Bromoacetic acid

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: supporting laboratory study to investigate stabiltiy in blood

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

In vitro study with whole rat blood and plasma using the radio labelled test item.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Mono bromo acetic acid

IUPAC Name:

Mono bromo acetic acid

Constituent 2

Reference substance name:

Bromoacetic acid

EC Number:

201-175-8

EC Name:

Bromoacetic acid

Cas Number:

79-08-3

Molecular formula:

C2H3BrO2

IUPAC Name:

2-bromoacetic acid

Details on test material:

Radiolabeled material: 442-128-050
Specification Radiochemical purity 99% (HPLC)
Radiolabeled material: 50 mCi.mmol-1
Purity Non-radiolabeled material: 99.6%
Radiolabeled material: 99% (HPLC)

Radiolabelling:

yes

Remarks:

[2-14C]Bromoacetic acid

Administration / exposure

Details on study design:

Rat blood was collected from the abdominal aorta into heparinised tubes. In addition, whole blood and plasma was obtained from a commercial source (Harlan, the Netherlands. Batch no.R24JUL07BLNL (whole blood), R24MEI0LNL (plasma)) for practical and logistic reasons. Plasma samples of sacrifice blood were prepared by centrifugation (10 minutes, 1200x g). The red blood cells were discarded.
Concentration of test substance
10 μM and 100 μM; control 0.9% NaCl.
Test condition A: 0.9 % NaCl in demineralised water (saline; control)
B: Whole blood and blood plasma from male rats
C: 25 mM glutathione (GSH) in 1 M phosphate buffer pH 7.6 (todetermine auto-reactivity towards GSH)
Determination of radioactivity
The radioactivity in the samples was determined using a LKB/WallacS1414 scintillation counter in presence of Ultima GoldTM scintillation liquid (Packard). For the whole blood and plasma pellets, Hionic FluorTM scintillation liquid (Packard) was added to a sub-sample of the samples that were digested using 1.5 M KOH in a 20 % aqueous ethanol solution.
Analysis HPLC analysis of [2-14C]Bromoacetic acid
Prior to the start of the study, the radiochemical purity of the tracer was verified using the following method:
Column Phenomenex Aqua C18, 250 x 4.6 mm, 5 μm
Mobile phase A: 0.1 % TFA in water
Gradient isocratic
Flow rate 1.0 mL.min-1
Detection on-line radioactivity detection
Breakdown of Bromoacetic acid was assessed using the same radio-HPLC with on-line radioactivity detection, but applying a linear gradient using 0.1 % TFA in acetonitrile (B) instead, for separation of parent Bromoacetic acid and possible breakdown products.G
LC-MS analysis
Adapted from HPLC described above. The gradient was slightly adapted and formic acid was used instead of TFA. Using formic acid instead of
TFA, retention times were found to shift backwards. The analysis was performed on a Thermo Finnigan LT-Q which is a linear iontrap directly
coupled to an LC system.
Sampling time 30, 60 and 120 min.
Sample preparation To samples of 225 μL (saline, plasma or whole blood) 25 μL 10-times stock [14C]- Bromoacetic acid was added. At the end of the incubation period, 25 μL 10 % TFA was added under constant stirring using a Vortex. Then, ca 5 mL MTBE was added while stirring and the tubes were placed on ice. Tubes were centrifuged (ca 5 min. at 3000 rpm, ca 4 °C). The MTBE fraction was transferred to a glass tube and evaporated
under a nitrogen gas stream in a water bath adjusted to approx. 30 °C. The wall of the tube was rinsed with a small volume of MTBE and
evaporated again. The residue was taken up in a small volume of HPLC eluent A. To identify the test compound related radio-activity in the whole blood pellets (remaining after MTBE extraction), a small volume of water (ca 200 μL) was added to this pellet and the sample was repeatedly frozen
and thawed to disrupt the blood cells. The sample was centrifuged and the supernatant used for radio-HPLC analysis. Additional incubations were performed using disrupted blood cells, whole blood was mixed 1:1 with water, repeatedly frozen and thawed (4 times) and disrupted using an UltraTurrax for ca 30 sec.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): low bioaccumulation potential based on study results
The results of this investigation show fast and almost complete removal of Bromoacetic acid from plasma and in particular from whole blood.
No unexpected metabolites are found; the observed strong binding to cellular proteins removes Bromoacetic acid from circulation. It can be
concluded that the reported results of Saghir et al. 2005 are realistic.

Executive summary:

As a recent paper (Saghir et al. 2005) has shown that Bromoacetic acid could not be detected in the plasma of male Fisher rats at any sample point after oral or intravenous administration, it was decided to investigate the stability of Bromoacetic acid in blood. In vitro incubations with whole blood and blood plasma obtained from rats have been performed using [14C]- Bromoacetic acid for analytical sensitivity. For comparison reasons the incubations were performed in the following conditions: A: 0.9 % NaCl in demineralised water (saline; control) B: Whole blood and blood plasma from male rats C: 25 mM glutathione (GSH) in 1 M phosphate buffer pH 7.6 (to determine auto-reactivity towards GSH) Condition A served as control, condition C was added to study the possible conjugation of Bromoacetic acid with glutathione. Condition B served to investigate the behaviour of Bromoacetic acid in plasma excluding the cells present in blood and whole blood, which represents the natural situation. In a preliminary study general condition were tested. In the main study two concentration of Bromoacetic acid (10 μM and 100 μM). These concentrations are below and slightly above the NOAEL of the subchronic 90-day oral rat study. 5.2 Results and discussion In plasma, radioactivity that could be extracted with MBTE (attributable to free Bromoacetic acid) decreased considerably with time to about 42 % at a concentration of 10 μg/mL and 50 % at a concentration of 100 μg/mL after 120 min. In whole blood, only very little extractable radioactivity was observed already after 30 min of incubation (1 % at 10 μg/mL, 2 % at 100 μg/mL). Radioactivity associated with the protein or blood pellet remaining after extraction was increasing simultaneously. This was most pronounced using whole blood were about 80 % of the total radioactivity added was associated with the blood pellet already after 15 minutes of incubation. Radio-HPLC analysis of the extract after incubation identified mainly Bromoacetic acid as the radioactive species in the extract. In the disrupted blood pellet, Bromoacetic acid was only detectable up to about 10 minutes of incubation. One peak was observed being most likely a conjugate of [14C]-Bromoacetic acid and glutathione. This assumption is based on additional incubations using [14C]-Bromoacetic acid and GSH in phosphate buffer, which showed rapid formation of a product with a comparable retention time in the radio-HPLC chromatograms. The results of this investigation show fast and almost complete removal of Bromoacetic acid from plasma and in particular from whole blood. No unexpected metabolites are found; the observed strong binding to cellular proteins removes Bromoacetic acid from circulation. It can be concluded that the reported results of Saghir et al. 2005 are realistic