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In vitro study: Bacterial systems

The test substance Bronopol (purity 99.7 %) was tested for mutagenicity in the reverse mutation assay on bacteria with and without metabolic activation (S9-mix) (Boots Company PLC Research Department, 1986/TX86004). Following Salmonella typhimurium tester strains were used in this assay: TA 1535, TA 1537, TA 1538, TA 98, TA 100. Following concentrations of Bronopol were used: 3.9, 7.8, 15.6, 31.2, 62.5 and 125 µg/plate; water was used as solvent. The test series were accompanied by a solvent control and by the following positive controls: Cyclophosphamide (250 µg/plate; TA 1535), Neutral red 25 µg/plate; TA 1537) and 2-Aminofluorene (50 µg/plate; TA 1538, TA 98 TA 100). Replicate assays were conducted, each with 3 plates per test concentration. The plates were incubated at 37° C for two days; thereafter the number of revertant colonies per plate was determined by manual counting or by means of an automated colony counter. The results were expressed as means of the 3 plates.

Up to the maximum dose levels allowed by bacterial toxicity (i.e. 125 µg/plate in presence of S9-mix and 62.5 µg/plate in absence of S9-mix), the numbers of revertants recorded were within the range of the negative control (distilled water); the increased numbers of revertants obtained within the positive controls confirmed the validity of the results of the present test. Bronopol showed no mutagenic potential in the Ames test.

This GLP-conform study is classified as acceptable (key study). No guideline mentioned; however the test was conducted according to the acknowledged method of Ames et al. (Proc. Nat. Acad. Sci. 70: 2281-2285 & 782-786, 1973 and Mutat. Res. 31: 347-364, 1975).

 

 

In vitro study: Mammalian cell gene mutation test

A mammalian cell forward gene mutation assay was conducted with Chinese hamster V79 cells with and without metabolic activation (S9-mix) (Boots Company PLC Research Department, 1986.TX86043).

Main assay: The main assay was conducted with 0.5, 1, 2, 4, 8 and 16 µg/mL Bronopol (purity 99.7%) in absence of S9 mix, and 1, 2, 4 and 8 µg/mL Bronopol in presence of S9 mix. The cells (10E6 cells/flask) were seeded 24 hours prior treatment with the test substance; the cells were treated with Bronopol for 3 hours; they were then rinsed and were further incubated in absence of test substance for a whole expression period of 7 days. The cytotoxicity was estimated by comparing the cloning efficiency of the different test groups after 24 hours following treatment. The mutagenic potential of the test substance was estimated as number of 6TG mutants/ 10E6 clonable cells at the end of the 7-days expression period; for this purpose, 10 flasks/ treatment group were seeded each with 10E5 cells in medium containing 6TG and the cells were incubated for 9 days for estimation of the numbers of mutants. For determination of the number of clonable cells at the end of the 7-days expression period, 5 flasks/treatment group were seeded each with 200 cells in medium and the cells were incubated for 6 days.

Additional assay: The test procedure was as described above, however with following exception: in order to compensate the reduction in cell numbers due to the toxicity of Bronopol, 6 x 10E6 cells were seeded 24 h prior treatment instead of 10E6 cells/flask. Following Bronopol concentrations were tested: 4, 5, 6, 7 and 8 µg/mL (with S9 mix).

Controls: N-methyl-N´-nitro-N-nitrosoguanidine (MNNG, 0.4 and 0.5 µg/mL) served for positive control in the absence of S9 mix. In the presence of S9 mix, the positive control was performed with 7,12-dimethylbenz(a)antracene (DMBA, 20 µg/mL). The negative controls were conducted without test substance. For all control groups the cell number was 10E6 cells/flask.

Cytotoxicity:

In absence of S9-mix, a clear cytotoxic effect of Bronopol was seen at a test concentration of 16 µg/mL; at this concentration, cloning efficiency was <1 % of control. In the presence of S9 mix, cloning efficiency was 1% at 8 µg/mL Bronopol. Therefore, 8 µg/mL was regarded as the maximum concentration for mutagenicity testing allowed by the cytotoxicity of Bronopol.

Genotoxicity:

In the absence of S9-mix, no evidence of mutagenicity was seen at test concentrations of Bronopol up to 8 µg/mL, which was the maximum test concentration allowed by cytotoxicity.

