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Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: No guideline was mentioned and the study did not follow GLP. The purity of the unlabelled bronopol was not specified. However the data are scientifically acceptable.
Objective of study:
absorption
Principles of method if other than guideline:
Method: other: no data
GLP compliance:
no
Radiolabelling:
yes
Remarks:
14C (located on C2)
Species:
mouse
Strain:
other: CFLP
Sex:
male
Details on test animals and environmental conditions:
Mice were obtained from Interfauna UK Ltd.
Age/weight at study initiation: The mice were approximatively 28 days old and weighed between 17 and 32 g
Route of administration:
dermal
Vehicle:
other: acetone:water (9:1)
Duration and frequency of treatment / exposure:
Frequency: Three times per week over a period of 30 days (13 applications).
Remarks:
Doses / Concentrations:
Males: 0.3 ml of 0.5% w/v solution
No. of animals per sex per dose:
4
Control animals:
no
Details on study design:
The mice obtained from Interfauna UK Ltd were allocated into groups of 4 animals each. Prior to the initial treatment, the dorsal skin of each mouse was closely shaved; this was repeated once weekly throughout the test period.The mice received repeated applications of 0.3 ml/mouse of 5 mg/ml adiolabelled bronopol (0.5% w/v test solution in 90% acetone/10% water), over a period of 30 days. The test solution was applied onto the shaved dorsum of each mouse by means of an automatic pipetting device on three days a week (i.e. Monday, Wednesday and Friday), resulting in a total of 13 applications.
Details on dosing and sampling:
Blood samples were collected from the mice following sacrifice; for details on sacrifice time point and sampling. Plasma was separated from whole blood by centrifugation, and aliquots were subjected to liquid scintillation in Optiphase Safe liquid scintillator in duplicate (LC).Pooled plasma samples were extracted and the organic phases were pooled and evapored to dryness under nitrogen at 40 °C. Following redissolution , 20 µl aliquots of the extracts were spotted onto thin layer chromatography plates (TLC); [14C] bronopol was used as marker. Development was conducted in chloroform/methanol (4:1). Areas of radioactivity were visualised by autoradiography, scraped at the amounts of radioactivity-related material for each area was measured by LC.Duplicate aliquots of dose material were retained prior and after each dosing period for verification of test substance contents.
Details on absorption:
The results indicated that [14C] bronopol was absorbed following application to the shaved dorsal skin of mice. Radioactivity was rapidly cleared from plasma after dosing with an apparent t1/2 of ca. 8 hrs, resulting in no accumulation potential at the dose regimen used (see table 1). The plasma profiles for day 1 and day 29 were similar. The observed maximum plasma levels of radioactivity were therefore not affected by an interval of 48 or 72 hours between consecutive dosing.
Toxicokinetic parameters:
half-life 1st: ca. 8 h
Metabolites identified:
not measured
Details on metabolites:
Thin layer chromatography revealed some radioactive material that could be associated to standard [14C] bronopol; the percentage of material however ranged from very low to not detectable. Further compounds that had resulted from the rapid and extensive metabolism of bronopol also were reported. These compounds were no further identified.

Table 1: Concentration of radioactivity in the plasma of mice (µg/ml bronopol equivalent +/- SEM) after repeated dermal applications of bronopol.

Day of Dosing

Doses (Number)

Time after dosing (hours)

0.5 h

1 h

3 h

6 h

8 h

24 h

1

1

9.1+/-2.2

13.0+/-1.8

8.2+/-1.0

5.5+/-0.5

3.7+/-0.2

1.5+/-0.1

3

2

11.4+/-1.3

1.4+/-0.1

5

3

8.1+/-0.6

0.8+/-0.1

8*

4

9.7+/-0.3

1.3+/-0.2

10

5

16.2+/-1.7

1.6+/-0.2

12

6

12.9+/-1.9

1.8+/-0.3

15*

7

12.8+/-1.3

1.2+/-0.1

17

8

11.6+/-1.5

1.6+/-0.1

19

9

17.0+/-2.0

1.3+/-0.2

22*

10

12.9+/-1.6

1.5+/-0.2

24

11

13.7+/-1.9

1.5+/-0.1

26

12

10.7+/-0.4

1.9+/-0.1

29*

13

6.7+/-1.8

10.7+/-1.0

6.7+/-0.7

2.2+/-0.5

2.2+/-0.2

1.4+/-0.1

*, indicated 3 day gap since previous dosing

Endpoint:
basic toxicokinetics
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted in accordance with GLP.
Objective of study:
metabolism
Qualifier:
according to
Guideline:
EPA OPP 85-1 (Metabolism and Pharmacokinetics)
GLP compliance:
yes
Radiolabelling:
yes
Remarks:
14C (located on C2)
Species:
mouse
Strain:
other: CFLP
Sex:
male
Details on test animals and environmental conditions:
Age/weight at study initiation: The mice were approximatively 4 weeks old and weighed between 13.3 and 19.5 g.
Mice were obtained from Interfauna UK Ltd.
Route of administration:
dermal
Vehicle:
other: acetone:water (9:1)
Duration and frequency of treatment / exposure:
First series: Sacrifice 0.5, 1, 2, 3, 6, 8, 24 and 48 hours after treatment.
Second series: Sacrifice 1, 6, and 24 hours after second dosing which was applied 48 hours after the first dosing.
Remarks:
Doses / Concentrations:
First series: 1.5 mg/animal (corresponds to aproximately 75 mg/kg bw)
Second series: two times 1.5 mg/animal
No. of animals per sex per dose:
4
Control animals:
no
Details on study design:
A total of 44 mice obtained from Interfauna UK Ltd was allocated into two test series. The first test series consisted of 8 groups of 4 mice each; the second test series consisted of 3 groups of 4 mice each. Prior to treatment, the dorsal skin of each mouse was closely shaved.
Details on dosing and sampling:
A 0.5% w/v test solution of [14C] bronopol in 90% acetone/10% water was prepared, which corresponded to a nominal concentration of 5 mg/ml. The mice of the first test series received a single application of 0.3 ml test solution/mouse on a dorsal skin area of 1.5 x 1.5 cm2, resulting in an applied dose concentration of 1.5 mg/animal; the application site was covered with a dressing. Groups of 4 mice were sacrificed after 0.5, 1, 2, 3, 6, 8, 24 and 48 hours following dosing. The mice of the second test series received two consecutive applications of respectively 0.3 ml test solution/mouse as described above; the animals received the second application after 48 hours following the first one. Groups of 4 mice were sacrificed after 1, 6 and 24 hours following second dosing. For both test series, blood samples were collected from the sacrificed mice and plasma was separated by centrifugation. Plasma samples from each group of 4 mice were pooled. The dressing material was collected. The shaved dorsal area of each mouse was wet swabbed (cotton wool swab with 5% detergent) and dry swabbed. The skin area was then excised. All plasma, dressings, swabs and skin samples were placed in vials and were stored at -20 °C until analysis.For analysis of total radiolabelled material, aliquots of plasma were subjected to liquid scintillation counting (LC). Skin samples from the first test series were chopped and pooled for each group; skin samples from the second test series also were chopped but were then analysed separately. The samples were subjected to oxygen combustion; the combustion products were prepared for LC counting. Whole dressing and swabs also were subjected to oxygen combustion and were prepared for and subjected to LC counting.For analysis for the metabolic profile, pooled plasma samples was acidified, extracted, and prepared for HPLC analysis For the skin samples, aliquots of the pooled samples (series 1) or of individual samples (series 2) were homogenised, acidified, extracted and prepared for HPLC analysis system. Sample peaks were identified by comparing the retention times with those of the authentic reference standards (bronopol, 2-nitropropan-1,3-diol).
Details on absorption:
Following a single application, the maximum concentration of 230 µg eq./g of radiolabelled material was recovered in the skin after 0.5 h and corresponded to 6% of total administered radiolabelled material; the maximum concentration in the plasma was reached after 1 hour and was about 1.4 µg eq./g. The concentrations in both, skin and plasma decreased with time, and after 48 h, the respective concentrations were 148 and 0.61 µg eq./g.
Following two consecutive applications at an interval of 48 h, the concentration level reached in skin after 1 hour following the second application was 351 µg eq./g (i.e. 5% of total administered radiolabelled material); the level was therefore about 2.5 fold the concentration measured after 1 hour following a single application (i.e. 138 µg eq./g). In contrast, for plasma, the concentration of radiolabelled material recovered after 1h hour following the second application was within the range of concentration obtained at the same time point after a single application (i.e. 1.52 µg eq./g versus 1.41 µg eq./g). After 24 hours, the decrease in concentration retained in the skin was slow and the final concentration of 332 µg eq. /g still amounted for 4% of the total administered dose. Decrease in plasma was more rapid and after 24 hours, about 0.85 µg eq./g was measured.
The results are indicative of a persistence of the radiolabelled material in skin, with only slow release into the plasma following an initial peak.
Metabolites identified:
yes
Details on metabolites:
HPLC analysis of the extracts yielded profiles with one major peak which was identified as 2-nitropropan-1,3-diol.
This accounted for 95 to 98.4% of radiolabel from skin samples following a single dose (in plasma: 70 to 88%).
No decline was seen over the 48 hrs observation period. After two doses, 2-nitropropan-1,3-diol accounted for 78% of radiolabel in plasma, but this declined to 15% after 24 hrs. The remainder was attributed to a second major metabolite which was not identified. In skin 2-nitropropan-1,3-diol represented 75 to 98% of the radioactivity extracted from skin.
Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: As the study was based of a method developed by Boots especially for bromide ion detection in the urine of rat, the study follows no acknowledged guideline. The data however are scientifically acceptable. The study followed GLP.
Objective of study:
metabolism
Principles of method if other than guideline:
Method: other: no data
GLP compliance:
yes
Radiolabelling:
no
Species:
rat
Strain:
other: CD
Sex:
male
Details on test animals and environmental conditions:
Animals were obtained from Charles River Laboratories.
Body weights: At test initiation, the body weights of the rats ranged between 140 and 146 g.
Route of administration:
oral: gavage
Vehicle:
water
Duration and frequency of treatment / exposure:
single treatment
Remarks:
Doses / Concentrations:
1 and 50 mg/kg bw
No. of animals per sex per dose:
4
Control animals:
no
Details on dosing and sampling:
In the present study, the presence of bromide ion as breakdown product of bronopol in the urine was examined in
rats following single oral dosage with 1 and 50 mg/kg bw bronopol. In a group of 8 male Charles River CD rats, four animals received a
single oral dose of 1 mg/kg bw whereas the remaining four animals received 50 mg/kg bw of bronopol.

