Ethylene oxide

Specific investigations: other studies

Administrative data

Endpoint:

biochemical or cellular interactions

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Unnamed

Year:

2017

Report date:

2017

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

The purpose of this study was to investigate the mode of action for ethylene oxide-induced lung mutations in male B6C3F1 mice. Inhalation is a probable route of human exposure during the use or manufacture of EO.

GLP compliance:

yes

Type of method:

in vivo

Endpoint addressed:

other: Mode of action

Test material

Test material information

Constituent 1

Reference substance name:

Ethylene oxide

EC Number:

200-849-9

EC Name:

Ethylene oxide

Cas Number:

75-21-8

Molecular formula:

C2H4O

IUPAC Name:

oxirane

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: ARC Specialty Products, New Hampton, New York, USA (Satellite Group), ARC Specialty Products, Belchem Corporation, Green Pond, South Carolina, USA (Experimental groups)
- Lot No.of test material: UTLX902827F10 (Satellite group); UTLX902111C11 (Experimental groups)
- Molecular weight: 44.1 g/mol
- Purity: 99.9%

Test animals

Species:

mouse

Strain:

B6C3F1

Remarks:

Tac and Big Blue hemizygous

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: Approximately 8 weeks
- Housing: individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 1 (maximum permissible excursion 3°C)
- Humidity (%): 40-70%
- Air changes (per hr): 12-15
- Photoperiod (hrs dark / hrs light): 12 h (5 am to 5 pm illumination)

OTHER SPECIFICS:
JUSTIFICATION FOR STRAIN SELECTION:
B6C3F1 mice were selected because of their general acceptance and suitability for toxicity testing, availability of hisorical gackground data, the reliability of the commercial supplier, and their use in previous studies on EO. Big Blue B6C3F1 mice were used for mutational analysis.

Administration / exposure

Route of administration:

inhalation: gas

Vehicle:

unchanged (no vehicle)

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 4 m³ stainless steel and glass exposure chambers. Whole body exposure
- Method of holding animals in test chamber: mice were housed singly
- Source and rate of air: Exposure atmospheres were generated by mixing Eo in nitrogen with HEPA-filtered breathing air
- Method of conditioning air: not specified
- Temperature, humidity, pressure in air chamber: 22 +/- 2°C, 40-60%, with the exception of the control chamber (slightly positive pressure), the exposure chambers were operated at a slightly negative pressure relative to the surrounding area.
- Air flow rate: 869.9 - 898.1 lpm (liters per minute)
- Air change rate: 12-15 per hour

TEST ATMOSPHERE
- Brief description of analytical method used: Test atmosphere concentration was determined approximately twice per hour with a Miran 1A infrared spectrometer
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Exposure chamber concentrations were assessed daily and the mean, standard deviation, a minimum and a maximum concentration were established.

Duration of treatment / exposure:

4, 8, 12 weeks

Frequency of treatment:

6 h/d, 5 d/w

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

10 ppm

Remarks:

4 weeks exposure

Doses / concentrations 2

Dose / conc.:

50 ppm

Remarks:

4 weeks exposure

Doses / concentrations 3

Dose / conc.:

100 ppm

Remarks:

4, 8, and 12 weeks exposure

Doses / concentrations 4

Dose / conc.:

200 ppm

Remarks:

4, 8, and 12 weeks exposure

No. of animals per sex per dose:

356 total animals

Control animals:

yes

Details on study design:

PROBE STUDY:
A satellite group of 10 male B6C3F1/Tac mice were exposed 6 h/d for 3 consecutive days to 0 and 200 ppm EO (n = 5/concentration). Spleen, Liver, and lung from these animals were isolated, processed, and analyzed for biomolecular analyses (DNA adducts and isoprostates, in all three tissues and 8-OHdG and GSH/GSSG only in the lung).

