Registration Dossier

Administrative data

Endpoint:
basic toxicokinetics
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Objective of study:
toxicokinetics
Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 417 (Toxicokinetics)
Deviations:
no
Remarks:
The study was conducted according to the guideline in effect at the time of study conduct.
Qualifier:
according to
Guideline:
EPA OPPTS 870.7485 (Metabolism and Pharmacokinetics)
Deviations:
no
Remarks:
The study was conducted according to the guideline in effect at the time of study conduct.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Purity: 99.97%
Radiolabelling:
no

Test animals

Species:
rat
Strain:
other: Crl:CD®(SD)IGS BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories International, Inc., Raleigh, North Carolina, U.S.A.
- Age at study initiation: 9 to 11 weeks
- Housing: group housed (non-cannulated) or housed individually (cannulated) in solid bottom caging
- Individual metabolism cages: yes
- Diet: PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002 ad libitum
- Water: tap water ad libitum
- Acclimation period: quarantined for at least 3 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26ºC
- Humidity (%): 30-70%
- Photoperiod: 12-hour light/dark cycle.

Administration / exposure

Route of administration:
inhalation: vapour
Vehicle:
other: air
Details on exposure:
TYPE OF INHALATION EXPOSURE: nose only

GENERATION OF TEST ATMOSPHERE / CHAMPER DESCRIPTION
- 150-liter Chamber:
The chamber atmospheres were generated by flash evaporation of the liquid test substance in air. The test substance liquid was metered to a heated 1000-mL 3-neck generation flasks using a Cole-Parmer Masterflex pump equipped with a Fluid Metering Inc. (FMI) piston. The flask was heated to 175ºC using Electrothermal Unimantle which was controlled by the Camile Inhalation Toxicology Automated Data System (CITADS). The generation air supply was metered to the generation flask by a Brooks model 5850E mass flow controller. The chamber dilution air was metered to the glass transfer tube leading to the exposure chamber by a Brooks model 5851E mass flow controller. The mass flow controllers were also monitored and controlled by CITADS. The vapour and air mixture entered the 150-liter chamber through a tangential turret at the top of the chamber. The test atmosphere was exhausted from the bottom of the chamber and discharged into the fume hood via a vacuum pump.

- 19-liter Chamber:
The chamber atmospheres were generated by flash evaporation of the liquid test substance in air. The test substance liquid was metered to a heated 1000-mL 3-neck generation flasks using a Harvard Apparatus model 22 infusion pump. The flask was heated to 175ºC using Electrothermal Unimantle which was controlled by the Camile Inhalation Toxicology Automated Data System (CITADS). The generation/chamber air supply was metered to the generation flask by a Brooks model 5850E mass flow controller. The mass flow controller was also monitored and controlled by CITADS. The vapour and air mixture was distributed throughout the chamber via a circular glass baffle. The test atmosphere was exhausted from the bottom of the chamber and discharged into the fume hood via a vacuum pump.
Duration and frequency of treatment / exposure:
Group 1: 6 hours, 1 exposure
Group 2: 6 hours, 1 exposure
Group 3: 6 hours, 1 exposure
Sub-Group 3: 1 or 4 hours, 1 exposure
Group 4: 6 hours, 14 days exposure
Doses / concentrations
Remarks:
Doses / Concentrations:
2500 ppm (Groups 2 and 4)
10000 ppm(Groups 1 and 3)
No. of animals per sex per dose:
Group 1: 8
Group 2: 4
Group 3: 24
Sub-Group 3: 4
Group 4: 4
Control animals:
no
Details on dosing and sampling:
PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, blood, heart, brain, liver, kidney, lung, spleen, muscle, fat, reproductive organs and bile
- Time and frequency of sampling:
Blood samples from 4 male and 4 female rats were collected at 0.25, 0.5, 0.75, 1, 1.5, 2, 4 and 18 hours (Group 2, 3, and 4).
Blood samples were collected during the exposure phase of the pilot experiment (Group 1) at 2, 4, and 5.75 hours.

Tissues were collected from 4 male and 4 female rats during exposure phase at two time points, after 1 hour and after 4 hours of exposure and tissues were collected 30 minutes after the animals were removed from the chamber. Tissues were also collected from 4 male and 4 female rats at 30 minutes, 3 hours, 8 hours and 18 hours post 6 hour exposure.