In the presence of S9-mix, an increase in mutant frequency was seen in the first test of the main assay, at the highest tested concentration of 8 µg/mL Bronopol (41.8 +/- 8.22 mutants/10E6 clonable cells versus 4.0 +/- 2.06 for control). No such increase was seen in the second test. In the additional assay, the mutant frequency in the Bronopol treated groups was within control range and therefore inconspicuous in the first test. In the second test, a slight increase in mutant frequency was seen at 8 µg/ml Bronopol (18.4 +/- 5.59 mutants/10E6 clonable cells, versus 10.0 +/- 3.76/10E6 clonable cells for control), which however did not satisfy the criterion for a positive result as defined by Mc Millan and Fox (1979). The increase seen in the first test of the main assay was therefore considered to be due to a random event in a total cell population, which was highly affected by the increased toxicity level of the test substance Bronopol. Bronopol was not mutagenic in Chinese hamster V79 cells.

This GLP-conform study is classified as acceptable (key study). No guideline was mentioned; however the study followed the acceptable method of Mc Millan and Fox (1979) with some modification (O´Donovan MR, The Boots Company Ltd., Report No: TX 83017, 1983).

 

Reference: Mc Millan S and Fox M (1979). Failure of caffeine to influence induced mutation frequencies and the independence of cell killing and mutation induction in V79 Chinese hamster cells. Mut. Res. 60: 91-107

 

 

In vitro study: Chromosome aberration test

In an in vitro mammalian chromosome aberration test (Boots Company PLC Research Department,1986/TX86049) the clastogenic potential of Bronopol on human lymphocytes was assessed in the absence and the presence of metabolic activation (S9-mix). A range-finding cytotoxicity test was performed prior to the main test. On the basis of the results of this test, the concentrations of Bronopol (purity 99.7%) chosen for the main test were 10, 20 and 30 µg/mL without S9-mix, and 20, 30, and 40 µg/mL with S9-mix. In an additional assay conducted without S9, Bronopol was tested at 20 and 30 µg/mL. The test concentrations were prepared by diluting the test substance in distilled water. The negative, vehicle control (with and without S9-mix) was conducted with distilled water; Mitomycin C (0.5 µg/mL) was used as positive control in absence of S9-mix whereas Cyclophosphamide (25 µg/mL) was used as positive control in the presence of S9-mix. The lymphocytes obtained from the heparinized aseptic blood samples were put in culture in adequate medium. After 48 hours following initiation of cell division in the culture medium containing phytohaemagglutinin, the test substance was added to the culture medium. The cells were incubated at 37 °C with the test substance for 24 hours in the absence of S9-mix and for 2 hours in the presence of S9-mix. The procedure was the same for the negative controls (untreated, distilled water control group) and the positive controls (Mitomycin C and Cyclophosphamide). After incubation, the metaphase chromosomes were examined for aberrations using a high-power light microscope. The statistical assessment of the results was based on the Fisher exact test. The maximum test concentration of Bronopol allowed by its cytotoxicity was 30 µg/mL. In the main test, a small but statistically significant increase in the incidence of cells showing chromosomal aberrations (with and without gaps) was evident at the maximum tested concentration of 30 µg/mL Bronopol. This finding was confirmed by the repeat assay conducted with 20 and 30 µg/mL test substance in absence of S9-mix. No increased incidence of cells with chromosomal aberrations was seen in the presence of S9-mix. A weak but reproducible clastogenic effect was seen in absence of S9-mix, but not in the presence of S9-mix. The authors of the study suggested that the observed clastogenic effect rather might have been due to formaldehyde liberated from Bronopol-degradation, than to Bronopol as such. In the methodological part of the study, the addition of colcemid to the culture medium to stop mitosis in the metaphase was not mentioned and no data were provided on the preparation of the cells for microscopical examination.

In the material part of the study, data from other studies referring to the stability of Bronopol were provided in a summarized form. The author reported that after a 2 hours incubation at 37 °C in lymphocyte culture medium (pH 7.0–7.2), recovery of Bronopol was 10 %; within the same sentence, a concentration of up to 4.2 µg/mL of formaldehyde was mentioned but without further information allowing allocation of this finding.

This GLP-conform study is classified as acceptable (key study). No guideline mentioned; however the study to a large extent was in accordance with the requirements of OECD guideline 473.