MAIN STUDY:
Animales were assigned to one of the following test groups:
- Satellite group: Probe study
- Group 1: Histopathoogy, cell proliferation, micronucleus, and comet:
A total of 91 mice were used of which 86 were exposed resulting in 10 mice per exposure level (4-week exposure). Six mice per exposure level were used for weeks 8 and 12.
- Group 2: Bioanalytical assays
A total of 160 mice were used. 20 mice per exposure level were used (4-week exposure). 10 mice were used for each exposure level (8-and 12-week exposure).
- Group 3: cII and K-ras mutations
A total of 110 mice were used of which 10 mice per exposure level were utilized (4-, 8-, 12-week exposure).

Examinations

Examinations:

Cage-side examination was conducted at least once a day. Animals were observed for morbifity, mortality, and the availability of feed and water at least twice daily. A hands-on evaluation of skin, fur, mucous membranes, respiration, and nervous system
function (including tremors, convulsions, diarrhea, and animal behavior) were conducted twice during the first week of exposure and weekly thereafter. Animals were weighed and visually examined prior to exposure to EO. All mice were weighed during the pre-exposure period and at least weekly during the exposure period. Feed consumed was determined at least weekly for all animals by weighing feed crocks
at the start and end of a measurement cycle.

Positive control:

Additional 5 mice were exposed to 300 mg/kg ethyl methanesulfonate in water by oral gavage and were used as a positive control.

Results and discussion

Details on results:

CHAMBER CONCENTRATIONS:
Mean analytically determined concentrations values were 0, 10.1 ± 0.4, 50.0 ± 1.6, 100.5 ± 4.6, and 199.2 ± 2.7 ppm,. The nominal concentrations were determined by the mass of EO divided by volume of gas delivered to each exposure chamber. Nominal concentrations for this study were 10.0 ± 0.2, 51.5 ± 0.4, 104.0 ± 7.7, and 206.5 ± 0.9 ppm,

MORTALITY:
No mortality was observed except for one mouse in the 50 ppm group which spontaneously died on the second day of exposure.

CAGE-SIDE OBSERVATIONS:
No effects observed

CLINICAL OBSERVATIONS:
No effect observed

BODY WEIGHTS7/BODY WEIGHT GAINS:
No effects observed except in one B6C3F1/Tac mice exposed to EO for 8 weeks at 200 ppm a treatment-related statistically-decreased mean body weight was observed when compared to the respective controls. In Big Blue B6C3F1 hemizygous mice, there were no statistically significant differences except in mice exposed to 100 ppm of EO for 12 weeks where a treatment-related statistically-decreased mean body weight when compared to the respective controls was observed.

FEED CONSUMPTION:
There was a statistically identified difference in feed consumption for the 200 ppm group of Big BLue B6C3F1 mice although there was an inconsistent temporal responsiveness to the alterations in feed consumption, i.e. not the same week in the three exposure periods was affected. A similar subtle and inconsistent increase in feed consumpion was noted in the B6C3F1/Tac mice.

ORGAN WEIGHTS:
In group 1, no consistent or remarkable changes were observed. The lung weight of the 200 ppm treated B6C3F1/Tac mice was statistically increased (after 8 weeks of exposure), this finding was not observed for the other two time periods.
In group 2, there was a consistent, statistically significant increase in the lung weights of the animals in the 8 and 12-week treatment group at an exposure concentration of 200 ppm. In addition, the liver weights were decreased at the 4-week treatment group at 200 ppm.
In Big Blue B6C3F1 mice, the only statistically increased change in organ weight was in the lungs at the 200 ppm exposure concentration treated for 12 weeks.

HISTOPATHOLOGY:
no effects observed.

CELL PROLIFERATION (KI-67) IN LUNG TISSUE:
Effects observed, non treatment-related.
Statistically identified decreases in labeling index and unit length labeling index were noted at 4 and 8 weeks at 200 ppm. A slight decrease was also observed in numeric density at 200 ppm at the 4 week exposure time only (B6C3F1/Tac mice), see table 1 in "any other information on results". Lung tissue from one control animal from the 4-week exposure group (animal #1877) was inadvertently not prepared for evaluation of proliferation in the terminal bronchioles. Another control animal in the 4-week exposure group (animal # 1876) as well as 200 ppm animal from the 4-week exposure group (animal # 2143) did not have any terminal bronchioles present in the prepared slide and hence were not available for evaluation. Two control animals (animal # 1876 and 1878), one 10 ppm animal (animal # 1921), two 50 ppm animals (animal # 1959 and 1960), three 100 ppm animals (animal # 2047, 2054, and 2055), and three 200 ppm animals (animal #2139, 2141, and 2143) from the 4-week exposure group did not have any terminal bronchioles present in the prepared slide and hence were not available for evaluation.

