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Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)

Data source

Reference
Reference Type:
publication
Title:
Ethylene Oxide in Blood of Ethylene-Exposed B6C3F1 Mice, Fischer 344 Rats, and Humans
Author:
Filser, J.G., Kessler, W., Artati, A., Erbach, E., Faller, T., Kreuzer, P.E., Li, Q., Lichtmannegger, J., Numtip, W., Klein, D., Pütz, C., Semder, B., Csanády, G.A.
Year:
2013
Bibliographic source:
Toxicological Sciences 136(2), 344-358 2013

Materials and methods

Objective of study:
metabolism
toxicokinetics
Test guideline
Qualifier:
no guideline followed
GLP compliance:
not specified

Test material

Constituent 1
Chemical structure
Reference substance name:
Ethylene
EC Number:
200-815-3
EC Name:
Ethylene
Cas Number:
74-85-1
Molecular formula:
C2H4
IUPAC Name:
ethylene
Constituent 2
Chemical structure
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
Details on test material:
- Name of test material (as cited in study report): Ethylene (ET) 3.5 and Ethylene Oxide (EO) 3.0
- Physical state: gas
- Other: purchased from Linde, Unterschleißheim, Germany.
Radiolabelling:
no

Test animals

Species:
other: mouse and rat
Strain:
other: B6C3F1 (mice) and Fischer 344 (rats)
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Species: Fischer 344/N rats
- Source: Charles River Wiga Deutschland, Sulzfeld, Germany
- Weight at study initiation: 230–290g

TEST ANIMALS
- Species: B6C3F1 mice
- Source: Charles River Wiga Deutschland, Sulzfeld, Germany
- Weight at study initiation: 23–30g

CONDITIONS (Rodents)
- Housing: 2 rats in a Macrolon type III cage in an IVC top flow system (Tecniplast, Buguggiate, Italy) which provided clean air
- Diet: ad libitum (chow Nr. 1324, Altromin, Lage, Germany)
- Water: ad libitum (tap)
- Acclimation period: 5+ days
- Photoperiod (hrs dark / hrs light): 12-h light/dark cycle

Other details
The animal tests followed the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996).

Human Volunteers
- General Description: Good physical condition, nonsmoking
- Sex: males
- Group size: 4 individuals
- Profession: toxicologists

Other details
The study protocol was reviewed and approved by the Ethics Committee of the Medical Faculty of the Technische Universität München.

Administration / exposure

Route of administration:
inhalation: gas
Vehicle:
other: Synthetic air 5.5
Details on exposure:
GENERATION OF TEST ATMOSPHERE
Animals were exposed in closed all-glass chambers (see Filser (1992) Arch. Toxicol 66, 1-10 for details). Exhaled carbon dioxide was trapped using soda lime, and replaced using pure oxygen.

Studies in rodents – EO measurements in exhaled air:
Animals exposed whole body for 6 hr
- mice: n = 5, chamber volume = 2.8 l
- rats: n = 2, chamber volume = 6.6 l.

Target concentrations of ET in air were:
- mice: 0, 1, 3, 10 or 30 ppm
- rats: 0, 1 or 3 ppm

Studies in rodents – EO measurements in blood:
Mice and rats were exposed either whole body (63 l glass sphere) or nose only (flow through chamber, mouse = 290 ml volume, rat = 750 ml volume)
Target concentrations of ET in air were:
- whole body = 0, 30, 100 (mice only), 130 (rats only), 300, 1000, 10000 ppm (6 hr exposure period)
- nose only = 0, 100 (mice only), 300, 1000 or 10000 ppm (45 min exposure period)

Studies in volunteers:

Three volunteers (A, B, C) were exposed to target concentrations of 5, 20 or 50 ppm ET for 4 hr; a fourth volunteer (D) was exposed to 5 or 20 ppm ET for the same period.
Volunteers were exposed to increasing concentrations of ET with a 2-d gap between exposures.
Methods described elsewhere (Filser et al. (2000) Toxicol. Appl. Pharmacol. 169, 40-51; Filser et al. (2008) Toxicol. Sci. 102, 219-231)
Duration and frequency of treatment / exposure:
Animals - 45 min or 6 h
Humans volunteers - 4h
Doses / concentrations
Remarks:
Doses / Concentrations:
Whole body : 30, 100 (mice only), 130 (rats only), 300, 1000, and 10 000 ppm.
Nose only : 100 (mice only), 300, 1000, or 10 000 ppm.
Volunteers A, B, and C to 3 ET target concentrations: 5, 20, and 50 ppm. Volunteer D to 2 ET target concentrations: 5 and 20 ppm.
No. of animals per sex per dose / concentration:
2 mice
5 rats
Control animals:
yes, concurrent no treatment
Details on dosing and sampling:
ET monitoring
- Sample taken from: Air in chamber and ambient air
- Frequency of measurement: 10-20 minutes
- Method type(s) for identification: Gas chromatograph (GC) with flame ionisation detector (FID)
- Limit of Detection: 0.07 ppm