Urine and faeces were collected from Group 3 animals only (4 male and 4 female rats, single exposure, 10000 ppm). Bile was collected from Group 3 animals only (3 male and 3 female rats, single exposure, 10000 ppm). Collection time points for bile, urine and feces were 0-4 hours, 4-8 hours and 8-18 hours.

- Method type(s) for identification: gas chromatography (GC) coupled with a mass selective detector (MSD)
- Limits of detection and quantification: The lower limit of quantitation (LLOQ) for the test item in blood and tissues was calculated based on the initial sample weight, thus, the LLOQ was different for each sample.
Statistics:
A Two Sample t-Test was used to generate the statistics used for the pharmacokinetic parameters. Group data were represented as a mean ± SD as appropriate. The concentration time course data for blood from each animal was analysed to determine the terminal half-life (T1/2, hr), area-under-the-curve (AUC, hr x ng/mL), peak concentration (Cmax, ng/mL), and time to peak concentration (Tmax, hr). These pharmacokinetic parameters were calculated by non-compartmental analysis using a commercially available software program.

Results and discussion

Preliminary studies:
Systemic uptake was rapid with blood reaching steady-state within 2 hours of the 6-hour exposure period during a single exposure at 10000 ppm (Group 1, pilot study).

Toxicokinetic / pharmacokinetic studies

Details on absorption:
Blood samples from 4 male and 4 female rats were collected at 0.25, 0.5, 0.75, 1, 1.5, 2, 4 and 18 hours in each of the definitive studies (Group 2, 3, and 4). The test substance was not detected in blood samples beyond the 0.75 hour time point in males nor in blood samples beyond 1.5 hours in females post 6 hour exposure at the 2500 ppm concentration (Group 2). The test substance was detected in 3 of 4 blood samples at the 4 hour time point in males but not in blood samples beyond the 2 hour time point in females post 6 hour exposure at the 10000 ppm concentration (Group 3). The test substance was not detected in blood samples beyond the 2-hour time point in males nor in blood samples beyond the 1-hour time point in females post 6-hour exposure at the 2500 ppm concentration (14-day repeat exposure, Group 4). Samples were collected during the exposure phase of the pilot experiment (Group 1) at 2, 4, and 5.75 hours and results indicate that the test substance had reached steady state in blood by 2 hours in both male and female rats.

Blood Kinetics:
- For the single exposure, 2500 ppm experiment (Group 2), the mean peak blood concentration (Cmax) was 1130 (± 603) and 1630 (± 725) ng/g in males and females, respectively. The mean terminal elimination half-life (T1/2) values were 0.189 (± 0.0276) and 0.314 (± 0.0943) hours with an area-under–the-curve (AUC) value of 418 (± 179) and 807 (± 325) hr x ng/g in males and females, respectively. There was a statistically significant sex difference observed in half-life between male and female animals. There was no statistically significant sex difference observed in AUC or Cmax.
- For the single exposure, 10000 ppm experiment (Group 3), the mean peak blood concentration (Cmax) was 4110 (± 2420) and 5240 (± 776) ng/g in males and females, respectively. The mean terminal elimination half-life (T1/2) values were 1.54 (± 0.613) and 0.323 (± 0.114) hours with an area-under–the-curve (AUC) value of 2180 (± 796) and 2310 (±565) hr × ng/g in males and females, respectively. There was a statistically significant sex difference observed in half-life between male and female animals. There was no statistically significant sex difference observed in AUC or Cmax.
- For the 14-day repeated exposure, 2500 ppm experiment (Group 4), the Cmax values were 1040 (± 821) and 1070 (± 1070) ng/g in males and females, respectively. The terminal elimination half-life (T1/2) was 0.537 (± 226) and 0.453 (± 0.144) hours with an area-under–the-curve (AUC) value of 669 (± 290) and 640 (± 428) hr x ng/g in males and females, respectively. There was no statistically significant sex difference observed in half-life, AUC or Cmax.
- Six hour inhalation exposure of rats to 2500 ppm compared to rats exposed to a single, 6 hour exposure of 10000 ppm indicated that the dose normalized AUC (AUCinf_D_obs) (0.164 ± 0.0701 and 0.221 ± 0.0807 hr × ng/g/ppm in male rats and 0.316 ± 0.127 and 0.234 ± 0.057 hr × ng/g/ppm in female rats) and the dose normalized Cmax (Cmax_D)(0.443 ± 0.237 and 0.417 ± 0.246 ng/g/ppm in male rats and 0.640 ± 0.284 and 0.531 ± 0.0786 ng/g/ppm in female rats) were not statistically significantly different . The similarity in these parameters, suggest that there is no difference in distribution and elimination of the test substance from blood over this range of exposure concentrations.
- The AUC and Cmax from a single 2500 ppm exposure and the 14-day repeated 2500 ppm exposure in both male and female rats were approximately equal (not statistically significantly different), indicating that there was no accumulation of the test item in rats following 14-day repeated inhalation exposure.
Details on distribution in tissues:
Tissues were collected from Group 3 animals only (4 male and 4 female rats, single exposure, 10000 ppm). Tissues were collected from 4 male and 4 female rats during exposure phase at two time points, after 1 hour and after 4 hours of exposure and tissues were collected 30 minutes after the animals were removed from the chamber. Tissues were also collected from 4 male and 4 female rats at 30 minutes, 3 hours, 8 hours and 18 hours post 6 hour exposure. The test substance was detected to some degree in all tissues types collected (fat, reproductive organs, muscle, spleen, lung, kidney, heart, brain and liver, listed in order of greatest amount of test substance detected to least amount detected per tissue). The fat was the only tissue with measurable test substance in the 18 hour, post exposure time point samples (3100 ± 3133 ng/g, males and 2266 ± 2262 ng/g females). Liver had the least amount of test substance detected (198 ng/g from liver collected 30 minutes post 6 hour exposure in 1 out of 4 males only and 187.3 ± 49.52 ng/g in 3 out of 4 female animals from livers collected 30 minutes after 4 hours of exposure and 455 ng/g detected in 1 out of 4 female animals from liver collected 30 minutes post 6 hour exposure).