MICRONUCLEATED PERIPHERAL BLOOD RETICULOCYTES:
no effects observed

IN VIVO COMET ASSAY IN LUNG TISSUE:
effects observed, treatment-related.
A statistically significant increase in the percent tail DNA intensity in lung tissue of animals treated for 4 weeks with EO at concentrations of 50, 100, or 200 ppm was observed when compared to control values (B6C3F1/Tac mice). Sections of the lung tissues were evaluated in order to determine whether the response was due to cytotoxicity or genotoxicity revealing no evidence of necrosis or apoptosis.

BIOANALYTICAL ASSAYS:
EO did not cause detectable ROS induction. Alkylated DNA adducts were substantially increased and were clear biomarkers of exposure. Specifically, in the lung N7-HEG was increased 499-, 1553-, 2296-, and 8692-fold at 10, 50, 100, and 200 ppm, respectively. Similarly, this biomarker of exposure was increased at least 10,800-fold in the spleen and at least 12,700-fold in the liver at 200 ppm. N1- and N6-HEdA were both qualitatively increased in a concentration-dependent manner. Analysis of the GSH-related biomarkers also indicated a concentration-related increase in biomarkers of exposure, i.e., HESG.

Any other information on results incl. tables

TABLE 1. Summary of Proliferation Observations (Ki-67) in Lung Tissue (B6C3F1/Tac Male Mice), Control and High Dose (4-,8-, and 12-week)

	4 Weeks		8 Weeks		12 Weeks
	0 ppm	200 ppm	0 ppm	200 ppm	0 ppm	200 ppm
Labeling Index	0.50 ± 0.31	0.21 ± 0.21	0.14 ± 0.06	0.03 ± 0.03	0.28 ± 0.12	0.35 ± 0.12
Unit Length Labeling Index	0.84 ± 0.53	0.32 ± 0.31	0.25 ± 0.11	0.05 ± 0.06	0.45 ± 0.19	0.58 ± 0.22
Numeric Density	166.7 ± 17.3	149.7 ± 9.5	172.4 ± 14.8	171.2 ± 8.1	164.7 ± 7.8	164.1 ± 9.8

Values are mean +/- standard deviation; bold values = significantly different than similarly exposed 0 ppm mice (p ≤ 0.05)

Table 2: Summary in vivo comet assay in lung tissue (B6C3F1/tac mice)

		Lung
Exposure (ppm)	Na	Mean % Tail Intensity
0	10	4.066
10	10	4.298
50	10	7.971^b
100	10	14.734^b
200	10	39.594^b
Positive Control (300 mg/kg EMS^c)	5	53.802^b

a N is the number of animals per dose group at the time of scheduled sacrifice. 100 cells were examined/animal for Comet analysis, and expressed as mean % tail intensity.

b  The values are significantly different from the negative control (alpha=0.05).

c  EMS = Ethyl Methanesulfonate (positivecontrol).