In vitro study: Chromosome aberration test on breakdown product of Bronopol

The testing of Bronopol for clastogenicity using the in vitro human lymphocyte chromosomes assay resulted in a weak clastogenicity at a concentration of 30 µg/mL in the absence of S9-mix (Boots Company PLC Research Department, 1986/TX86050). However, the release of formaldehyde during decomposition of Bronopol was shown (Boots Company PLC Research Department, 1986, DT 86030) under conditions similar to those of the in vitro human lymphocyte chromosomes assay. From an initial Bronopol concentration of 30 µg/mL in chromosome medium, a maximum concentration of formaldehyde of 4.2 µg/mL was released after 2 h of incubation; thereafter the concentration tended to decrease.Thus, the clastogenic potential of Formaldehyde (purity: 38 % w/v; 0-14% methanol) in human lymphocytes was assessed in the absence of metabolic activation (Boots Company PLC Research Department, 1986/TX86050) at a concentration range covering the 4.2 µg/mL Formaldehyde released by Bronopol under the test conditions of the in vitro human lymphocyte chromosomes assay.The selected test concentrations of formaldehyde were as follows: 0.5, 1.0, 2.0, 4.0, 6.0 and 8.0 µg/mL. The test conditions were similar to those for Bronopol, except for the metabolic activation and the positive controls. In order to simulate conditions similar to those used for Bronopol testing, which resulted in a positive finding, the present test was conducted in the absence of metabolic activation, i.e. without S9-mix. No positive controls were included in the test. Formaldehyde is known to be clastogenic in vitro. The negative controls were treated with the solvent only, i.e. distilled water. After incubation, the metaphase chromosomes were examined for aberrations using a high-power light microscope. The statistical assessment of the results was based on the Fisher exact test.A statistically significant increase in chromosomal aberrations, with and without gaps, was seen at both highest test doses of 6 and 8 µg/mL.Percentages of cells with aberrations (including gaps) of 8 % and 21 % were reported for 6 and 8 µg/mL respectively, versus 1 % in the negative control. 15 % of cells with aberrations (excluding gaps) were reported for 8 µg/mL, versus 0 % in the negative control. The remaining tested concentrations were inconspicuous. Moreover, the extent and quality of the findings seen at 8 µg/mL were very similar to those reported for Bronopol at 30 µg/mL. In the absence of S9-mix, a significant clastogenic effect was reported for formaldehyde at test concentrations of 6 and 8 µg/mL, which were slightly above the peak of 4.2 µg/mL released by decomposition of 30 µg/mL Bronopol (see Boots Company PLC Research Department, 1986, DT86030). However, the extent and quality of the findings seen at 8 µg/mL were very similar to those reported for Bronopol 30 µg/mL, supporting the assumption that the clastogenicity reported for Bronopol rather was due to released formaldehyde than to the parent compound as such, even if there was a comparatively reduced activity of formaldehyde under direct testing conditions (i.e. direct addition to the cells).

This GLP-conform study is classified as acceptable (key study). No guideline mentioned; however the study to a large extent was in accordance with the requirements of OECD guideline 473.

 

 

In vivo study: Mouse micronucleus assay

In an in vivo micronucleus test (MNT) in mice the in vivo genotoxic potential of Bronopol was investigated (Boots Company PLC Research Department, 1986/TX86001). CD1 mice received single oral application of the test substance by gavage. Bronopol was tested at following doses: 80 mg/kg bw (12 animals/sex) and 160 mg/kg bw (24 animals/sex), the latter dose being the maximum dose tolerated by the mice. A negative control group consisting of 12 animals/sex was treated with the purified water whereas a positive control group, which also comprised 12 animals per sex, was treated with the clastogenic substance cyclophosphamide (75 mg/kg bw). Following dosage, the animals were observed for overt signs of toxicity and mortality. After 24, 48 and 72 hours following treatment, 4 animals/sex and 8 animals/sex, respectively from the groups with a total of 24 mice and the group with 48 mice, were sacrificed, and the femoral bone marrow was extracted, prepared and examined for following parameters: the polychromatic/normochromatic erythrocytes ratio and the number of micronuclei in 1000 polychromatic erythrocytes per animal. The statistical assessment of the findings was based on the Analysis of variance, the Freeman-Tukey transformation and the use of F-distribution tables.

Overt signs of toxicity: In the 160 mg/kg bw group, 4 males and 4 females died within 48 hours whereas in the 80 mg/kg bw group, one female died within 72 hours.