Tissue Kinetics:
- Elimination of the test substance was rapid from all tissues collected (fat, reproductive organs, muscle, spleen, lung, kidney, heart, brain and liver). No toxicokinetic parameters were obtained for 6 of 9 tissues collected (spleen, lung, kidney, heart, brain, and liver) due to the lack of concentration data beyond the 3 hour time point. Although tissue extract samples were not analysed for the 8 and 18 hour samples for these tissues, the pilot study results indicated that concentrations of test substance were below LOD for extracts at time points beyond 4 hours.
- Fat peak tissue concentration (Cmax) was 567000 and 622000 ng/g in males and females, respectively. The mean terminal elimination half-life (T1/2) values were 2.28 and 2.27 hours with area-under–the-curve (AUC) values of 2480000 and 2210000 hr × ng/g in males and females, respectively. Although statistics could not be performed on this data set, the values for male and female rats were similar, which would suggest that there was no sex difference.
- Reproductive organ peak tissue concentration (Cmax) was 30200 and 111000 ng/g in males and females, respectively. The mean terminal elimination half-life (T1/2) values were 1.79 and 1.99 hours with area-under-the-curve (AUC) values of 90100 and 245000 hr × ng/g in males and females, respectively.
- Muscle peak tissue concentration (Cmax) was 3700 and 9040 ng/g in males and females, respectively. The mean terminal elimination half-life (T1/2) values were 1.53 and 0.775 hours with area-under-the-curve (AUC) values of 17200 and 15800 hr × ng/g in males and females, respectively.
Details on excretion:
Urine and faeces were collected from Group 3 animals only (4 male and 4 female rats, single exposure, 10000 ppm). Bile was collected from Group 3 animals only (3 male and 3 female rats, single exposure, 10000 ppm). Collection time points for bile, urine and faeces were 0-4 hours, 4-8 hours and 8-18 hours. Relatively small amounts of test item were detected in urine, faeces and bile through the 4-8 hour time point but no test substance was detected in the 8-18 hour samples except for 236 ± 20.5 ng/g detected in faeces from 2 out of 4 female animals. The low levels of the test item in the urine, faeces and bile are likely due to volatility of the test substance during sample collection.
Toxicokinetic parametersopen allclose all
Test no.:
#2
Toxicokinetic parameters:
Cmax: Blood - Group 2, single 6-hour exposure to 2500 ppm; Peak concentrations (Cmax) in blood were 1130 (± 603) and 1630 (± 725) ng/g in males and females, respectively.
Test no.:
#2
Toxicokinetic parameters:
half-life 1st: Blood - Group 2, single 6-hour exposure to 2500 ppm; The mean terminal elimination half-life (T1/2) values in blood were 0.189 (± 0.0276) and 0.314(± 0.0943) hours in males and females, respectively.
Test no.:
#2
Toxicokinetic parameters:
AUC: Blood - Group 2, single 6-hour exposure to 2500 ppm; The area-under–the-curve (AUC) values for blood were 418 (± 179) and 807 (±325) hr × ng/g in males and females, respectively.
Test no.:
#3
Toxicokinetic parameters:
Cmax: Fat - Group 3, single 6-hour exposure to 10000 ppm; Peak concentrations (Cmax) in fat were 567000 and 622000 ng/g in males and females, respectively.
Test no.:
#3
Toxicokinetic parameters:
half-life 1st: Fat - Group 3, single 6-hour exposure to 10000 ppm; The mean terminal elimination half-life (T1/2) values for fat were 2.28 and 2.27 hours in males and females, respectively.
Test no.:
#3
Toxicokinetic parameters:
AUC: Fat - Group 3, single 6-hour exposure to 10000 ppm; The area-under–the-curve (AUC) values for fat were 2480000 and 2210000 hr × ng/g in males and females, respectively.
Test no.:
#3
Toxicokinetic parameters:
Cmax: Blood - Group 3, single 6-hour exposure to 10000 ppm; The mean peak blood concentration (Cmax) was 4110 (± 2420) and 5240 (± 776) ng/g in males and females, respectively.
Test no.:
#3
Toxicokinetic parameters:
half-life 1st: Blood - Group 3, single 6-hour exposure to 10000 ppm; The mean terminal elimination half-life (T1/2) values in blood were 1.54 (± 0.613) and 0.323 (± 0.114) hours in males and females, respectively.
Test no.:
#3
Toxicokinetic parameters:
AUC: Blood - Group 3, single 6-hour exposure to 10000 ppm; The area-under–the-curve (AUC) values for blood were 2180 (± 796) and 2310 (±565) hr × ng/g in males and females, respectively.
Test no.:
#4
Toxicokinetic parameters:
Cmax: Blood - Group 4, daily 6-hour exposure for 14-days to 2500 ppm; Peak concentrations (Cmax) in blood were 1040 (± 821) and 1070 (± 1070) ng/g in males and females, respectively.
Test no.:
#4
Toxicokinetic parameters:
half-life 1st: Blood - Group 4, daily 6-hour exposure for 14-days to 2500 ppm; The mean terminal elimination half-life (T1/2) values in blood were 0.537 (± 0.226) and 0.453 (± 0.144) hours in males and females, respectively.
Test no.:
#4
Toxicokinetic parameters:
AUC: Blood - Group 4, daily 6-hour exposure for 14-days to 2500 ppm; The area-under–the-curve (AUC) values for blood were 669 (± 290) and 640 (±428) hr × ng/g in males and females, respectively.