Urine samples were collected pre-dose over 0 to -24 and -24 to -48 hours. Urine samples were collected over the time periods 0 to 8, 8 to 24, 24 to 48 and 48 to 120 hours following dosing. The samples were examined for the presence of bromide ion by means of the capillary electrophoresis method developed by Thakrar H (Boots Report No: DT 93015, 1993). The method allows the quantification of bromide ion in rat urine, the limit of quantification being 10 µg/ml when 0.1 ml urine was used. A linear response is obtained from 10 µg/ml up to 150 µg/ml. The assay accuracy was -10% at 10 µg/m level and -1.5% at 60 µg/ml. Recovery of bromide ion from rat urine was 98% and was considered to be good. The specificity of the assay was checked for any possible interference from chloride, formate and nitrate ions. These ions were chromatographically well-separated from the ions of interest. The measurement of urine samples collected prior dosing allows to account possible small quantities of endogenous bromide ion (e.g. arising from diet) in urine.
Details on absorption:
The treatment with 1 mg/kg bw of bronopol resulted in concentrations of bromide ion which were not above the endogenous level.
The treatment with 50 mg/kg bw of bronopol resulted in an excreted amount of about 17% of the initial dose as bromide ion over the period of 120 hours.
Toxicokinetic parameters:
half-life 2nd:
Toxicokinetic parameters:
half-life 1st:
Toxicokinetic parameters:
half-life 3rd:
Metabolites identified:
no
Endpoint:
basic toxicokinetics
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted in accordance with GLP.
Objective of study:
metabolism
Qualifier:
according to
Guideline:
EPA OPP 85-1 (Metabolism and Pharmacokinetics)
GLP compliance:
yes
Radiolabelling:
yes
Remarks:
14C, located on C2
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Source: Animals delivered from Charles River Laboratories, UK Ltd., Margate, Kent, UK.
Age/weight: At test initiation, the body weights of the rats ranged between 119 and 156 g.
Route of administration:
oral: gavage
Vehicle:
other: 0.4% hydroxyethylcellulose
Details on exposure:
Specific activity of test substance: 65.9 MBq/g (13.2 GBq/mol)
Volume applied: 1 ml/100g bw
Duration and frequency of treatment / exposure:
single dose
Dose / conc.:
10 mg/kg bw (total dose)
No. of animals per sex per dose:
5
Details on study design:
In the present study, the excretion and distribution of bronopol in rat were examined over a period of 7 days
following a single oral dosage with 10 mg/kg bw radioactive labelled compound. A single dose of 10 mg/kg of [2-14C] Bronopol was given to 5 male and 5 female Charles River CD rats.
Details on dosing and sampling:
Urine, faeces and respired carbon dioxide were collected at 4, 8, 12, 24, 36, 48, 72, 96, 120, 144 and 168 hrs
after dosing with the exception of carbon dioxide which was collected at 4, 8, 12, 24 and 48 hrs after dosing.
Washing sampling: washings were added to the urine samples at sampling time point 72 h.
At 168 hrs the animals were killed and blood was collected for the determination of radioactivity in blood and
plasma; tissue samples also were sampled and prepared for analysis.
For air sampling, the expired gases were passed through two CO2 traps containing a mixture of 2-ethoxyethanol:ethanolamine (2:1 v/v). Aliquots were taken from these traps for analysis. The samples were stored at ca. 4 °C until analysis.
Urine and faeces samples were stored at –20 °C until analysis
All samples were assayed for 14C-radioactivity by liquid scintillation counting. Prior to measurement, urine, 2-ethoxyethanol:ethanolamine and plasma samples were mixed with 10 ml of Optiphase “safe” scintillant. Faeces samples were homogenised in distilled water, air-dried and were subjected to oxygen combustion; the combustion products were absorbed in Carbo-sorb E and were mixed with Permafluor E+. Tissue samples were chopped and aliquots were air-dried and subjected to oxygen combustion.

Preliminary studies:
Samples of the test solution were taken immediately prior and after dosing and were subjected to HPLC. The content of bronopol in the test solution was found to be within 10% of nominal value.
Liquid scintillation counting revealed a dose content within 1% of the nominal value.
Details on absorption:
Following single oral dosage with 10 mg/kg bw of radiolabelled bronopol, total recovered radioactive material for the males ranged between 76 and 92% whereas for the females, total recovery ranged between 75 and 89%.
Details on distribution in tissues:
For the males, the mean concentration of radioactive material in whole blood and plasma respectively was 0.072 µg/g and 0.021 µg/g, resulting in a ratio of 3.4. For the females, the mean concentrations respectively were 0.036 µg/g and 0.016 µg/g, resulting in a whole blood to plasma ratio of 2.3.
Recovery of radioactivity in tissues and carcass was greater than in plasma. In fact, the highest concentrations of radioactive material were reported for the liver (male: 0.236 µg/g; female: 0.108 µg/g) and the lung (male: 0.199 µg/g; female: 0.087 µg/g).
Details on excretion:
Following single oral dosage with 10 mg/kg bw of radiolabelled bronopol, about 72% of radioactive material for males and 73% for females was recovered in the urine over a period of 168 h (i.e. 7 days); about 70% there from, was excreted during the first 24 hours following dosage. Excretion of radioactive material via faeces was about 11% for males and 10% for females. The percentages recovered in the expired air were about 4% for males and 2% for females.
Metabolites identified:
not measured

Table 1: Recovery of radiolabelled material in urine, faeces and CO2, and remaining radioactivity in tissues and carcass after single oral dosage of 10 mg/kg bw 14C bronopol.

 Sample              Percentage of administered radiolabelled material  
 Sex Animal number  Urine(including cage washing)  Faeces   CO2 (sum of the highestrecovery from each pair of traps)  Tissue and carcass Total radiolabel(%) 
 Male  73.5 15.4  2.1  0.9  91.9 
   2  66.2 9.3  not measured  0.9  76.4 
   3  71.8 11.5  6.3  0.9  90.5 
   4  78.3 10.3  3.2  0.7  92.5 
   5  68.0 10.8  4.6  1.2  84.6 
   Mean  71.6 11.4  4.1  0.9  87.2 
   +/- SD  4.8 2.4  1.8  0.2  6.8 
 Female  1  76.5 8.5  1.9  0.4  87.3 
   2  65.5 9.0  not measured  0.7  75.3 
   3  70.1 12.0  2.6  0.8  85.4 
   4  77.0 10.6  0.9  0.3  88.8 
   5  76.1 10.7  1.6  0.3  88.6 
   Mean  73.1 10.2  1.8  0.5  85.1 
   +/- SD  5.0 1.4  0.7  0.2  5.6 

Table 2: Recovery of radiolabelled material in tissues after 168 h following single oral dosage of 10 mg/kg bw 14C bronopol.

 Tissue  Concentration of radiolabelled material +/- SD (µg/g of bronopol)   
   Male  Female
 Plasma 0.021 ± 0.005 0.016 ± 0.009 
 Bone 0.137 ± 0.013 0.093 ± 0.015 
 Brain 0.045 ± 0.013 0.030 ± 0.015 
 Fat 0.216 ± 0.083  0.086 ± 0.039 
 Heart 0.105 ± 0.048 0.047 ± 0.030 
 Kidney 0.129 ± 0.042 0.074 ± 0.032 
 Liver 0.236 ± 0.074 0.108 ± 0.056 
 Lung 0.199 ± 0.078 0.087 ± 0.046 
 Muscle 0.091 ± 0.032 0.046 ± 0.028 
 Ovary  not applicable 0.058 ± 0.032 
 Spleen 0.145 ± 0.040 0.047 ± 0.028 
 Testes 0.092 ± 0.038 not applicable 
 Whole blood 0.072 ± 0.030 0.036 ± 0.015 
 Carcass 0.036 ± 0.008 0.020 ± 0.009 
Conclusions:
CL-Freetext:
Following single oral dosage, bronopol was extensively absorbed in male and female rats (> 75%) and was rapidly excreted, mainly via urine (>= 70% during the first 24 h), followed by the faeces (10-11% over 168 h); contribution of expired air to the excretion pathways was low (max. 4% during the first 48 h). About 0.5 to 0.9% of radioactive material was bound to tissue after a period of 7 days following dosage; the highest concentrations of radioactive material were found in liver and lung.
Endpoint:
basic toxicokinetics
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted in accordance with GLP.
Objective of study:
metabolism
Qualifier:
according to
Guideline:
EPA OPP 85-1 (Metabolism and Pharmacokinetics)
GLP compliance:
yes
Radiolabelling:
yes
Remarks:
14C (located on C2)
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Source: Charles River UK Ltd., Margate, Kent, UK
Weight at test initiation: At test initiation, the body weights of the rats ranged between 146.5 and 181.2 g.
Route of administration:
oral: gavage
Vehicle:
other: 0.4% hydroxyethylcellulose
Details on exposure:
A single dose of 50 mg/kg of [2-14C] Bronopol was given to 6 male and 5 female Charles River CD rats.
Duration and frequency of treatment / exposure:
Single dose
Dose / conc.:
50 mg/kg bw (total dose)
No. of animals per sex per dose:
Males: 6 Females: 5
Control animals:
no
Details on study design:
In the present study, the excretion and distribution of bronopol in rat were examined over a period of 7 days following a single oral dosage with 50 mg/ kg bw radioactive labelled compound.
The animals received a single oral dose of radiolabelled bronopol as a 5 mg/ml suspension in 0.4% Cellosize in purified water BT .
Specific activity of test substance: 32.0 MBq/g (6.4 GBq/mol)
Volume applied: 1 ml /100 g bw

In a first approach, 5 male rats were given a single oral dose of 200 mg/kg bw of radioactive-labelled bronopol. After 4 hours following dosage, one animal died and the other had to be sacrificed in extremis because of moribundity.
In a second approach, 5 male and 5 female rats were given a single oral dose of 100 mg/kg bw of radioactive-labelled bronopol. Following dosage and up to 48 h, two females and 3 males suffered from respiratory distress; this findings was seen as treatment-related.
In a third approach, 5 male rats were given a single oral dose of 50 mg/kg bw of radioactive-labelled bronopol. One of them showed respiratory distress; necropsy revealed no lesion in the lung, therefore, the distress was brought into relation with an irritant effect of the test substance on the trachea. An additional male rat was used, which however showed similar symptoms as described above and therefore was sacrificed in extremis. Therefore, only 4 males were used for evaluation of absorption, excretion and distribution.
Details on dosing and sampling:
Urine, faeces and respired carbon dioxide were collected at 4, 8, 12, 24, 36, 48, 72, 96, 120, 144 and 168 hrs
after dosing with the exception of carbon dioxide which was collected at 4, 8, 12, 24 and 48 hrs after dosing.
After 168 hours, the rats were sacrificed. Whole blood and plasma, as well as tissues were collected and prepared for determination of radioactivity. The carcasses also were kept for analysis. All samples were stored at –20 °C until analysis.
For air sampling, the expired gases were passed through two CO2 traps containing a mixture of 2-ethoxyethanol:ethanolamine (2:1 v/v). Aliquots were taken from these traps for analysis. The samples were stored at ca. 4 °C until analysis.
Urine and faeces samples were stored at –20 °C until analysis.
The samples were prepared for and subjected to radioactivity measurement by scintillation counting.
Prior to measurement, urine, 2-ethoxyethanol:ethanolamine and plasma samples were mixed with 10 ml of Optiphase “safe” scintillant. Faeces samples were homogenised in distilled water, air-dried and were subjected to oxygen combustion; the combustion products were absorbed in Carbo-sorb E and were mixed with Permafluor E+. Tissue samples were chopped and aliquots were air-dried and subjected to oxygen combustion.
Preliminary studies:
Samples of the test solution were taken immediately prior and after dosing and were subjected to HPLC. The content of bronopol in the test solution was found to be within 10% of nominal value.
Liquid scintillation counting revealed a dose content within 1% of the nominal value.
Details on absorption:
Following single oral dosage with 50 mg/kg bw of radiolabelled bronopol, total recovered radioactive material for the males ranged between 87 and 93% whereas for the females, total recovery ranged between 85 and 108%.
Details on distribution in tissues:
For the males, the mean concentration of radioactive material in whole blood and plasma respectively was 0.420 µg/g and 0.149 µg/g, resulting in a ratio of 2.8. For the females, the mean concentrations respectively were 0.425 µg/g and 0.232 µg/g, resulting in a whole blood to plasma ratio of 1.8
Recovery of radioactivity in tissues and carcass was greater than in plasma. In fact, the highest concentrations of radioactive material were reported for the lung (male: 0.951 µg/g; female: 1.175 µg/g), ), fat (male: 0.990 µg/g; female: 0.834 µg/g) and kidney (male: 0.814 µg/g; female: 0.921 µg/g).
Details on excretion:
Following single oral dosage with 50 mg/kg bw of radiolabelled bronopol, about 68% of radioactive material for males and 79% for females was recovered in the urine over a period of 168 h (i.e. 7 days); about 64 to 75% there from, was excreted during the first 24 hours following dosage. Excretion of radioactive material via faeces was about 14% for males and 12% for females. The percentages recovered in the expired air were about 7% for males and 6% for females.
Metabolites identified:
not measured

Table 1: Single oral dosage of 50 mg/kg bw 14C bronopol, recovery of radiolabelled material in urine, faeces and CO2, and remaining radioactivity in tissues and carcass.