Ratio of polychromatic to normochromatic erythrocytes in the femoral bone marrow of Bronopol treated CD1-mice: After 72 h, the polychromatic/normochromatic erythrocytes ratio for the males of the 160 and the 80 mg/kg bw groups as well as for the females of the 80 mg/kg bw group were reduced compared to the normal ration of ca. 1:1; this effect indicated a decrease in haemopoiesis.

Incidence of micronuclei in polychromatic erythrocytes: The incidence of micronuclei in polychromatic erythrocytes of Bronopol-treated mice was within the range of negative control. In contrast, in the Cyclophosphamide-treated mice (positive control), statistically significant increases in the incidence of micronuclei in polychromatic erythrocytes were reported after 24 and 48 hours; after 72 hours, the incidence of micronuclei in polychromatic erythrocytes was increased in the males but without being statistically significant.

Bronopol at doses up to 160 mg/kg bw, which is the maximum dose tolerated by mice, was not clastogenic in the in vivo mouse micronucleus test.

This study is classified as acceptable (key study). The test was conducted according to OECD 474 and followed GLP.

 

In-vivo study: Unscheduled DNA Synthesis

The in vivo genotoxic potential of Bronopol was investigated using the UDS assay in hepatocytes of rat (Covance Laboratories Limited, 1998). The test substance was Myacide AS (Bronopol) with a purity of 99.5%.

On the basis of the results of a range finding pre-test, 150 mg/kg bw was selected as maximal tolerable dose for male Wistar rats, and the UDS assay was performed using following two test concentrations: 60 and 150 mg/kg bw. The assay comprised two experiments with different post-treatment period: 12–14 hours and 2–4 hours; each test group comprised 5 male rats. The test doses were prepared using purified water as vehicle; the rats received single oral application of test solution at an application volume of 10 ml/kg bw. Purified water was tested as negative control; 2-acetylaminofluorene (2-AAF; 75 mg/kg bw suspended in corn oil) and Dimethylnitrosamine (DMN; 10 mg/kg bw suspended in water were tested as positive controls, respectively within experiment 1 and 2. The test animals were examined for clinical signs of toxicity; body weights were recorded. At the end of the respective post-treatment periods, the rats were sacrificed and the hepatocytes were isolated from the liver of each rat. The cells were examined for cell viability as measured by the trypan blue exclusion technique. DNA damage and repair was measured by incorporation of 3H-thymidine using autoradiography technique. For evaluation and quantification of UDS, a total of 100 cells/animal was examined and following parameters were considered: net nuclear grain (NNG) count/cell, group mean net nuclear grain (NNG) count, mean net grain (NG) count of cells in repair, percentage of cells in repair (cells with NNG >= 5).

The acceptance criteria were as follows:

-Clearly negative results in the untreated and in the vehicle controls in the range of historical control data.

-Clearly positive results in the positive control group (NNG >= 5, with 50% or more cells having NNG >= 5).

The evaluation criteria were as follows:

-Positive response: a positive response implicates a dose-related increase in mean number of NNG counts (> 0 at one of the test points) and in percentage of cells in repair (i.e. cells with NNG >= 5), which must be >= 20%.

-Negative response: a negative response implicates that both, the NNG counts and the percentage of cells in repair are within the range of negative control.

The main findings of the UDS assay can be summarized as follows:

Toxicity: abnormal gait and abnormal breathing were reported for the animals of the 150 mg/kg bw group; in the 60 mg/kg bw group, only one animal showed similar symptoms. No mortality was seen, and body weights were inconspicuous.

UDS testing: hepatocyte viability in the Bronopol-treated animals was within the range of the negative control. The group mean net grain count for the negative control respectively was –2.5 and –2.6 for the first and the second experiment and therefore was less than the upper limit of the historical control range. Furthermore, the positive control substances 2-AAF and DMN resulted in increased group mean net grain count values (respectively 8.8 and 15.2), and respectively 67.7 % and 78.3 % cells had net grain count >= 5; this indicates that the test system was sensitive to the two positive control substances and the experiment was valid. The mean net grain count values for the Bronopol treated groups in both experiments ranged between –1.6 and –2.0 and were therefore < 0, i.e. below the threshold value indicative of a positive response; furthermore, the percentage of cells in repair with NNG >= 5 ranged between 1.3 and 3 % (i.e. < 20%). This clearly indicates that the oral treatment of male rats with Bronopol at doses up to 150 mg/kg bw, which was the maximum tolerable dose, did not induced increased UDS in the hepatocytes of the liver.