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
A total of 9 metabolites were tentatively identified in blood, tissues, bile, urine, and faeces samples based on accurate mass and parent ion fragmentation spectra. There were no standards available for quantitation of the identified metabolites. The primary biotransformation steps for the test item appear to be either oxidation or direct conjugation with glutathione. Additional secondary metabolism resulted in phase I and phase II metabolites including oxidation to a ketone, formation of a second glutathione conjugate, or further hydrolysis to dihydroxyl-test item and glucuronidation. The glutathione conjugates were degraded to cysteine conjugates and N-acetylated cysteine conjugates. The metabolic profile appears to be qualitatively similar between male and female rats.

Any other information on results incl. tables

The mean chamber test item vapour concentration for the 14-day study was 2500 ± 236 ppm (mean ± mean standard deviation) for all 14 exposures. The mean concentration for the single exposure to a design concentration of 2500 ppm was 2550 ± 284 ppm. The mean concentration for the 10000 ppm exposure with bile cannulated rats was 10100 ± 632 ppm. The mean concentration for the single 6-hour exposure to a design concentration of 10000 ppm was 9870 ± 732 ppm. The mean concentration for the entire exposure in which rats were exposed for 1 or 4 hours to a design concentration of 10000 ppm was 10500 ± 1810 ppm. The chamber atmospheres were considered adequate for inhalation testing in rats.