Sample

Percentage of administered radiolabelled material

Total radiolabel (%)

Sex

Animal Number

Urine (including cage washing)

Faeces

CO2 (sum of the highest recovery from each pair of traps)

Tissue & carcass

Male

1

69.3

10.9

8.9

1.1

90.2

2

72.2

11.2

9.1

0.8

93.3

3

58.5

22.2

5.6

0.9

87.2

4

70.5

13.1

6.1

1.0

90.7

Mean

67.6

14.4

7.4

1.0

90.4

+/- SD

+/- 6.2

+/- 5.3

+/- 1.8

+/- 0.1

+/- 2.5

Female

1

72.3

8.1

4.4

0.8

85.6

2

82.4

9.6

4.6

0.8

97.4

3

83.5

9.0

6.5

0.9

99.9

4

64.0

22.7

7.3

0.9

94.9

5

90.6

9.3

8.1

0.6

108.6

Mean

78.6

11.7

6.2

0.8

97.3

+/- SD

+/- 10.4

+/- 6.2

+/- 1.6

+/- 0.1

+/- 8.3

Table 2: Single oral dosage of 50 mg/kg bw 14C bronopol, recovery of radiolabelled material in tissues after 168 h following oral dosage.

Tissue

Concentration of radiolabelled material +/- SD (µg/g of bronopol)

Male

Female

Plasma

0.149 +/- 0.030

0.232 +/- 0.039

Bone

0.761 +/- 0.013

0.861 +/- 0.071

Brain

0.260 +/- 0.076

0.286 +/- 0.043

Fat

0.990 +/- 0.108

0.834 +/- 0.126

Heart

0.613 +/- 0.134

0.708 +/- 0.077

Kidney

0.814 +/- 0.166

0.921 +/- 0.157

Liver

0.665 +/- 0.099

0.690 +/- 0.103

Lung

0.951+/- 0.137

1.175 +/- 0.161

Muscle

0.708 +/- 0.173

0.765 +/- 0.100

Ovary

Not applicable

0.675 +/- 0.157

Spleen

0.628 +/- 0.144

0.757 +/- 0.124

Testes

0.528 +/- 0.133

Not applicable

Whole blood

0.420 +/- 0.106

0.425 +/- 0.119

Carcass

0.337 +/- 0.078

0.269 +/- 0.052

Endpoint:
basic toxicokinetics
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted in accordance with GLP.
Objective of study:
metabolism
Qualifier:
according to
Guideline:
EPA OPP 85-1 (Metabolism and Pharmacokinetics)
GLP compliance:
yes
Radiolabelling:
yes
Remarks:
14C (located on C2)
Species:
rat
Strain:
other: CD (Sprague Dawley derived)
Sex:
male/female
Details on test animals and environmental conditions:
Source: Charles River UK Ltd., Margate, Kent, UK
Route of administration:
oral: gavage
Details on exposure:
In the initial studies the animals either received single oral dosage of 10 or 50 mg/kg bw of radiolabelled test
substance (DT93045, DT93046) or they received 14 consecutive oral dosages of unlabelled bronopol followed
by a last dosage with radiolabelled test substance on the 15th day of treatment (DT93043).
Remarks:
Doses / Concentrations:
Single oral dosage: 10 and 50 mg/kg bw
Repeated oral dosages: 10 mg/kg bw
Details on study design:
The aim of the present study was to identify bronopol metabolites in the urine of rats having received oral dosages of bronopol. For this purpose, urine samples obtained from 3 previous studies dealing with the absorption, excretion and tissue distribution pathways of bronopol in rats following single or repeated administration were used (Boots reports DT93045, DT93046 and DT93043).
Details on dosing and sampling:
In all three initial studies, urine was sampled at following time points: 4, 8, 12, 24, 36, 48, 72, 96, 120, 144 and 168 h after dosing. Urine samples were stored at -20 °C until analysis; the samples were pooled to provide 0 to 24 hour samples for each animal. For the purpose of high pressure liquid chromatography (HPLC) and liquidchromatography - mass spectrometry (LC-MS), 1 ml samples of pooled urine of individual animals were filtered (0.2 µm membrane filter unit) and centrifuged. The supernatant (300 to 400 µl) was transferred to a plasticautosampler vial for HPLC or LC-MS. For the pooled samples from all animals of the same sex and group, urines were pooled after filtration. For thin-layer chromatography (TLC), 10 ml of pooled urine from animals of thesame sex and group were placed into round-bottomed flasks and were freeze-dried overnight. For removal of ureawhich may interfere with the development of the TLC plates, the residues were suspended in and well mixed withacetone; acetone was then discarded. Liquid scintillation counting prior and after acetone wash indicated thatonly minimal radiolabelled material was lost. Following resuspension in methanol and centrifugation, theobtained supernatant was evapored to dryness under nitrogen, and the residue was then dissolved in methanolfor application onto the TLC plates.Urine samples from individual animals were used in HPLC system 1 and LC-MS analysis; pooled samples from animals of the same sex and group were used in HPLC system 2 and the TLC analysisMonitoring of the test substance: Samples of the daily test solution were taken immediately prior and after dosing and were subjected to HPLC. The concentration of bronopol in the dosing formulations ranged between 1.78 and 2.25 mg/ml, resulting a an actual dosage of 8.9 to 10.1 mg/kg bw.
Preliminary studies:
The aim of the present study was to identify bronopol metabolites in the urine of rats having received oral dosages of bronopol. For this purpose, urine samples obtained from 3 previous studies dealing with the absorption, excretion and tissue distribution pathways of bronopol in rats following single or repeated administration were used. In fact, the urine samples were obtained from following three studies:
(1) Fidoe SJ, Hewertson SJ (1993) [14C] Bronopol: Excretion of radiolabelled material and terminal tissue distribution in tissues from male and female Charles River CD rats after administration of a single oral dose of [14C] Bronopol (10 mg/kg). Boots Pharmaceuticals Research Department, Nottingham, UK, Report No: DT93045 (Unpublished)
(2) Garrett AL, Hewertson SJ (1993) [14C] Bronopol: Excretion of radiolabelled material and terminal tissue distribution in tissues from male and female Charles River CD rats after administration of a single oral dose of [14C] Bronopol (50 mg/kg). Boots Pharmaceuticals Research Department, Nottingham, UK, Report No: DT93046 (Unpublished)
(3) Crawley FEH (1993) Bronopol: Repeated oral administration: distribution and excretion in the rat. Pharmaco-LSR Ltd, Eye, Suffolk, England, Report No: 93/BHR006/0075, Unpublished, (sponsor: Boots Company plc, Nottingham, UK, Report No: DT93043)
Metabolites identified:
yes
Details on metabolites:
High pressure liquid chromatography: HPLC revealed two major peaks; one of them was tentatively identified as 2-nitropropan-1,3-diol by comparison of retention times with authentic standard and accounted for a mean of about 50.2% (see table 1, 2). No parent compound was detected. Further detected radioactivity was not resolved into peaks. The HPLC profiles for male urine and female urine following the different dosing regimes were quite similar, excepted for the urine of female treated with a single oral dose of 50 mg/kg bw bronopol. In this case, the concentration of the peak tentatively identified as 2-nitropropan-1,3-diol was lower in measurement than the second, non-identified peak.

Thin-layer chromatography: TLC revealed a single major peak which accounted for a mean of 45% of the total radioactivity in the lanes on the TLC plate. The peak was tentatively identified as 2-nitropropan-1,3-diol by comparison of retention times with authentic standard. Further detected radioactivity was not resolved into peaks. The TLC profiles for male urine and female urine following the different dosing regimes were quite similar, even for the urine of female treated with a single oral dose of 50 mg/kg bw bronopol (see table 3).

Liquid chromatography - mass spectrometry: The urine of two male and two female rats having received repeated doses of 10 mg/kg bw bronopol and which showed elevated concentrations of major metabolite, were selected for LC-MS examination. LC-MS results however showed interference and poor response and could not be used for confirmation of the tentatively identified major metabolite 2-nitropropan-1,3-diol.

Table 1: Relative amount of the major metabolite (% of counts in the region of interest) in HPLC radio-chromatograms of filtered urine from male and female rats treated with single or repeated oral doses of bronopol.

Treatment

Sex

Rat Number

Major metabolite (%; identified as 2-nitropropan-1,3-diol)

Single oral, 10 mg/kg bw

Males

1

54.9

2

16.5

3

51.8

4

62.4

5

45.5

Females

1

60.8

2

23.4

3

30.3

4

47.6

5

63.6

Single oral, 50 m/kg bw

Males

1

75.5

2

76.4

Females

1

4.9

2

11.0

3

12.1

4

13.2

Repeated oral, 10 mg/kg bw

Males

1

64.4

2

68.2

3

66.2

4

67.1

5

52.4

Females

1

64.8

2

80.9

3

61.1

4

65.9

5

64.4

Means +/- SD

50.2 +/- 23.0

Table 2: Relative amount of the major metabolite (% of counts in the region of interest) in HPLC radio-chromatograms of pooled filtered urine from male and female rats treated with single or repeated oral doses of bronopol.

Treatment

Sex

Major metabolite (%; identified as 2-nitropropan-1,3-diol)

Single oral, 10 mg/kg bw

Males

48.1

Females

51.1

Single oral, 50 m/kg bw

Males

63.2

Females

19.3

Repeated oral, 10 mg/kg bw

Males

61.6

Females

57.9

Means +/- SD

50.2 +/- 16.2

Table 3: Relative amount of the major metabolite (% of counts on TLC plates) in TLC radio-chromatograms of pooled filtered urine from male and female rats treated with single or repeated oral doses of bronopol.

Treatment

Sex

Major metabolite (%; identified as 2-nitropropan-1,3-diol)

Single oral, 10 mg/kg bw

Males

52.1

Females

34.9

Single oral, 50 m/kg bw

Males

47.5

Females

25.6

Repeated oral, 10 mg/kg bw

Males

55.1

Females

55.0

Means +/- SD

45.0 +/- 12.1

The aim of the present study was to identify bronopol metabolites in the urine of rats having received oral dosages of bronopol. For this purpose, urine samples obtained from 3 previous studies dealing with the absorption, excretion and tissue distribution pathways of bronopol in rats following single or repeated administration were used (Boots reports DT93045, DT93046 and DT93043).

Test substance: Bronopol (batch No: 889002, from Boots MicroCheck) and [14C] Bronopol (batch 5A, obtained from Boots Pharmaceuticals); purity of bronopol >= 98%, radiochemical purity of [14C] Bronopol > 97% (HPLC)

The test followed US EPA Guideline No. 85-1 and GLP.

In the initial studies the animals either received single oral dosage of 10 or 50 mg/kg bw of radiolabelled test substance (DT93045, DT93046) or they received 14 consecutive oral dosages of unlabelled bronopol followed by a last dosage with radiolabelled test substance on the 15th day of treatment (DT93043). In all three initial studies, urine was sampled at following time points: 4, 8, 12, 24, 36, 48, 72, 96, 120, 144 and 168 h after dosing. Urine samples were stored at –20 °C until analysis; the samples were pooled to provide 0 to 24 hour samples for each animal. For the purpose of high pressure liquid chromatography (HPLC) and liquid chromatography - mass spectrometry (LC-MS), 1 ml samples of pooled urine of individual animals were filtered (0.2 µm membrane filter unit) and centrifuged. The supernatant (300 to 400 µl) was transferred to a plastic autosampler vial for HPLC or LC-MS. For the pooled samples from all animals of the same sex and group, urines were pooled after filtration. For thin-layer chromatography (TLC), 10 ml of pooled urine from animals of the same sex and group were placed into round-bottomed flasks and were freeze-dried overnight. For removal of urea which may interfere with the development of the TLC plates, the residues were suspended in and well mixed with acetone; acetone was then discarded. Liquid scintillation counting prior and after acetone wash indicated that only minimal radiolabelled material was lost. Following resuspension in methanol and centrifugation, the obtained supernatant was evapored to dryness under nitrogen, and the residue was then dissolved in methanol for application onto the TLC plates.