Thus, under the conditions of this UDS test, Bronopol (Myacide AS) did not induce DNA-damage leading to repair synthesis in the hepatocytes of the treated rats (= showed no genotoxic potential within the in vivo UDS assay performed with rats).

This study is classified as acceptable (key study). The test was conducted according to OECD 486 and followed GLP.

In vivo study: Dominant lethal assay in mice

In a dominant lethal mouse assay Bronopol was tested for cytogenicity in mouse meiotic and post-meiotic sperm stages (Boots Company Limited Research Department, 1974/TX74034).

Ten male mice were used per test group and for positive control. Twenty males served as negative control. The test groups either received Bronopol at 20 and 100 mg/kg bw by gavage once daily for 6 consecutive days, or received single i.p. injection of 10 mg/kg bw Bronopol in 0.9% saline. Negative controls were treated orally with water. Positive controls received a single i.p. injection of 25 mg/kg bw tris(2-methyl-1-aziridinyl)-phosphine oxide (METEPA) in 0.1 mL/10 g bw of 0.9% physiological saline. Four hours after completion of the dosing, each male was housed with 3 females for mating, which were replaced at weekly intervals for 4 weeks. The females were killed 14 days from the midpoint of the mating week, corresponding to gestation days 9 to 16. Numbers of pregnancies, and live and dead implants were recorded.

Mortalities in treated males were seen at the highest orally applied dose of Bronopol (100 mg/kg bw) and following i.p. injection. The remaining test groups were inconspicuous. For the highest orally applied dose of Bronopol (100 mg/kg bw, six times) and for the single i.p. injection, a conspicuous decrease in pregnancy rate was observed, which was indicative of a reduced fertility related again to the toxicity of the tested doses of Bronopol observed for the males in these groups.

Implantation rates were significantly reduced in week 2 and 3 for the group treated orally with 100 mg/kg bw of Bronopol; a similar reduction also was observed for the group having received i.p. injection of Bronopol, however in week 4. This effect appears to be a consequence of the toxic effect of Bronopol on the treated males of these two groups, which resulted, as mentioned above, in a decreased fertility and rate of pregnancy. A significant increase in the frequency of dead implants was reported for the i.p. treated group in week 4. In all remaining treated groups, the frequency of dead implants was within control range.

The results of the positive control were as expected, with significant decrease in live implants and increase in dead implants when compared to negative control; this indicates a clear dominant lethal effect for METEPA when applied at 25 mg/kg bw intraperitoneally in mouse.

Reduction in implantation and pregnancy rate observed in the 100 mg/kg bw group (oral treatment) and the 10 mg/kg bw group (i.p. injection) clearly was a consequence of the toxicity of the tested concentrations on the male mice. Therefore, these effects rather were seen as non-genetic anti-fertility effects and were not indicative of a cytogenic effect of Bronopol on the meiotic and post-meiotic sperm stages. Thus, the test substance lacks cytotoxicity for mouse meiotic and post-meiotic sperm stages.

This study is classified as acceptable (key study). The study was conducted according to the method of Bateman AJ (1958; Heredity 12: 213-232) and Bateman AJ and Epstein SS (1971; in Chemical Mutagens, Vol. 2, ed. Hollaender, Plenum Press). The study did not follow GLP as GLP was not compulsory at the time the study was performed; test procedure and results obtained were considered as scientifically acceptable.


Justification for selection of genetic toxicity endpoint
All listed key studies were selected, which includes In vitro and in vivo assays performed to estimate the potential of Bronopol to cause genetic toxicity (e.g. mutagenicity or clastogenicity).

Short description of key information:
Bronopol did not reveal any indication for mutagenicity in the Ames test or in a gene mutation test in cultured mammalian cells (HGPRT assay). In an in vitro chromosomal aberration test in cultured V79 cells, positive effects were noted in the absence of metabolic activation due to a breakdown product of Bronopol. In an in vivo mouse micronucleus assay Bronopol does not induce cytogenetic damage in bone marrow cells of mice. In addition in an UDS assay in vivo the test substance did not induce DNA-damage leading to repair synthesis in the hepatocytes of treated rats. Moreover, Bronopol lacks cytotoxicity for mouse meiotic and post-meiotic sperm stages (dominant lethal assay). In conclusion, the test substance is not considered to be mutagenic in vivo.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available results of the three different in vitro and three vivo genetic toxicity studies, Bronopol has not to be classified and labelled as genotoxic according to Directive 67/548/EEC (DSD) and Regulation (EC) No 1272/2008 (CLP).