Table 3: Summary of alkylated DNA adduct biomarkers

Biomarke rs	Alkylate d DNA Adduct Biomarke rs
LC/MS-MS assay	Assay 1						Assay 2
Biomarke r type s	O6-HEdG		N1-HEdA		N6-HEdA		N7-HEG
Biomarke r le vel units	Adducts/dG [10 (e 6)]		Adducts/dA[10 (e 6)]				Adducts/NT [10 (e9)]
Sample Name	Ave rage	SD	Ave rage	SD	Ave rage	SD	Ave rage	SD
4W spleen (0 ppm)	0.130	0.0690	<LLQ (0.431)	NA	<LLQ (0.431)	NA	<LLQ (4.83)	NA
4W spleen (10 ppm)	0.129	0.0666	<LLQ (0.359)	NA	<LLQ (0.359)	NA	872	85.6
4W spleen (50 ppm)	0.152	0.0918	<LLQ (0.335)	NA	<LLQ (0.335)	NA	4024	926
4W spleen (100 ppm)	0.147	0.0329	0.412	0.103	<LLQ (0.360)	NA	8419	571
4W spleen (200 ppm)	0.410	0.0635	1.47	0.149	0.596	0.0433	27112	5936
4W liver (0 ppm)	0.518	0.797	<LLQ (0.364)	NA	<LLQ (0.349)	NA	<LLQ (6.84)	NA
4W liver (10 ppm)	0.611	0.761	<LLQ (0.340)	NA	<LLQ (0.340)	NA	606	49
4W liver (50 ppm)	0.214	0.193	<LLQ (0.307)	NA	<LLQ (0.307)	NA	2547	553
4W liver (100 ppm)	0.0936	0.0224	0.351	0.0384	<LLQ (0.306)	NA	9255	2825
4W liver (200 ppm)	0.193	0.0696	1.70	0.119	0.680	0.0380	31807	7392
4W lung (0 ppm)	0.0977	0.0160	<LLQ (0.448)	NA	<LLQ (0.448)	NA	4.74	1.61
4W lung (10 ppm)	0.183	0.125	<LLQ (0.410)	NA	<LLQ (0.410)	NA	2342	1651
4W lung (50 ppm)	0.140	0.0339	<LLQ (0.383)	NA	<LLQ (0.367)	NA	7285	3072
4W lung (100 ppm)	0.234	0.0599	0.765	0.131	<LLQ (0.392)	NA	10769	1178
4W lung (200 ppm)	0.848	0.540	2.84	0.330	1.19	0.0442	40766	1950
8W lung (0 ppm)	0.111	0.0251	<LLQ (0.585)	NA	<LLQ (0.585)	NA
8W lung (100 ppm)	0.279	0.0207	2.03	0.163	1.02	0.132	Not Analyzed
8W lung (200 ppm)	1.02	0.0989	5.64	0.502	2.90	0.254
12W lung (0 ppm)	0.179	0.164	<LLQ (0.403)	NA	<LLQ (0.403)	NA
12W lung (100 ppm)	0.467	0.365	1.96	0.152	1.39	0.0897	Not Analyzed
12W lung (200 ppm)	0.743	0.118	6.97	1.79	4.27	0.540

LLQ = Lower limit of quantitation

Table 4: Summary of GSH-related biomarkers

Biomarke rs	GSH Relate d Biomarke rs
LC/MS-MS assay	Assay 3
Biomarke r types	GSH		GSSG		HESG		Ratio
Biomarke r level units	µg/g tissue
Sample Name	Average	SD	Ave rage	SD	Average	SD	GSH/GSSG	HESG/GSH
4W spleen (0 ppm)	1001	84.2	35.8	7.75	LLQ< (8.37)	NA	28.0	NA
4W spleen (10 ppm)	958	100	27.8	4.47	5.24	0.975	34.5	0.0055
4W spleen (50 ppm)	882	67.0	28.1	4.24	14.8	3.65	31.4	0.0168
4W spleen (100 ppm)	894	37.3	26.4	2.33	39.7	16.6	33.9	0.0444
4W spleen (200 ppm)	824	74.7	23.8	6.40	67.3	15.6	34.6	0.0817
4W liver (0 ppm)	2006	352	57.3	8.03	LLQ< (4.87)	NA	35.0	NA
4W liver (10 ppm)	1776	88.1	46.0	3.35	12.1	4.08	38.6	0.00683
4W liver (50 ppm)	1879	219	52.7	11.4	57.7	13.1	35.7	0.0307
4W liver (100 ppm)	1438	146	33.9	8.01	102	56.7	42.4	0.0710
4W liver (200 ppm)	935	435	25.8	11.7	351	178	36.2	0.376
4W lung (0 ppm)	702	59.0	53.4	4.78	LLQ< (5.53)	NA	13.2	NA
4W lung (10 ppm)	717	29.5	41.8	3.16	14.3	3.75	17.1	0.020
4W lung (50 ppm)	687	38.1	49.0	6.23	84.6	23.8	14.0	0.123
4W lung (100 ppm)	590	40.6	38.8	6.15	206	69.8	15.2	0.349
4W lung (200 ppm)	432	222	27.3	4.37	440	75.5	15.9	1.02
8W lung (0 ppm)	850	115	50.5	16.6	LLQ< (5.39)	NA	16.8	NA
8W lung (100 ppm)	661	114	51.3	19.4	131	27.6	12.9	0.198
8W lung (200 ppm)	489	83.8	31.0	9.4	338	95.5	15.8	0.691
12W lung (0 ppm)	758	35.8	65.5	9.01	LLQ< (5.35)	NA	11.6	NA
12W lung (100 ppm)	603	54.9	49.6	6.67	107	20.1	12.2	0.177
12W lung (200 ppm)	368	51.0	33.0	6.10	263	100	11.1	0.715