Urine samples from individual animals were used in HPLC system 1 and LC-MS analysis; pooled samples from animals of the same sex and group were used in HPLC system 2 and the TLC analysis

Monitoring of the test substance: Samples of the daily test solution were taken immediately prior and after dosing and were subjected to HPLC. The concentration of bronopol in the dosing formulations ranged between 1.78 and 2.25 mg/ml, resulting a an actual dosage of 8.9 to 10.1 mg/kg bw.

High pressure liquid chromatography: HPLC radiochromatograms revealed two major peaks; one of them was tentatively identified as 2-nitropropan-1,3-diol by comparison of retention times with authentic standard (see representative figure 1) This peak accounted for a mean of about 50.2% (range from 4.9 to 80.9% for all samples taken together). No parent compound was detected. Further detected radioactivity was not resolved into peaks. The HPLC profiles for male urine and female urine following the different dosing regimes were quite similar, excepted for the urine of female treated with a single oral dose of 50 mg/kg bw bronopol. In this case, the concentration of the peak tentatively identified as 2-nitropropan-1,3-diol was lower in measurement than the second, non-identified peak.

Thin-layer chromatography: TLC radiochromatograms revealed a single major peak which accounted for a mean of 45% of the total radioactivity in the lanes on the TLC plate (see representative figure 2). The peak was tentatively identified as 2-nitropropan-1,3-diol by comparison of retention times with authentic standard. Further detected radioactivity was not resolved into peaks. The TLC profiles for male urine and female urine following the different dosing regimes were quite similar, even for the urine of female treated with a single oral dose of 50 mg/kg bw bronopol.

Liquid chromatography - mass spectrometry: The urine of two male and two female rats having received repeated doses of 10 mg/kg bw bronopol and which showed elevated concentrations of major metabolite, were selected for LC-MS examination. LC-MS results however showed interference and poor response and could not be used for confirmation of the tentatively identified major metabolite 2-nitropropan-1,3-diol.

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was conducted prior to implementation of appropriate guideline; GLP was not compulsory at the time the study was conducted. However the methods and results are scientifically acceptable.
Objective of study:
metabolism
Principles of method if other than guideline:
Method: other: no data
GLP compliance:
no
Radiolabelling:
yes
Remarks:
14C (located on C2)
Species:
rat
Strain:
other: CFY
Sex:
male/female
Details on test animals and environmental conditions:
Source: Carworth Europe
Weight: At test initiation, the mean body weight of the rats was about 200 g.
Route of administration:
dermal
Vehicle:
other: acetone
Details on exposure:
Skin areas of the backs of 6 rats were clipped free from hair and 0.05 ml of an acetone solution of [14C] bronopol
(4.8 mg/ml) was applied to the skin; the application sites were maintained under occlusive conditions as
described above. The applied test concentration was 1.2 mg/kg bw; the exposure period was 4 days.
Specific activity of test substance: 21 µCi/mg.
Duration and frequency of treatment / exposure:
4 days
Dose / conc.:
1.2 mg/kg bw (total dose)
Details on study design:
Preliminary test: A preliminary test was conducted for determination of the influence of the vehicle (water, acetone, 9:1 acetone/water mixture) on the dermal absorption of 4 mg/ml [14C] bronopol following skin application. For this purpose, groups of 6 rats each received topical application of 0.05 ml test solution (i.e 1 mg/kg bw of [14C] bronopol in either water, acetone or in an acetone/water mixture) on an area of ca. one cm2. Prior to treatment, the application sites were clipped free of hair. The test material was applied to the skin under occlusive conditions (polythene sheet and “Sleek” adhesive dressing).
Main test: Skin areas of the backs of 6 rats were clipped free from hair and 0.05 ml of an acetone solution of [14C] bronopol (4.8 mg/ml) was applied to the skin; the application sites were maintained under occlusive conditions as described above.
Details on dosing and sampling:
Urine and faeces were sampled at 24 h intervals during 5 days; expired air was conducted through a CO2 trapping system (20% ethanolamine/2-ethoxyethanol solution). The animals were sacrificed at various time point and the amounts of radioactivity in carcass, skin (application site) and dressing were determined. The samples were prepared for and subjected to radioactivity measurement by liquid scintillation analysis. Faeces and minced carcasses and organs were extracted with methanol. Following centrifugation, the radioactivity was measured in the supernatants and the residues. Urine and contents of the expired air traps were mixed with toluene-Triton X-100-based scintillator. Residues were subjected to oxygen combustion; the combustion products were absorbed into a ß-phenylethylamine-based scintillator. Radioactivity in the dressings was extracted with toluene,
and the toluene solutions were mixed with toluene-Triton X-100 scintillator. For monitoring of metabolites, freeze-dried urine samples were extracted with methanol. The extracts were concentrated and applied to thin layer plates for thin layer chromatography TLC. The radiolabelled metabolites were detected by autoradiography. Areas of silica corresponding to zones of radioactivity were removed, divided and eluted with methanol and water. The concentrations of radioactivity in the eluates were measured.
Preliminary studies:
A preliminary test was conducted for determination of the influence of the vehicle
(water, acetone, 9:1 acetone/water mixture) on the dermal absorption of 4 mg/ml [14C] bronopol following skin
application. Groups of 6 rats each received topical application of 0.05 ml test solution (i.e 1 mg/kg bw of
[14C] bronopol in either water, acetone or in an acetone/water mixture) on a clipped skin area of ca. 1 cm2.
The test material was applied to the skin under occlusive conditions. Urine and faeces were sampled from all
animals/group at 24 h intervals during 5 days; the expired air from two animals/group was conducted through a
CO2 trapping system (20% (v/v) ethanolamine/2-ethoxyethanol solution). On the basis of the results of this test,
acetone was retained as solvent for the main test.
Details on absorption:
Main test: Following dermal application, percutaneous absorption of bronopol in rat under occlusive conditions was low.
Details on excretion:
Preliminary test: When acetone was used as solvent, ca. 12% of the initial applied dose of [14C] bronopol was excreted via urine during 5 days; in contrast, when either water or the acetone/water mixture was used, excretion via the urine only amounted for 4% of the initial dose.
When acetone was used as solvent, ca. 12% of the initial applied dose of [14C] bronopol was excreted via urine during 5 days; in contrast, when either water or the acetone/water mixture was used, excretion via the urine only amounted for 4% of the initial dose. These results indicated that (1) the dermal absorption of bronopol was less than the absorption via the gastro-intestinal tract (i.e. following oral administration) and (2) the dermal absorption was higher when acetone as such was used as vehicle instead of water or an acetone/water mixture.
Measurement of radioactivity in the expired air of treated rats revealed a comparatively increased amount of radioactivity trapped in CO2; in fact, amounts of radioactivity in the air were within the range of a third to one amount excreted via the urine. The authors suggested that this finding either may indicate more extensive metabolism of bronopol when absorbed slowly through the skin or may be seen as a result of skin bacterial degradation of bronopol to 14CO2. The latter explanation was seen as the more probable one.
Acetone was selected as solvent for the main test.

About 11% of radioactivity was excreted via the urine, mainly during the first 24 hours following application.
Most of the radioactivity remained on and in the skin at the application site, and only less than 3% of radioactivity was found in the carcass (without the skin application site).
Metabolites identified:
yes
Details on metabolites:
Main test:
Similar as in the oral dosing study, TLC of urine samples from rats having received dermal applications of [14C] bronopol revealed five
metabolites, defined as A, B, C, D and E; metabolite A being the major identified metabolite. No parent compound (i.e. bronopol) was detected. Metabolite A isolated by TLC from rat urine was identified as 2-nitropropane-1,3-diol.

Table 1: Recovery of radioactivity in rats having received dermal applications of [14C] bronopol (main test, table extracted from the report):

% of dose in

Rats sacrificed after (hours)

6

12

24

48

72

96

Mean

Urine (total)

1.83

3.87

4.82

10.75

5.21

11.12

Urine (24 h)

9.05

3.38

7.51

Faeces

0.02

0.07

0.32

0.35

0.27

1.27

Expired air

0.41

0.60

1.76

4.31

3.84

6.53

Carcass (without application site)

2.26

2.73

2.05

2.65

1.70

2.21

Application site (skin)

77.12

88.50

96.40

90.84

78.90

106.24

Skin, methanol extract

50.63

37.80

15.70

12.93

21.71

10.44

Skin, residue

26.49

50.70

80.70

77.91

57.19

95.80

Adhesive dressing

1.72

2.48

1.49

1.88

2.60

0.88

Recovery of radioactivity

83.36

98.25

106.84

110.78

92.52

128.25

103.33 +/- 5.84

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was conducted prior to implementation of appropriate guideline; GLP was not compulsory at the time the study was conducted. However the methods and results are scientifically acceptable.
Objective of study:
metabolism
Principles of method if other than guideline:
Method: other: no data
GLP compliance:
no
Radiolabelling:
yes
Remarks:
14C (located on C2)
Species:
dog
Strain:
Beagle
Sex:
male/female
Details on test animals and environmental conditions:
Weight: 8-10 kg
Route of administration:
other: oral, aqueous solution in gelatine capsules
Vehicle:
other: aqueous solution buffered to pH 5
Duration and frequency of treatment / exposure:
single treatment
Dose / conc.:
1 mg/kg bw (total dose)
No. of animals per sex per dose:
Males: 3 Females: 1
Control animals:
no
Details on study design:
The animals received single oral doses of 1 mg/kg bw of [14C] Bronopol and the test substance was offered as aqueous solution in gelatin capsules.
Specific activity of test substance: 21 µCi/mg.

Measurement of radioactivity:
The samples were prepared for and subjected to radioactivity measurement by liquid scintillation analysis. Faeces and minced carcasses and organs were extracted with methanol. Following centrifugation, the radioactivity was measured in the supernatants and the residues. Urine and plasma samples as well as samples of the solvent extracts were mixed with toluene-Triton X-100-based scintillator according to Patterson MS and Greene RC (Analyt. Chem. 37: 854, 1965).
Residues were subjected to oxygen combustion by means of a modified plastic bag technique (Lewis JD, Internat. J. Appl. Radiot. Isotopes, 23: 39, 1972); the combustion products were absorbed into a ß-phenylethylamine-based scintillator (Dobbs HE, Analyt. Chem. 35: 783, 1963). For measurement of radioactivity, a Philips Liquid Scintillation Analyser was used.
Details on dosing and sampling:
Urine and faeces were collected daily from two animals (one male and one female) during 5 days. Blood samples were collected from the cephalic vein at intervals. Organs were sampled from animals sacrificed after 1.5 and 6 hours post dosing, respectively. All samples were
prepared for and subjected to radioactivity measurement by liquid scintillation analysis.