There were no animal deaths, no adverse clinical signs, and no significant effect on body weight observed in any exposure group.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): no bioaccumulation potential based on study results
This study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).
The absorption of the test item was rapid with steady-state in whole blood occurring by 2 hours during the 6-hour exposure phase. The whole blood elimination half-lives ranged from 0.19–1.5 hours. The test item was distributed throughout the tissues evaluated and there was no indication of accumulation potential. The pattern of distribution and excretion was similar across sex and dose. Excretion was substantially complete by 18 hours post exposure.
Executive summary:

In the present study, the absorption, distribution, metabolism and excretion of the test item was studied in male and female Sprague-Dawley, Crl:CD®(SD)IGS BR rats. Experiments were performed to evaluate: 1) the steady-state properties of the test substance via inhalation exposure; 2) the distribution of the test substance among whole blood, tissues and bile; 3) the pharmacokinetic behaviour of the test substance in whole blood and tissues (fat, reproductive organs and muscle); and 4) the elimination properties of the test substance in urine and faeces. The metabolite profile was characterized in whole blood, tissues, bile, urine and faeces by HPLC mass spectrometry. Four exposure groups were included: single exposures for 1, 4 or 6 hours at 2500 or 10000 ppm and a 14-day/6 hours/day exposure at 2500 ppm.

Systemic uptake was rapid with blood reaching steady-state within 2 hours of the 6-hour exposure period during a single exposure at 10000 ppm (Group 1, pilot study). Blood concentrations in male and female rats during the exposure period (2, 4, and 5.75 hours) were approximately 19100 ± 3070 ng/g. The test item was present in blood samples and all collected tissues (fat, reproductive organs, muscle, spleen, lung, kidney, heart, brain and liver) 30 minutes after a single 6-hour exposure to 10000 ppm of test substance. By 18 hours after the end of the single 6-hour exposure, the test substance concentrations in blood, bile, and tissues (except fat) were less than the level of detection (<LOD). The concentration in fat of male and female rats at 18 hours after the single 6-hour exposure to 10000 ppm was 3100 ± 3133 ng/g and 2266 ± 2262 ng/g, respectively. Peak concentrations (Cmax) of the test item in fat were 567000 and 622000 ng/g in males and females, respectively. The mean terminal elimination half-life (T1/2) values were 2.28 and 2.27 hours with area-under–the-curve (AUC) values of 2480000 and 2210000 hr × ng/g in males and females, respectively.

The pharmacokinetic behaviour of the test item in blood was similar between male and female rats following a single 6-hour, 2500 ppm exposure (Group 2), single 6-hour, 10000 ppm exposure (Group 3) and 14-days of repeated 2500 ppm exposure (Group 4) with blood half-lives ranging from 0.19–1.5 hours. After a single 6-hour exposure to 2500 ppm and a single 6-hour exposure to 10000 ppm, the blood AUC and Cmax values were proportional to the exposure concentration. The single 2500 ppm exposure group (Group 2) and the single 10000 ppm exposure group (Group 3) dose-normalized AUC and Cmax values in males and females were not statistically significantly different. These findings indicate similar kinetic behaviours for the distribution and elimination of the test substance from blood at these exposure concentrations. The AUC and Cmax values in blood in both male and female rats were not statistically significantly different between the single 2500 ppm exposure group (Group 2) and the 14-day repeated 2500 ppm exposure group (Group 4) indicating the absence of accumulation of the test item in rats following 14 consecutive days of exposure. The amount of test item detected in urine and faeces was minimal and decreased with time. There was likely some unmeasured loss of the test item from urine and faeces during collection due to volatility, and elimination of the test item via exhalation could not be determined under the conditions of this study.

A total of 9 metabolites of the test item were tentatively identified in blood, tissues, bile, urine, and faeces samples based on accurate mass and parent ion fragmentation spectra. There were no standards available for quantitation of the identified metabolites. The primary biotransformation steps for the test item appear to be either oxidation or direct conjugation with glutathione. Additional secondary metabolism resulted in phase I and phase II metabolites including oxidation to a ketone, formation of a second glutathione conjugate, or further hydrolysis to dihydroxyl-test item and glucuronidation. The glutathione conjugates were degraded to cysteine conjugates and N-acetylated cysteine conjugates. The metabolic profile appears to be qualitatively similar between male and female rats.

In summary, the absorption of the test item was rapid with steady-state in whole blood occurring by 2 hours during the 6-hour exposure phase. The whole blood elimination half-lives ranged from 0.19–1.5 hours. The test item was distributed throughout the tissues evaluated and there was no indication of accumulation potential. The pattern of distribution and excretion was similar across sex and dose. Excretion was substantially complete by 18 hours post exposure. The relatively small amounts of the parent test substance detected in the urine and faeces was likely due to volatility during collection. Metabolism of the absorbed dose was extensive and characterized by numerous identified components in blood, tissues, bile, urine and faeces.