EO monitoring
- Sample taken from: Air in chamber and ambient air
- Frequency of measurement: Graphical information indicates that levels of EO in exhaled air were monitored every 30-60 minutes
- Method type(s) for identification: GC/FID and GC coupled with mass-selective detector (MSD)
- Limit of Detection: 0.2 ppm (GC/FID); 0.45 ppb (mice), 4 ppb (rats) (GC/MDS)

Humans
ET monitoring
- Samples taken from: Air at the point of flow into the mask and exhaled air
- Method for identification: GC/FID
- Measurement time points: 1 minute after exposure initiation, then every 10 to 15 minutes (inflowing air); every 0.5h (gas bag air)
- Limit of Detection: 0.07 ppm

EO monitoring
- Samples taken from: Exhaled air
- Method for identification: GC/MSD
- Measurement time points: before initiation of exposure to ET and every hour until exposure termination
- Limit of Detection: 0.8 ppb

METABOLITE CHARACTERISATION STUDIES
EO detection
Rodents
- Tissues and body fluids sampled: blood
- Time and frequency of sampling: Graphical information indicates that levels of EO in blood were monitored 15, 30, 45 and 60 minutes after the start of exposure, and also at 2 and 6 hours
- Method type(s) for identification: GC/MSD
- Limits of detection and quantification: EO = 80 nmol/l
- Other: Predicted levels of EO in blood following ET exposure were calculated using the function: EO concentrations at plateau (ppm) x blood-to-air partition coefficient of EO/ 25.13 (the molar volume [l] of an ideal gas at 25°C and 740 torr)

Humans
- Tissues and body fluids sampled: blood
- Time and frequency of sampling: before initiation of exposure and every hour until exposure termination
- Method type(s) for identification: GC/MSD
- Limits of detection and quantification: 3-5 nmol/l
Statistics:
Linear regression analysis and standard errors of the means of slopes (SEMs), arithmetic means and SDs, as well as one-way ANOVA followed by Bonferroni’s post test for multiple comparisons were calculated using Prism 5 for Mac OSX (GraphPad Software, San Diego, California). A statistically significant difference was defined as p ≤ .05.

Results and discussion

Main ADME results
Type:
metabolism
Results:
Formation of ethylene oxide from ethylene followed the relationship rat>mouse>human

Any other information on results incl. tables

 Conversion of ethylene to ethylene oxide in vivo

 

Studies in rodents

No background EO was detected in blood from unexposed rats and mice (limit of detection = 80 nmol/l).

 

Graphical data indicated that at lower ET exposures (<30 ppm), levels of EO in blood and exhaled air increased over time and reached a plateau after about 2 hr exposure; half-lives of approx. 8.9 min and 15 min were calculated for mice and rats, respectively, exposed to 30 ppm ET.

 

At higher exposures (>30 ppm ET), levels of EO in blood peaked following approx. 30 min exposure and then decreased to a plateau after around 2 hr. (No information collected on levels of EO in exhaled air at these higher ET exposure.)

 

Steady state concentrations of EO in blood (calculated from EO exhalation data) were approx. 2-fold higher in rats compared to mice:

 

ET in air

ppm

EO in blood (umol/l)

Mean ± SD

Mouse

Rat

1

0.007 ± 0.002

0.016 ± 0.001

3

0.018 ± 0.003

0.044 ± 0.004

10

0.077 ± 0.004

not done

30

0.198 ± 0.024

not done

 

A similar relationship was apparent for the direct estimation of levels of EO in blood for mice (0.203 ± 0.026 umol/l) and rats (0.469 ± 0.074 umol/l) exposed to 30 ppm ET.

