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Specific investigations: other studies

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specific investigations: other studies
Type of information:
experimental study
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Scientifically acceptable data.
Reason / purpose:
reference to same study

Data source

Reference Type:
study report

Materials and methods

Principles of method if other than guideline:
other: no data
GLP compliance:

Test material

Test material form:
solid: particulate/powder
migrated information: powder
Details on test material:
Purity 99.7%

Test animals

other: none
other: not relevant
not specified

Administration / exposure

Route of administration:
other: addition of 30 µg/ml bronopol in chromosome medium 1A
Duration of treatment / exposure:
Exposure period: 24 hour(s)
Doses / concentrations
Dose / conc.:
30 other: µg/ml
Control animals:
Details on study design:
A working solution of 30 µg/ml bronopol was obtained by adding 50 µl of a 3 mg/ml aqueous solution of bronopol to 5 ml of lymphocyte culture medium (chromosome medium 1A; ex Gibco Ltd.) containing 8% whole human blood. The mixture was incubated in capped sterile culture tubes at 37 °C; two separate tubes were removed for analysis of bronopol content after 2 and 24 hours respectively.
The tubes were subjected to centrifugation (2,500 rpm for 8 min.) for separation of the red cells. One ml of the resultant clear supernatant was supplemented with 100 µl of an internal standard solution consisting of about 7mg/100 ml aqueous caffeine. The mixture was filtered (0.2 µm filter) and the filtrate was subjected to high pressure liquid chromatography according to the Drug Metabolism Physical Methods Standard Operation Procedure DMPM/92A. The samples were measured against a standard reference of 30 µg/ml which was prepared as described above but without whole human blood, and was analyzed immediately after preparation. Owing to the considerable amount of background interference resulting from the large excess of the medium, peak height ratios were measured automatically using a computing integrator as well as manually to obtain accurate measurements.


Estimation of formaldehyde release from bronopol in chromosome medium:
A working solution of 30 µg/ml bronopol was obtained by adding 50 µl of a 3 mg/ml aqueous solution of bronopol to 5 ml of lymphocyte culture medium (chromosome medium 1A; ex Gibco Ltd.). The mixture was incubated in capped sterile culture tubes at 37 °C for 24 hours; two separate tubes, each containing 5 ml of the mixture were used. One ml of mixture was collected from the tubes at following time points: 0.5, 1, 2, 4, 6 and 24 h. The samples were subjected to analysis for formaldehyde, according to the method described by Ashby J and Lefevre PA (Formaldehyde generators: relationship between stability, lipophilicity and carcinogenic potency. Carcinogenesis Vol. 3. 1273-1276, 1982). Briefly, 1 ml of mixture was incubated with 2.5 ml Nash reagent (30% w/v ammonium acetate in distilled water containing 0.6% v/v glacial acetic acid and 0.6% v/v acetylacetone) for one hour at 37 °C. Thereafter the absorbance of the resultant yellow coloured solution was measured at 415 nm in 10 mm cells in an ultraviolet spectrophotometer. The concentrations of formaldehyde in the samples were calculated relative to a standard calibration curve prepared by spiking blank medium at concentrations of 0, 1, 2, 3, 4 and 5 µg/ml formaldehyde.

Results and discussion

Any other information on results incl. tables

Decomposition of bronopol in lymphocyte culture medium: Taking into account the fact that the large excess of medium used resulted into a considerable amount of interference, the results of the manual measurements were considered as maximum values. After 24 hours, 4.2µg/ml bronopol was measured. Formaldehyde-release during bronopol decomposition in lymphocyte culture medium:

Time point (h)   Formaldehyde concentration

0.5       3.6 µg/ml

1          4.1 µg/ml

2          4.2 µg/ml

4          3.7 µg/ml

6          3.3 µg/ml

24        3.4 µg/ml


The results of the tests are indicative of a rapid and extensive decomposition of bronopol in culture medium, with only about 10% of the initial concentration of parent compound remaining after 2 and 24 hours of incubation at 37°C. Furthermore, it could be shown that at an initial concentration of 30µg/ml bronopol, a maximum concentration of 4.2µg/ml formaldehyde was present in the test medium after 2 h following addition of bronopol to the medium. Thereafter, the concentration of formaldehyde tended to slightly decrease. The authors suggested that the decrease either was related to the loss of formaldehyde after prolonged exposure to the test conditions, or to its reaction with bronopol to form 2-hydroxymethyl-2-nitro-1,3-propanediol.

Additional test:

Following incubation of 1 ml of 30µg/ml bronopol in 2.5 ml Nash reagent for 1 h at 37°C, no formaldehyde could be detected by absorbance measurement, hence excluding the possibility of formaldehyde release by the Nash reagent alone.

Applicant's summary and conclusion

Bronopol in lymphocyte culture medium and under conditions used for genotoxicity testing is rapidely and extensively decomposed, resulting in formaldehyde liberation in the medium. This supports the assumption that the observed clastogenic effect of bronopol in the absence of S9 mix (Everest RP, and Williams CV, The Boots Company PLC Research Department, Report No: TX 86049, 1986; rather was due to formaldehyde liberated from bronopol-degradation, than to bronopol as such.