LLQ = Lower limit of quantitation

Applicant's summary and conclusion

Conclusions:

The test substance, when administered by inhalation (6 hours/day, 5 days/week) at exposure concentrations of 0, 10, 50, 100, or 200 ppm (4 weeks) or 0, 100, or 200 ppm (8 or 12 weeks), did not result in significant phenotypic apical alterations of the lung. This was demonstrated by a lack of both histopathological alterations and altered cell proliferation in terminal bronchioles as measured by Ki-67 nuclear antigen. Furthermore, reactive oxygen species-related adducts (i.e., biomarkers of effect: 8-OHdG, Croton dG, M1dG) were only minimally affected, whereas alkylated DNA adducts (i.e., biomarkers of exposure: O6-HEdG, N1-HEdA, N6-HEdA, and N7-HEG) were increased more robustly. There were no treatment-related changes in the MN-RET frequency or % RET. Comet analysis revealed a dose-dependent, statistically significant increase in %DNA in tail at 50 ppm and above. While a clear NOEL was identified for all apical endpoints examined, the molecular analyses reveal a complex biological outcome of inhalation
exposure to EO.

Executive summary:

The test substance, a reactive industrial chemical, is known to induce alveolar/bronchiolar adenomas and carcinomas in the lungs of B6C3F1 male mice at atmospheric concentrations of 50 and 100 ppm. Previous work has identified a specific mutation in the K-ras oncogene that appears to be associated with the tumors. The purpose of the current study was to investigate the mode of action (MoA) of these test substance-induced lung mutations in male B6C3F1 mice. Mice were exposed via whole body inhalation for 6 hours/day, five consecutive days/week, for 4, 8, or 12 weeks (a total of 20, 40, or 60 days of exposure, respectively) to target concentrations of 0, 10, 50, 100, or 200 ppm (4 weeks) or 0, 100, or 200 ppm (8 or 12 weeks). Tissues from these animals were examined for incidence of micronuclei in the

peripheral blood (MNT), DNA damage (Comet assay), histopathology of the lung, and characterization of DNA- and glutathione-adducts and lipid peroxidation in the tumor target and non-target tissues. In general, reactive oxygen species-related adducts (8-OHdG, CrotondG, M1dG) were only minimally affected, whereas alkylated DNA adducts (O6-HEdG, N1-HEdA, N6-HEdA, and N7-HEG) were increased more robustly. There was a dose-dependent increase in glutathione adducts (HESG) in all tissues, although severe GSH depletion was not noted. There were no treatment-related changes in the MN-RET

(reticulocyte) frequency or % RET. Comet analysis of the lung revealed a dose-dependent, statistically significant increase in DNA damage at 50 ppm and above. There were no treatment-related alterations in the lung at any concentration or exposure duration, and there was no consistent change in the proportion of Ki-67 positive cells in the terminal bronchioles of the animals examined. In summary, these observations revealed a complex sequela of biomarkers of exposure and effect following inhalation exposure to the test substance with a clear no observable effect level (NOEL) for all apical endpoints studied.