Necropsy was performed on all animals, including those dead unless they were excessively autolyzed or cannibalized, missexed, or found missing. Thus, the number of animals from which particular organs or tissues were examined microscopically varies and is not necessarily equal to the number of animals that were placed on the study.
During necropsy, all organs and tissues were examined for grossly visible lesions.
Slides/tissues are generally not evaluated in a blind fashion (i.e., without knowledge of dose group) unless lesions in question are subtle or unless there is inconsistent diagnosis of lesions by the pathologist.

Statistics:

Analysis of tumor incidence: Three statistical methods are used to analyze tumor incidence data. Tests of significance included pairwise comparisons of high dose and low dose groups with control and tests for overall dose-response trends.

Details on results:

Body weights: Mean body weights of dosed mice were not adversely affected by exposure to EO. No compound-related clinical signs were observed. Survival: No significant differences in survival were observed between any groups of either sex.
Pathology and Statistical Analyses of Results: Lung: Alveolar/bronchiolar carcinomas in male and female mice and alveolar/bronchiolar adenomas in female mice occurred with significant positive trends. The combined incidences of the benign and malignant lung tumors occurred with positive trends in both male and female mice (male: control, 11/50; low dose, 19/50; high dose, 26/50; female: 2/49; 5/48; 22/49). The combined incidences in the 100-ppm exposure groups were significantly greater than those in the controls. The incidences of adenomas and carcinomas in the high dose groups, except for adenomas in male mice, were significantly greater than those in the controls. Harderian gland: Papillary cystadenomas in dosed male and female mice occurred with significant positive trends, and the incidences in dosed males and dosed females were significantly greater than those in the controls. In addition, one papillary cystadenocarcinoma was observed in a high dose male mouse and one in a low dose female mouse. Hematopoietic system: Malignant lymphomas occurred with a positive trend in female mice and the incidence in the high dose group was greater than that in the controls. The incidence in the low dose group was slightly lower than that in the controls. Uterus: Adenocarcinomas occurred in female mice with a positive trend and the incidence in high dose female mice was marginally increased compared with that in the controls. Mammary gland: The incidences of adenocarcinomas and adenocarcinomas or adenosquamous carcinomae (combined) in low dose female mice were greater than those in the controls. Liver: The incidence of hepatocellular adenomas in low dose female mice was greater than that in the controls (control, 1/49; low dose, 8/48; high dose, 3/49; P = 0.021). Hepatocellular carcinomas occurred with a negative trend (5/49; 1/48, 0/49; P < 0.01). The incidence of adenomas or carcinomas (combined) in dosed female mice was not significantly different from that in the controls (6/49; 9/48; 3/49).

Dose descriptor:

NOAEC

Effect level:

< 0.09 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable

Remarks:

no NOAEC identified

Critical effects observed:

yes

Lowest effective dose / conc.:

50 ppm

System:

hepatobiliary

Organ:

liver

lungs

mammary gland

uterus

other: harderian gland, hematopoietic system

Treatment related:

yes

Dose response relationship:

yes

Conclusions:

There was clear evidence of carcinogenic activity for mice as indicated by dose-related increased incidences of benign or malignant neoplasms of the lung and benign neoplasms of the harderian gland in both male and female mice following exposure to the test substance' vapors at 50 and 100 ppm. In female mice, the test substance caused additional malignant neoplasms of the uterus, mammary gland, and hematopoietic system (lymphoma).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEC

18 mg/m³

Study duration:

chronic

Species:

rat

Quality of whole database:

Several scientifically acceptable studies in rats and mice are available.

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

​The substance requires classification as a carcinogen of category 1B, H350 (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).