Plasma samples were extracted twice with 3 volumes of ethyl acetate; the combined acetate portions were evapored under a N2 stream to a small volume and were then applied to thin layer plates for thin layer chromatography (TLC).
Urine samples were freeze-dried and the residues were extracted with methanol. The extracts were concentrated and applied to thin layer plates for TLC.
TLC system, characteristics:
Pre-layered Kieselgel F254 plates, layer thickness of 2 or 0.25 mm;
Developing solvents: chloroform/methanol (4:1 v/v), chloroform/methanol/acetic acid (90:16:8 v/v), butanol: acetic acid: water (2:1:1, v/v)
Detection of 14C-labelled metabolites:
The radiolabelled metabolites were detected by autoradiography using Kodak Kodirex X-ray film. Areas of silica corresponding to zones of radioactivity were removed, divided and eluted with methanol and water. The concentrations of radioactivity in the eluates were measured. The non-radioactive reference compounds were detected by their quenching of the TLC plate fluorescence at 254 nm.
Details on absorption:
Peak plasma concentrations were reached in 0.5 to 2 hrs after dosing and declined with a t1/2 of about 4 hrs. It was estimated that the total plasma radiolabel content accounted for 6 to 9% of the administered dose.
Details on distribution in tissues:
Tissue distribution in the dog revealed highest concentrations in plasma with the exception of kidney at 1.5 and 6 hrs after dosing. Little variation between organs was seen, with highest values in kidneys and lowest in fatty tissue (see table 1).
Details on excretion:
Following oral administration of radiolabelled bronopol to Beagle dogs, mean recovery of radioactivity in urine reached 81% of the applied amount after 5 days, with up to 64% having been excreted during the first 12 hours following dosing. Excretion via the faeces reached about 3% of the initial radioactivity after 5 days; some radioactivity probably was excreted as 14CO2, expired air however was not monitored for radioactivity content.
The findings reported above indicate rapid absorption and excretion, mainly via urine.
Toxicokinetic parameters:
half-life 1st: 4h
Metabolites identified:
yes
Details on metabolites:
Five metabolites were identified in urine, which were more polar than Bronopol. Bronopol as such was not detected. Metabolite A accounted for more than 40% (see table 2). The metabolite had been identified in the urine of rat as as 2-nitropropane-1,3-diol.

Table 1: Distribution of radioactivity in dogs following oral dosing with 1 mg/kg bw of 14C-bronopol (extract from the appendix in the report):

Percentage of dose in

Dog sacrificed after

1.5 hours

6 hours

Total

Per g of tissue

Total

Per g of tissue

Brain

0.439

0.00533

0.317

0.00422

Eyes

0.0573

0.00587

0.0493

0.00511

Fatty tissue*

4.28

0.00340

1.03

0.00082

Heart

0.387

0.00518

0.245

0.00333

Kidneys

0.930

0.0168

0.549

0.00910

Liver

3.10

0.00717

2.00

0.00427

Lungs

0.471

0.00594

0.306

0.00325

Muscle

27.06

0.00771

12.18

0.00347

Testes

0.0338

0.00751

0.0185

0.00405

Plasma*

4.46

0.0099

2.52

0.0056

Urine

22.04

-

31.57

-

Faeces

0

-

0.623

-

*, Assuming these represent 14%, 39% and 5% of bw respectively (referring to 9kg bw)

Table 2: Metabolites identified in the urine of dogs following oral dosing with 1 mg/kg bw of 14C-bronopol (extract from table 7 in the report):

Compound

Rf values in solvent systems*

Mean % of dose in the urine of dogs (0 - 24 h)

Ch/Me

Ch/Me/AA

But/AA/W

Metabolites

A

0.47

0.41

0.61

43.7

B

0.03

0.13

0.55

9.7

C

0.00

0.04

0.43

D

0.00

0.00

0.34

11.3

E

0.00

0.00

0.22

Parent compound

Bronopol

0.64

0.62

0.72

None detected

*, Solvent system Ch/Me = chloroform/methanol (4:1 v/v); Ch/Me/AA = chloroform/methanol/acetic acid (90:16:8 v/v); But/AA/W = butanol: acetic acid: water (2:1:1, v/v)

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was conducted prior to implementation of appropriate guideline; GLP was not compulsory at the time the study was conducted. However the methods and results are scientifically acceptable.
Objective of study:
metabolism
Principles of method if other than guideline:
Method: other: no data
GLP compliance:
no
Radiolabelling:
yes
Remarks:
14C (located on C2)
Species:
rabbit
Strain:
New Zealand White
Sex:
male/female
Details on test animals and environmental conditions:
Weight: The rabbits had a mean body weight of 3 kg.
Route of administration:
dermal
Vehicle:
other: acetone
Details on exposure:
Skin areas of the backs of 4 New Zealand White rabbits were clipped free from hair. 0.2 to 0.4 ml of an acetone solution of [14C] bronopol (4.8 mg/ml) was applied to the skin under occlusive conditions. The applied test concentration of bronopol was about 0.72 mg/kg bw; the treated skin area was ca. 10 cm2. The exposure period was 4 days.
Specific activity of test substance: 21 µCi/mg

Duration and frequency of treatment / exposure:
4 days
Dose / conc.:
0.72 mg/kg bw (total dose)
No. of animals per sex per dose:
Males: 2 Females: 2
Control animals:
no
Details on study design:
Measurement of radioactivity:
The samples were prepared for and subjected to radioactivity measurement by liquid scintillation analysis.
Faeces and minced carcasses and organs were extracted with methanol. Following centrifugation, the radioactivity was measured in the supernatants and the residues.
Urine, plasma and contents of the expired air traps were mixed with toluene-Triton X-100-based scintillator according to Patterson MS and Greene RC (Analyt. Chem. 37: 854, 1965).
Residues were subjected to oxygen combustion by means of a modified plastic bag technique (Lewis JD, Internat. J. Appl. Radiot. Isotopes, 23: 39, 1972); the combustion products were absorbed into a ß-phenylethylamine-based scintillator (Dobbs HE, Analyt. Chem. 35: 783, 1963).
Radioactivity contained in the dressings was extracted with toluene, and the toluene solutions were mixed with toluene-Triton X-100 scintillator.
For measurement of radioactivity, a Philips Liquid Scintillation Analyser was used.
Microautoradiography:
Pieces of treated and untreated skin (10 mm x 5 mm) were taken from the rabbits immediately after sacrifice. The skin samples were quenched in isopentane precooled with liquid N2 (-196 °C) and then were sectioned (8 µm-sections) in a cryostat at – 30 °C. The sections were picked up onto Kodak AR-10 stripping film, and were mounted on cold coverslips under darkroom conditions. Further sections were picked up on Ilford G.S liquid emulsion. The sections were exposed at –30 °C for 7 to 14 days, fixed in acid/alcohol (5%), developed and mounted in DPX for photography.
Metabolite monitoring in urin:
Urine samples were freeze-dried and the residues were extracted with methanol. The extracts were concentrated and applied to thin layer plates for TLC.
TLC system, characteristics:
Pre-layered Kieselgel F254 plates, layer thickness of 2 or 0.25 mm;
Developing solvents: chloroform/methanol (4:1 v/v), chloroform/methanol/acetic acid (90:16:8 v/v), butanol: acetic acid: water (2:1:1, v/v)
Detection of 14C-labelled metabolites:
The radiolabelled metabolites were detected by autoradiography using Kodak Kodirex X-ray film. Areas of silica corresponding to zones of radioactivity were removed, divided and eluted with methanol and water. The concentrations of radioactivity in the eluates were measured. The non-radioactive reference compounds were detected by their quenching of the TLC plate fluorescence at 254 nm.
Details on dosing and sampling:
Urine, faeces, carcass, skin, plasma, dressings, organs and tissues were sampled. Urine and faeces were
sampled at 24 h intervals during the exposure period whereas the various organs of the rabbits were dissected,
prepared for and subjected to radioactivity measurement following sacrifice of the animals at the end of the test
period. The samples were prepared for and subjected to radioactivity measurement by liquid scintillation analysis.
Faeces and minced carcasses and organs were extracted with methanol and the radioactivity was measured in the
supernatants and the residues. Urine, plasma and contents of the expired air traps were mixed with toluene-Triton
X-100-based scintillator. Residues were subjected to oxygen combustion and the combustion products were absorbed
into a ß-phenylethylamine-based scintillator. Radioactivity contained in the dressings was extracted with
toluene, and the toluene solutions were mixed with toluene-Triton X-100 scintillator.For microautoradiography,
pieces of treated and untreated skin (10 mm x 5 mm) were taken from the rabbits immediately after sacrifice.
The skin samples were quenched in isopentane precooled with liquid N2 (-196 °C) and were then sectioned
(8 µm sections) in a cryostat at - 30 °C. The sections were picked up onto Kodak AR-10 stripping film,
and were mounted on cold coverslips under darkroom conditions. Further sections were picked up on Ilford G.S
liquid emulsion. The sections were exposed at -30 °C for 7 to 14 days, fixed in acid/alcohol (5%), developed
and mounted in DPX for photography. For metabolite monitoring, freeze-dried urine samples were extracted with
methanol. The extracts were concentrated and applied to thin layer plates for thin layer chromatography TLC.
The radiolabelled metabolites were detected by autoradiography. Areas of silica corresponding to zones of
radioactivity were removed, divided and eluted with methanol and water. The concentrations of radioactivity in
the eluates were measured.
Preliminary studies:
An additional study was conducted in order to investigate the restriction of penetration of radioactive labelled bronopol within the areas surrounding hair follicles observed in rabbits. For this purpose, rats received dermal application of radioactive labelled bronopol dissolved in water or acetone on their shaved back. Furthermore, quantities of rat fur were treated in vitro with the same test substance. A significant proportion of radioactivity remained in and on the skin, especially after longer application duration. No significant binding of radioactivity to the fur alone could be evidenced.
Details on absorption:
Most of the applied radioactivity remained within the skin application site. Recovery in the carcass did not exceed 5% of the applied amount of radioactivity (see table 1). The highest concentrations of radioactivity were found in the kidney. The tissue to plasma concentration ratios were < 1 except for liver, lungs and kidney (i.e. excretion organs); moreover, no specific localisation of the radioactive material could be evidenced.The comparison of the one rabbit which had its fur clipped and received an additional epilation, with the remaining, only clipped rabbits revealed a more increased dermal absorption of [14C] bronopol for the first animal, with increased urinary excretion. This animal furthermore showed the highest plasma concentrations of radioactivity.Microautoradiography of skin samples revealed presence of radioactivity within the epidermis, with superficial penetration being restricted to the areas surrounding hair follicles. No radioactivity could be evidenced in the dermis, muscle layers or in untreated skin areas.
Metabolites identified:
yes
Details on metabolites:
TLC of urine revealed five metabolites, defined as A, B, C, D and E; metabolite A being the
major identified metabolite. No parent compound (i.e. bronopol) was detected. metabolite A isolated by TLC from
rat urine was identified as 2-nitropropane-1,3-diol.Percutaneous absorption of bronopol in rabbit was similar
to that seen in rats, i.e., percutaneous absorption of bronopol in rabbit under occlusive conditions rather was
low.

Table 1: Distribution of radioactivity after application of [14C]-bronopol to the shaved backs of rabbits.