 

The extent of conversion of ET to EO was calculated as:

Mouse = 0.041 ± 0.001 umol EO/l blood per ppm ET in air

Rat = 0.092 ± 0.001 umol EO/l blood per ppm ET in air

 

 

Studies in humans

There was no statistically significant difference between the concentration of ET in inhaled and exhaled air for 4 volunteers exposed to 5, 20 or 50 ppm ET, however, the level of ET in exhaled air was generally around 6% lower than that of inhaled air.

 

Levels of EO in human venous blood were as follows:

 

Volunteer

ET exposure (ppm)

EO in blood (nmol/l)

Mean ± SEM

Calculated

Measured

A

5

10 ± 1.5

7 ± 0.8

20

25 ± 5.7

24 ± 0.3

50

59 ± 10.1

58 ± 3.4

B

5

8 ± 1.1

7 ± 0.5

20

28 ± 4.0

36 ± 7.3

50

97 ± 8.2

81 ± 2.9

C

5

5 ± 0.6

6.5 ± 0.6

20

27 ± 1.7

33 ± 0.9

50

58 ± 9.4

71 ± 12.2

D

5

6 ± 0.1

7 ± 0.7

20

33 ± 4.3

42 ± 9.6

 

Concentrations of metabolically formed EO after approx. 2 hr (pseudo-steady state) were used to calculate the extent of conversion of ET to EO. Results were as follows:

 

Volunteer

nmol EO/l blood

per ppm ET in air

A

1.16 a

B

1.74 b

C

1.34 a

D

1.75 b

Mean

1.431 ± 0.002

 

a = results not statistically significantly different

b = results not statistically significantly different

Values for volunteers A and C differed significantly from those of volunteers B and D.

Haemoglobin and DNA adduct comparison:

In mice, the model underestimated the HB adducts by approximately 2-4 fold, and 2 out of 4 DNA adduct levels were underestimated by up to 1.3 fold.

In rats, all of the HB adduct levels were underestimated by approximately 1-6 fold. The DNA adduct levels were all overestimated by approximately 1.6 fold.

In humans, the calculated value was approximately 1.2 times higher than the single reported measured value. DNA adduct levels could not be compared due to lack of reported data.

Summary

At ET concentrations up to 30 ppm, EO concentrations in blood from rodents (quantified in the umol/l range) were approx. 9-fold higher in rats, and approx. 4-fold higher in mice, than in the volunteer with the greatest EO burden (quantified in the nmol/l range).

At higher exposures of 50 ppm ET, EO formation in blood was approx. 6-fold higher in rats, and approx. 3-fold greater in mice, than in humans.

Measured and calculated adduct levels were higher in rats than in mice. The difference was more apparent in the calculated values, which varied by 2-5 orders of magnitude. The measured values were 1-2 times higher in rats for HB adducts and 1-3 times for DNA adducts. Comparison of rodent and human adducts is not possible due to the significant differences in ET exposure levels.

Applicant's summary and conclusion

Conclusions:
At ET concentrations up to 30 ppm, formation of EO was approx. 9-fold higher in rats, and approx. 4-fold higher in mice, than in human volunteers. At exposures of 50 ppm ET and above, EO formation was approx. 6-fold higher in rats, and approx. 3-fold greater in mice, compared to humans. Overall, formation of EO from ET followed the relationship rat>mouse>human.
Executive summary:

Formation of ethylene oxide (EO) from ethylene (ET) was quantified in blood and exhaled air from groups of mice, rats and human volunteers exposed to defined concentrations of ET (1 – 10000 ppm). EO formation was measured using GC-FID and/or GC-MSD. At ET concentrations up to 30 ppm, formation of EO was approx. 9-fold higher in rats, and approx. 4-fold higher in mice, than in human volunteers. At exposures of 50 ppm ET, EO formation was approx. 6-fold higher in rats, and approx. 3-fold greater in mice, compared to humans. Measured levels of EO in blood were also used to calculate formation of the adducts N-(2-hydroxyethyl)valine in haemoglobin and N7-(2-hydroxyethyl)guanine in DNA; this predicted higher levels of adduct formation in rats compared to mice, but there were inconsistencies between the predictions and measured values obtained by others. Overall the results demonstrate clear species differences, with humans being less efficient than rodents in converting ET to EO in vivo. They also suggest that ethylene oxide formed in vivo during the metabolism of ethylene has some capacity to bind to cellular macromolecules such as haemoglobin and DNA.