Percentage of applied radioactivity found  Animals killed at following times after application                     
  Rabbit 1   6 hours Rabbit 2   12 hours Rabbit 3   24 hours Rabbit 4   48 hours *
   Total  per g Total  per g  Total  per g  Total  per g 
 Brain  0.0066 0.00080  0.0146  0.00173  0.0062  0.00079  0.0078  0.00097 
 Eyes  0.0042 0.00094  0.0087  0.00185  0.0038  0.00081  0.0028  0.00066 
 Fatty tissue +  0.143 0.00034  0.315  0.00075  0.080  0.00019  1.092  0.0026 
 Gastrointestinal tract  0.51   1.40    0.84    2.89   
 Heart  0.0049 0.00089  0.0150  0.00183  0.0099  0.00097  0.0160  0.0024 
 Kidneys  0.049 0.0024  0.079  0.0031  0.032  0.0016 3  0.098  0.0059 
 Liver  0.156 0.00138  0.563  0.00344  0.286  0.00153  0.459  0.0016 
 Lungs  0.0140 0.0010  0.046  0.0021  0.020  0.00118  0.072  0.0010 
 Muscle +  1.111 0.00095  2.118  0.00181  0.737  0.00063  1.474  0.00126 
 Ovaries  - 0.00025  0.00138  0.0027  0.0044 
 Plasma +  0.176 0.00117  0.474  0.00316  0.197  0.00131  0.413  0.00275 
 Skin  - 0.00066  0.00118  0.00133  - 0.0042 
 Testes  0.0092 0.00096  0.0050  0.00068 
 Urine  0.27 3.92  6.62  25.37 
 Faeces  0.0057 0.244  0.256  0.819 
 Skin of site of application**  61.24 36.91  48.50  59.60 
 Adhesive dressing ?  11.54 6.22  9.16  2.62 
 Rest of organs  2.01 0.00115  4.06  0.0023  1.94  0.0010  2.86  0.00217 

* Since considerable amounts of fur were left on this animal´s back by the hair clippers, the appropriate area of skin was treated with depilating cream which was removed before applicationof [14C]-bronopol solution. ** Not including skin taken for autoradiography + Assuming these represent 14%, 39% and 5% of bodyweight (3 kg) respectively

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was conducted prior to implementation of appropriate guideline; GLP was not compulsory at the time the study was conducted. However the methods and results are scientifically acceptable.
Objective of study:
metabolism
Principles of method if other than guideline:
Method: other: no data
GLP compliance:
no
Radiolabelling:
yes
Remarks:
14C (located on C2)
Species:
rat
Strain:
other: CFY
Sex:
male/female
Details on test animals and environmental conditions:
Source: Carworth Europe
Weight: At test initiation, the mean body weight of the rats was about 200 g.
Route of administration:
oral: gavage
Vehicle:
other: aqueous solution buffered to pH 5
Dose / conc.:
1 mg/kg bw (total dose)
No. of animals per sex per dose:
Three males and three females for excretion study
Three males for plasma concentration measurements
One male and one female for biliary excretion study
6 males for whole body autoradiographic study
Details on study design:
For metabolism studies single doses of 1 mg/kg bw of [14C]Bronopol was given to 6 male and female rats of the CFY strain. Urine and faeces were collected every 24 hrs after dosing. Expired carbon dioxide was collected. The animals were killed after 5 days.
For the plasma concentration measurements 3 male rats received the same oral dose. Blood samples were taken from the tail vein. Biliary excretion was studied in 1 male and 1 female rat with cannulated bile ducts at the same oral dose. Bile, urine and faeces were collected for 48 hrs. For whole-body autoradiographic studies male rats were used which received the same oral dose.

Measurement of radioactivity:
The samples were prepared for and subjected to radioactivity measurement by liquid scintillation analysis. Faeces and minced carcasses and organs were extracted with methanol. Following centrifugation, the radioactivity was measured in the supernatants and the residues. Urine, plasma, solvent extracts, contents of the expired air traps as well as cage washings were mixed with toluene-Triton X-100-based scintillator according to Patterson MS and Greene RC (Analyt. Chem. 37: 854, 1965). Residues were subjected to oxygen combustion by means of a modified plastic bag technique (Lewis JD, Internat. J. Appl. Radiot. Isotopes, 23: 39, 1972); the combustion products were absorbed into a ß-phenylethylamine-based scintillator (Dobbs HE, Analyt. Chem. 35: 783, 1963). For measurement of radioactivity, a Philips Liquid Scintillation Analyser was used.

Metabolite monitoring in plasma and urine:
Plasma samples were extracted twice with 3 volumes of ethyl acetate; the combined acetate portions were evapored under a N2 stream to a small volume and were then applied to thin layer plates for thin layer chromatography (TLC). Urine samples were freeze-dried and the residues were extracted with methanol. The extracts were concentrated and applied to thin layer plates for TLC.
TLC system, characteristics:
Pre-layered Kieselgel F254 plates, layer thickness of 2 or 0.25 mm;
Developing solvents: chloroform/methanol (4:1 v/v), chloroform/methanol/acetic acid (90:16:8 v/v), butanol: acetic acid: water (2:1:1, v/v)

Detection of 14C-labelled metabolites:
The radiolabelled metabolites were detected by autoradiography using Kodak Kodirex X-ray film. Areas of silica corresponding to zones of radioactivity were removed, divided and eluted with methanol and water. The concentrations of radioactivity in the eluates were measured. The non-radioactive reference compounds were detected by their quenching of the TLC plate fluorescence at 254 n.m.

Enzyme treatment:
For identification of glucuronides or sulphates, urine extracts were treated with a glucuronidase/sulphatase mixture (at 37 °C for 18 hours) after evaporation to dryness and dissolution in acetate buffer (pH 5); the samples were then compared by chromatography with untreated extracts.
Mass spectrometry and infra-red spectrometry:
Mass spectrometry and infra red spectrometry were used for additional characterisation and/or confirmation of the identity of detected metabolites.
Details on dosing and sampling:
Excretion study:
Urine and faeces were collected at 24 h intervals during 5 days. Expired air was conducted throughout a CO2 trapping system consisting of a 20% (v/v) ethanolamine/2-ethoxyethanol solution. After 5 days, the rats of the excretion study were sacrificed and the carcasses were stored at –20°C.

Plasma concentration measurement:
Three males were treated as described in 3.3.1 and blood samples were collected from the tail veins at intervals. The plasma was separated from the whole blood by centrifugation. An aliquot of plasma was counted for radioactivity and a portion was extracted with ethyl acetate.

Bile excretion study:
Prior to gavage, the animals designed for bile excretion study (i.e one male and one female rat) were anesthetized and cannulated (bile ducts); they were subjected to gavage immediately after recovery. Bile , urine and faeces were collected for 48 hours.

Whole-body auroradiographic study:
Following gavage with 1 mg/kg bw [14C] bronopol (5 µCi), the animals were sacrificed at defined intervals and were the rapidly frozen in an acetone/solid CO2 mixture at –70°C. The frozen animals were prepared and embedded for microtome sectioning; sagittal, 20 µm sections were cut, which were then freeze-dried and placed on Kodak Kodirex X-ray films. The autoradiographs were developed after 19 days of exposure. One animal was considered at each time interval.
Details on absorption:
In two rats, the peak plasma concentrations of radioactivity of 0.2 to 0.3% per ml plasma were reached within 2 hours following dosing, whereas in the third rat, the peak of 0.3% already was reached after 30 minutes. For all three animals, the peak was followed by a tenfold decrease during the first 24 hour post dosing (0.03% per ml plasma). The initial half-life ranged between 3 and 5 hours.
Considering a plasma volume for the rats of about 5% of bodyweight, the peak concentrations of radioactivity in the total plasma would then amount for about 2.5 to 3.1% of the initial radioactivity.
Details on distribution in tissues:
Whole-body autoradiography of rats showed that radioactivity was rapidly absorbed within 15 to 60 minutes, and revealed no persistence of bronopol and/or metabolites in the organs (see table 1).
Details on excretion:
Following oral administration of radiolabelled bronopol to rats, mean recovery of radioactivity in urine reached 80% of the applied amount within the first day following dosing. Excretion via the faeces reached about 5% within two days following dosing, and about 6.3% of the initial radioactivity was recovered in the expired air (14CO2) sampled during the first two days. The recovered percentages of radioactivity within 24 to 48 h following dosing indicate rapid absorption and rapid elimination, mainly via the urine , during the first 24 hours.
The percentage of radioactivity excreted via the urine remained elevated throughout the post dosing period, and 83.3% was reached measured after 5 days. Excretion via the faeces remained stable throughout the 5 days post dosing (5.8%), and recovery in the expired air (14CO2) reached about 8.4%. Carcass analysis revealed a retention of 1.6% of the initial radioactivity.
There were no differences between males and females.
Examination of the biliary excretion revealed a recovery of up to 7% of initial radioactivity in the bile; the main recovery pf radioactivity was found in the urine of the cannulated rats (about 80% reached after 48 hours following dosing). As evidenced in figure 2, there was no major sex-related difference. The present results confirmed the complete absorption of bronopol administered by gavage to the rats, as well as the main excretion via the urine; furthermore, recovery of radioactivity in the bile was in accordance with recovery in the faeces, indicating that the radioactivity measured in the faeces had been secreted through the bile.
Rate and extent of excretion was similar in both male and female rats. In animals with cannulated bile ducts up to 7% of the radiolabel was excreted in the bile and up to 80.5% in the urine during 48 hrs.
Toxicokinetic parameters:
half-life 1st: 3-5 hrs
Metabolites identified:
yes
Details on metabolites:
In the 24 h urine of the rats having received oral doses of 1 mg/kg bw 14C bronopol, TLC revealed five metabolites, defined as A, B, C, D and E; metabolite A being the major identified metabolite (see table 2). Metabolite A also was detected in the plasma of the dosed rats, where it was the only extractable radioactive substance; metabolite A accounted for 45 to 66% of the plasma radioactivity up to 4 hours following dosing; thereafter, the amount decreased to about 10 – 12% after 24 hours. No parent compound (i.e. bronopol) was detected. By means of the infra-red spectrometry, metabolite A isolated by TLC from rat urine was identified as 2-nitropropane-1,3-diol as both, metabolite A and the reference standard 2-nitropropane-1,3-diol showed similar chromatographic properties in the three solvent systems used. This was further confirmed by the mass spectrometry analysis.
The remaining metabolites identified in the urine of the rats could not be characterized. However, the treatment of urine extracts with the glucuronidase/sulphatase mixture (37 °C, 18 hours) and the comparative chromatography revealed that the metabolites neither were glucuronides nor sulphates.

Table 1: Distribution of radioactivity in rats following oral dosing with 1 mg/kg bw of 14C-bronopol (extract from the appendix in the report).

Time

Relative concentrations of radioactivity in the tissues

High

Medium

Low

15 min.

Stomach, kidney (medulla), small intestine

Large intestine

General

1 h

Stomach, small intestine, bladder (wall)

Kidney

Brain, spinal cord, general

4 h

Stomach, large intestine, penis, fur

Small intestine

Brain, spinal cord

8 h

Large intestine

Small intestine

24 h

-

-

-

72 h

-

-

Trace (large intestine ?)

Table 2: Metabolites identified in the urine of rats following oral dosing with 1 mg/kg bw of 14C-bronopol (extract from table 7 in the report).

Compound

Rf values in solvent systems*

Mean % of dose in the urine of rats

(0 - 24 h)

Ch/Me

Ch/Me/AA

But/AA/W

Metabolites

A

0.47

0.41

0.61

42.6 +/- 2.2

B

0.03

0.13

0.55

11.7 +/- 0.8

C

0.00

0.04

0.43

4.2

D

0.00

0.00

0.34

2.7

E

0.00

0.00

0.22

3.0

Parent compound

Bronopol

0.64

0.62

0.72

None detected

*, Solvent system Ch/Me = chloroform/methanol (4:1 v/v); Ch/Me/AA = chloroform/methanol/acetic acid (90:16:8 v/v); But/AA/W = butanol: acetic acid: water (2:1:1, v/v)

Endpoint:
basic toxicokinetics
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted in accordance with GLP.
Objective of study:
metabolism
Qualifier:
according to
Guideline:
EPA OPP 85-1 (Metabolism and Pharmacokinetics)
GLP compliance:
yes
Radiolabelling:
yes
Remarks:
14C (located on C2)
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Source: Charles River UK Ltd., Margate, Kent, UK
Weight at study initiation: At test initiation, the body weights of the rats ranged between 204 and 239 g.

Route of administration:
oral: gavage
Vehicle:
not specified
Duration and frequency of treatment / exposure:
15 days
Remarks:
Doses / Concentrations:
Males: Single doses 10 and 50 mg/kg, repeated dose 10 mg/kg for 15 days Females: Single doses 10 and 50 mg/kg, repeated dose 10 mg/kg for 15 days
No. of animals per sex per dose:
Males: 14 Females: 15
Details on dosing and sampling:
Administration of the test substance: The animals received single oral dose of 10 mg/kg bw non-radiolabelled bronopol on 14 consecutive days. The 15th dosage consisted of 10 mg/kg bw 14C-labelled bronopol; prior to this last dosage, food was withdrawn overnight and was restored after 4 hours following dosage.
Dosage : 10 mg/kg bw (as 2mg/ml suspension in aqueous 0.4% Cellosize)
Specific activity of test substance: 170.2 MBq/g
Volume applied: 5 ml /kg bw
Five dosed animals per sex were used for sampling. Urine, faeces and respired carbon dioxide were collected
at 4, 8, 12, 24, 36 (excepted for expired air), 48, 72, 96, 120, 144 and 168 hrs after last dosing.
For air sampling, the expired gases were passed through two CO2 traps containing a sodium hydroxide solution (2.0 M, 200 ml). Aliquots were taken from these traps for analysis. The samples were stored at ca. 4 °C until analysis.
Urine and faeces samples were stored at –20 °C until analysis
After 168 hours, the rats were sacrificed. Whole blood and plasma, as well as tissues were collected and prepared for determination of radioactivity. The carcasses also were kept for analysis. All samples were stored at –20 °C until analysis.
All samples were assayed for 14C-radioactivity by liquid scintillation counting.
Prior to measurement, urine, plasma, sodium hydroxide and cage washing samples were mixed with scintillant. Faeces samples were homogenised in distilled water, air-dried and were subjected to oxygen combustion; the combustion products were absorbed in Carbo-sorb E and were mixed with Perma-fluor V scintillation fluid. Large organs and tissued were mixed with water and were homogenised; this was followed by digestion in Soluene-350 and, where necessary, by discolouration using hydrogen peroxide. Small tissues as well as samples of skin and fat were digested in total. Dried bones were crushed and digested in formic acid.
Preliminary studies:
Samples of the daily test solution were taken immediately prior and after dosing and were subjected to HPLC. The concentration of bronopol in the dosing formulations ranged between 1.78 and 2.25 mg/ml, resulting a an actual dosage of 8.9 to 10.1 mg/kg bw.
Liquid scintillation counting revealed a dose content within 1 % of the nominal value.
Details on distribution in tissues:
The comparison of the concentrations of 14C respectively in whole blood and in plasma resulted in a whole blood to plasma ratio of 1.5, indicating the uptake of 14C in blood cells.
Tissue concentrations of 14C after 168 hours following last dosage ranged between 173 and 531 ng eq/g in males and between 157 and 434 ng eg/g in females. The highest concentrations for both sexes were found in skin, followed by bones and gonads. The tissue concentrations indicated that small parts of the 14C persisted in the tissues.
Details on excretion:
Urine: At the end of the 15 days test period which consisted of 14 consecutive oral dosages with 10 mg/kg of unlabelled bronopol and one last dosage (i.e. 15th dosage) with 14C-bronopol, about 76% and 67% of the administered 14C was excreted via urine over 7 days, for the males and females respectively. Therefrom, about 90% was excreted during the first 12 hours following the last dosage; a half-life of 3 hours was determined.
Faeces: About 3 % of the 14C was excreted via the faeces for both, males and females.
Expired air: About 9 % of the 14C was recovered in CO2 for both, males and females.
Metabolites identified:
not measured

Description of key information

KINETICS AND METABOLISM OF BRONOPOL

The pharmacokinetic profile and metabolism of Bronopol were studied in different species after single and repeated oral or dermal administration. The available studies in rat demonstrate that Bronopol is rapidly and quantitatively absorbed per os. Bronopol has been reported to be metabolized in rodents, rabbits and dogs. Reports also suggested that Bronopol is preferentially distributed to the kidney and excreted via urine. Recovered percentages of radioactivity within 24 to 48 hours following dosing indicate rapid absorption and rapid elimination of Bronopol, mainly via the urine, during the first 24 hours. As major metabolite in urine 2-nitropropane-1,3-diol has been identified. Thus, no accumulation potential has been identified for Bronopol by oral or dermal application in animals. Following dermal application, percutaneous absorption of Bronopol in rat and rabbits under occlusive conditions was low. Human absorption data indicates that the percutaneous absorption of Bronopol is also low. Only about 2 - 5 % of the available dose of radioactivity was detected in the urine of volunteers and no radioactivity was found in the faeces following dermal application. On the contrary, recovery of applied Bronopol ranged between 72 % (males) and 80 % (females) after washing of the dermal application site after an exposure period of 8 hours.

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential

Additional information

Absorption

Toxicity experiments on rodents have shown that Bronopol is absorbed after oral and dermal exposure (cf. Boots Pharmaceuticals Research Department, 1993/DT93045; Boots Pharmaceuticals Research Department, 1993/DT93046; Huntingdon Research Centre, 1974; The Dow Chemical Company and LANXESS K-081547-045, 2007).

 

Distribution

The distribution of 14C-labelled Bronopol was determined in dogs (Huntingdon Research Centre, 1974). The animals received single oral doses of 1 mg/kg bw of [14C] Bronopol. Peak plasma concentrations were reached in 0.5 to 2 hours after dosing and declined with a t1/2 of about 4 hours. It was estimated that the total plasma radiolabel content accounted for 6 to 9% of the administered dose. Tissue distribution in the dog revealed highest concentrations in plasma with the exception of kidney at 1.5 and 6 hours after dosing. Little variation between organs was seen, with highest values in kidneys and lowest in fatty tissue.

The distribution of 14 C-labelled Bronopol to tissues was determined in rats after single application of 1 mg/kg bw at TCmax in blood (2 h) and TCmax/2 (6 h) (The Dow Chemical Company and LANXESS K-081547-045, 2007).

Bronopol was widely distributed with highest levels detected in liver and kidney (involved in absorption and excretion) and lowest levels detected in fat. The total tissue residues at TCmax/2 including the carcass and gastrointestinal tract (about 29% of absorbed dose) were about half of the values at TCmax, (~61% of dose) revealing an equally fast elimination from tissues than from blood. Residue values in the gastrointestinal tract (16% and 8.6% at 2 and 6 hours after application, respectively) confirmed that a bioavailability >80% can be considered for Bronopol.

 

 

Metabolism

Bronopol is eliminated mainly in a metabolized form in the urine of the rat, mouse, dog and rabbit (Boots Pharmaceuticals Research Department, 1993/DT93063; Boots Pharmaceuticals Research Department, 1993/DT 93046; Huntingdon Research Centre, 1974). Furthermore, the occurrence of the bromide ion (as metabolite of Bronopol) was investigated in rat (Boots Pharmaceuticals Research Department, 1993/DT93018).

 

 

Elimination and excretion

Expired air:

Following oral application in rats, the contribution of expired air to the excretion pathways was low (max. 8.4 %) during 5 days (Boots Pharmaceuticals Research Department, 1993/DT93045). In a metabolism study (Boots Pharmaceuticals Research Department, 1993/DT93046) the percentages recovered in the expired air were about 7 % for males and 6 % for females over a period of 7 days. Following repeated oral administration of 10 mg/kg bw Bronopol in rat for 14 consecutive days, followed by a single dosage with 10 mg/kg bw of [14C] – Bronopol on the 15th day (guideline study, Pharmaco-LSR Ltd. 1993), about 9 % of the 14C was recovered in CO2 for both, males and females.

Urine:

Following repeated oral administration of 10 mg/kg bw Bronopol in rat for 14 consecutive days, followed by a single dosage with 10 mg/kg bw of [14C] – Bronopol on the 15th day, 14C was found to be extensively absorbed and rapidly excreted, mainly via the urine. In fact, more than 90 % of the 14C was absorbed and a half-life of ca. 3 hours was determined (Pharmaco-LSR Ltd, 1993). Bronopol was rapidly absorbed after oral administration to rat, and peak plasma concentrations were reached 2 hours, with an initial half life ranging between 3 and 5 hours. Plasma concentrations decreased during 24 hours following dosing. Bronopol was rapidly eliminated and the major excretion pathway was the urine with more than 80% of the applied dose being excreted within 24 hours following treatment (Huntingdon Research Centre, 1974).

Faeces:

Following oral administration, biliary excretion of Bronopol in rats amounted for 7 % of the initial dose and was quite similar to the excreted amount of Bronopol via faeces (about 6 %), indicating that the radioactivity measured in the faeces had been secreted through the bile (Huntingdon Research Centre, 1974). According to Boots Pharmaceuticals Research Department (1993/DT93045), 10 - 11 % of the initial oral dose was excreted via faeces over a period of 168 hours.

 

 

Retention and turnover

The distribution of 14C-labelled Bronopol was determined in rats (Huntingdon Research Centre, 1974). The animals received single oral doses of 1 mg/kg bw of [14C] Bronopol. Urine and faeces were collected every 24 hours after dosing. Expired carbon dioxide was collected. The animals were killed after 5 days. Following oral administration of radiolabelled Bronopol to rats, mean recovery of radioactivity in urine reached 80 % of the applied amount within the first day following dosing. Excretion via the faeces reached about 5 % within two days following dosing, and about 6.3% of the initial radioactivity was recovered in the expired air (14CO2) sampled during the first two days. The recovered percentages of radioactivity within 24 to 48 hours following dosing indicate rapid absorption and rapid elimination, mainly via the urine, during the first 24 hours. The percentage of radioactivity excreted via the urine remained elevated throughout the post dosing period, and 83.3 % was reached measured after 5 days. Excretion via the faeces remained stable throughout the 5 days post dosing (5.8%), and recovery in the expired air (14CO2) reached about 8.4 %.Carcass analysis revealed a retention of 1.6 % of the initial radioactivity. There were no differences between males and females.

 

 

 

References / Conclusions

Boots Pharmaceuticals Research Department (1993) [14C] Bronopol: Excretion of radiolabelled material and terminal tissue distribution in tissues from male and female Charles River CD rats after administration of a single oral dose of [14C] Bronopol (10 mg/kg). Boots Pharmaceuticals Research Department, Nottingham, UK, Report No: DT93045;

Following single oral dosage, Bronopol was extensively absorbed in male and female rats (> 75%) and was rapidly excreted, mainlyviaurine (>= 70% during the first 24 hours), followed by the faeces (10-11% over 168 hours); contribution of expired air to the excretion pathways was low (max. 4% during the first 48 hours). About 0.5 to 0.9% of radioactive material was bound to tissue after a period of 7 days following dosage; the highest concentrations of radioactive material were found in liver and lung.

 

 

Boots Pharmaceuticals Research Department (1993) [14C] Bronopol: Excretion of radiolabelled material and terminal tissue distribution in tissues from male and female Charles River CD rats after administration of a single oral dose of [14C] Bronopol (50 mg/kg). Boots Pharmaceuticals Research Department, Nottingham, UK, Report No: DT93046 (Unpublished);

Following single oral dosage, Bronopol was extensively absorbed in male and female rats (> 76%) and was rapidly excreted, mainly via urine (> 60% during the first 24 hours), followed by the faeces (12-14% over 168 h); contribution of expired air to the excretion pathways was low (max. 7.5% during the first 48 hours). About 0.8 to 1.0% of radioactive material was bound to tissue after a period of 7 days following dosage; the highest concentrations of radioactive material were found in lung, fat and kidney.

 

 

Boots Pharmaceuticals Research Department (1993) An investigation of the metabolites of bronopol in urine from male and female CD rats after administration of a single oral dose of [14C] bronopol (10 or 50 mg/kg ) or fourteen repeated daily doses of bronopol followed by a single dose of [14C] bronopol (10 mg/kg). Boots Pharmaceuticals Research Department, Nottingham, UK, Report No: DT93063, (Unpublished);In the urine samples obtained from rats of both sexes, having either received single oral doses of Bronopol (10, 50 mg/kg bw) or having received repeated doses of bronopol over a 15 days period (10 mg/kg bw), 2-nitropropan-1,4-diol was tentatively identified as major metabolite; no parent compound could be evidenced.

 

 

Pharmaco-LSR Ltd (1993) Bronopol: Repeated oral administration: distribution and excretion in the rat. Pharmaco-LSR Ltd, Eye, Suffolk, England, Report No: 93/BHR006/0075, Unpublished, Sponsor: Boots Company plc, Nottingham, UK, Report No: DT93043;

Following repeated oral administration of 10 mg/kg bw Bronopol in rat for 14 consecutive days, followed by a single dosage with 10 mg/kg bw of [14C] – Bronopol on the 15thday, 14C was found to be extensively absorbed and rapidly excreted, mainlyviathe urine. In fact, more than 90% of the 14C was absorbed and a half-life of ca. 3 hours was determined. About 9% of the dose was recovered as 14CO2. Tissue concentrations of 14C were higher than plasma concentrations.

 

 

Huntingdon Research Centre (1974) The metabolism of Bronopol (2-bromo-2-nitropropane-1,3-diol) after oral administration to rats and dogs, and application to the skins of rats and rabbits. Huntingdon Research Centre, Huntingdon, UK, Report No: BTS31/74149, (Sponsor: The Boots Co. Ltd., Pharmaceutical Research, Nottingham, UK)

Bronopol was rapidly absorbed after oral administration to rat, and peak plasma concentrations were reached after 2 hours, with an initial half life ranging between 3 and 5 hours. Plasma concentrations decreased during 24 hour following dosing. Bronopol was rapidly eliminated and the majorexcretion pathway was the urine with more than 80% of the applied dose being excreted within 24 hours following treatment. Biliary excretion of bronopol amounted for 7% of the initial dose and was quite similar to the excreted amount of Bronopol via faeces (about 6%), indicating that the radioactivity measured in the faeces had been secreted through the bile. Expired CO2 amounted for 6 to 8% of the initial applied dose. No persistence of Bronopol and/or metabolites could be evidenced in the organs; in fact, the highest amounts of bronopol and/or metabolites were found in kidney , stomach and small intestine (i.e, organs involved in excretion) after 15 minutes following dosage. In the carcass, only little Bronopol and/or metabolite was retained (< 2 %). A main metabolite accounting more than 40% of the initial dose was detected in the urine whereas no unchanged bronopol was found; the metabolite was identified as 2-nitropropane-1,3-diol.

Following single oral dosage, Bronopol was extensively absorbed in male and female Beagle dogs and was rapidly excreted, mainlyviaurine (up to 64% during the first 12 h). Peak plasma concentrations of radioactivity were reached within 30 minutes to 2 hours, and the half-life of Bronopol and/or metabolites in plasma was ca. 4 hours. Persistence of Bronopol and/or metabolites in organs/tissues was low, and the highest concentrations were found in the kidney. Similar as in rats, a main metabolite accounting more than 40% of the initial dose was detected in the urine whereas no unchanged Bronopol was found; in rat study, metabolite A had been identified as 2-nitropropane-1,3-diol.

Absorption of Bronopol through rat skin under occlusive conditions was low and most of the applied radioactivity remained on and in the treated skin area. Excretionviaurine amounted for 11% of the applied dose and only small amounts of test material reached and were retained in the carcass. Metabolic pathways for Bronopol applied to the skin were similar to those following oral administration, with no parent compound found in urine but five metabolites, the major one having been identified as 2-nitropronane-1,3-diol.

Absorption of Bronopol through rabbit skin under occlusive conditions was low. Most of the applied radioactivity remained in the epidermis of the treated skin area, with superficial penetration being restricted to the areas surrounding hair follicles. Metabolic pathways for Bronopol applied to the skin were similar to those following oral administration, with no parent compound found in urine but five metabolites, the major one having been identified as 2-nitropronane-1,3-diol.

 

 

Boots Company(1993) An investigation of the metabolic fate of [14C] bronopol in the skin following one or two topical applications of [14C] Bronopol to the shaved dorsal skin of male CFLP mice. The Boots Company plc, Nottingham, UK, Report No: DT93090 (Unpublished);

The results of present study indicated a persistence of the radiolabelled material in skin, with only slow release into the plasma following an initial peak. When application was repeated, the amount of radiolabelled material retained in skin increased whereas release into the plasma remained similar as after a single application.Considering the metabolic fate of Bronopol, 2-nitropropan-1,3-diol was identified as main metabolite in skin and plasma; no parent compound was found. A further metabolite occasionally was seen, e.g. in skin samples of animals having received 2 consecutive applications of the test substance, as well as in some plasma samples at later time points; this metabolite was not identified. In skin 2-nitropropan-1,3-diol represented 75 to 98% of the radioactivity extracted.

 

 

Boots Company(1987) The absorption of [14C] material following the repeated topical application of 0.5% w/v solution of [14C] Bronopol in 90% acetone:10% water to a shaved area on the dorsum of male CFLP mice. The Boots Company plc, UK, Report No: DT87001 (Unpublished);

[14C] Bronopol was absorbed following application to the shaved dorsal skin of mice; this indicates a potential systemic exposure to significant levels of Bronopol and its metabolites following dermal application. The absorbed material was rapidly and extensively metabolised and cleared from plasma, indicating no accumulation potential in the plasma.

The Dow Chemical Company and LANXESS (2007)Bronopol: Oral absorption, distribution and elimination in Crl:CD(SD) - Sprague Dawley derived rats.K-081547-045, (Unpublished).

An investigation of the oral absorption, distribution and eliminationof bronopol in male CD rats after administration of a single oral dose of [14C] bronopol (1 or 30 mg/kg ) or fourteen repeated daily doses of bronopol followed by a single dose of [14C] bronopol (1 mg/kg).Bronopol was absorbed rapidly from the gastrointestinal tract with maximum levels of 17.5 and 22.8 ppm in blood cells and plasma, respectively, reached within 1 hour at 30 mg/kg bw. Elimination from blood was also fast with initial half life times of 3-4 hour and terminal half life times of 36 h (plasma) and 93 (blood cells). Distribution to tissues was determined after single application of 1 mg/kg bw at TCmax in blood (2 h) and TCmax/2 (6 h). Bronopol was widely distributed with highest levels detected in liver and kidney (involved in absorption and excretion) and lowest levels detected in fat. The total tissue residues at TCmax/2 including the carcass and gastrointestinal tract (about 29% of absorbed dose) were about half of the values at TCmax, (~61% of dose) revealing an equally fast elimination from tissues than from blood.

Residue values in the gastrointestinal tract (16% and 8.6% at 2 and 6 hours after application, respectively) confirmed that a bioavailability >80% can be considered for Bronopol.

The differences between tissue residues at 5 days after application between the low and high dose level were about proportional to the dose. Beside slightly higher levels in kidney and liver, tissue residues 5 days after multiple application were not different than after single application at the low dose level. Therefore no relevant accumulation potential is considered for Bronopol. At 5 days after application total tissue/carcass residues corresponded to 2.9-3.1% of the applied dose after single or multiple application of the low dose and single application of the high dose. Bronopol is also rapidly excreted mainly via urine (64-70% of applied dose within 12 hours) and to a lower extent via faeces (8-15% within 24 hours) independent of dose level and pre-treatment. Total excretion was 88-90% of applied dose.

 

 

Boots Pharmaceuticals Research Department (1993) Investigation of the metabolite, bromide ion, in male Charles River CD rats urine following single oral doses of Bronopol (1 mg/kg and 50 mg/kg). Boots Pharmaceuticals Research Department, Nottingham, UK, Report No: DT93018 (Unpublished);

Bromide ion was detected after a 50 mg/kg bw dose confirming that Bronopol undergoes some metabolism with the elimination of bromine. Peak excretion rate of bromide ion was observed between 8 and 24 hours and excretion was essentially complete at 120 hours after dosing. In urine the mean recovery of bromide ion was low, reaching 16.9 %of the dose. Following the single oral dose of 1 mg/kg bw of Bronopol, bromide ion was not detected above the endogenous level in rat urine. Thus,Bronopol undergoes metabolism with the elimination of bromide ion in rat urine.The percentage of bromine excreted as bromide ion was low. Excretion was essentially complete at 120 hours after dosing.

 

 

Boots Company (1984) (see chapter 7.10.3). An investigation of the absorption and excretion of [14C] Bronopol in volunteers following the application to the skin of Soltan 3 cream containing 0.1% [14C] Bronopol (preliminary report). The Boots Company, Research Department, Drug Metabolism and Medical Science, UK, DT84004, (Unpublished);

In a dermal study on human volunteers (Boots Company, 1984/DT84004) the absorption and excretion of 14C-labelled Bronopol was examined in two volunteers following application of a cream containing 0.1% of the test material (Soltan 3 cream) to the skin. The selected concentration was as used in a commercial product. An amount of 40 mg of radioactive test substance was dissolved in 400 µL distilled water, and 4000 mg of Soltan cream was added and mixed for one minute. Further 4000 mg of Soltan cream was added and mixed until the required concentration of 0.1% Bronopol was reached. The finally obtained radiolabelled test material was tested for homogeneity. Radioactivity content was assessed by means of liquid scintillation counting. About 6 µCi of the radioactive material was applied to the abdominal skin of each volunteer under occlusive conditions (application site: 200 cm²). After 8 hours following application, the abdominal application site was swabbed with water for removal of residual test material. Losses of test material upon application as well as contamination of the occlusion material resulted in 29.6% and 39.9% of the total applied dose, which was unavailable to volunteer A and B, respectively. About 1.8 % and 5 % of the available dose of radioactivity was detected in the urine of volunteer A and B, respectively; no radioactivity was found in the faeces. After 8 hours following application, 65.7% and 92% of the available dose was removed from the abdominal skin of volunteer A and B, respectively by swabbing, The maximum amount of 14C-labelled Bronopol, which could have been available systematically to the volunteers was about 34 % for volunteer A and 8 % for volunteer B. Metabolites were not measured in this study classified as key study.

 

 

Boots Company (1984) (see chapter 7.10.3) An investigation of the absorption of Bronopol in volunteers following the application to the skin of Soltan 3 cream containing 0.1% Bronopol. The Boots Company, Research Department, Drug Metabolism and Medical Science, UK, DT84044, (Unpublished);

In a further dermal study on human volunteers (Boots Company, 1984/DT84044) Bronopol was examined in 2 volunteers following application of a cream (Soltan 3 cream)containing 0.1% of the test material to the skin. The selected concentration was as used in a commercial product. The test material consisted of 5000 mg of Soltan cream containing 5 mg Bronopol. For the determination, all items used for application and removal of the test material from the skin of the treated volunteers were collected and the cream adhering to them was washed off into distilled water. The cream was broken down by agitation to allow dissolution of the Bronopol. The combined washings from each volunteer were diluted to a standard volume of 500 mL for analysis. Four mL aliquots were prepared and subjected to high performance liquid chromatography; the assay method was sufficiently sensitive for detection of Bronopol at the concentration levels used. After 8 hours, 52 to 87 % (mean: 72 %) of applied Bronopol was recovered after washing of the application site. Recovery in females ranged between 71 % and 87% (mean: 80 %) whereas in males, recovery ranged between 57% and 67%. The difference was statistically significant (Students´t-test, p<0.001). Metabolites were not measured in this study classified as key study.

 

Further references named in the conclusions:

 

Boots Pharmaceuticals Research Department (1993) The measurement of bromide ion in rat urine using capillary electrophoresis. Boots Pharmaceuticals Research Department, Nottingham, UK, Report No: DT93015 (Unpublished)

 

Boots Pharmaceuticals Research Department (1993) Study on the excretion, distribution and metabolism of bronopol in the rat. Boots Pharmaceuticals Research Department, Nottingham, UK, Report No. DT93077 (Unpublished);

This study summarizes the findings of following 3 studies:

- Boots Pharmaceuticals Research Department (1993), DT93045;

- Boots Pharmaceuticals Research Department (1993), ST93046;

- Pharmaco-LSR Ltd (1993), DT93043