Bronopol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria

Ames test, S. typhimurium TA 1535, TA 1537, TA 98, TA100 and TA 102; with and without metabolic activation: negative (GLP, OECD 471; Lanxess, 2000/PH 30497)

 

Gene mutation in mammalian cells

HPRT assay, CHL V79 cells, with and without metabolic activation: positive without metabolic activation (GLP, OECD 476; Lanxess, 2000/PH 30670)

 

Cytogenicity in mammalian cells

Chromosome aberration assay, CHL V79 cells, with and without metabolic activation: positive (GLP, OECD 473; Lanxess, 2000/PH 30397)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

8 August - 22 September 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian cell transformation assay

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

V79 cells were obtained from Dr. Utesch, Merck AG, Darmstadt. The cells arrived at the Toxicology of Bayer AG, Bayer AG, Wuppertal, on November 8, 1993. Chinese hamster V79 cells can be kept in culture as established cell lines (Kao and Puck, 1967). The mean generation time of the used cell line is approximately twelve hours.
Prior to the start of the study Chinese hamster V79 cells were normally grown in 20 ml medium and 75 cm2 flasks or under comparable conditions. Incubation of the cells was always performed at 37°C in a CO2-incubator (5% CO2). Unless reported otherwise, cells were grown in medium containing 10% fetal calf serum.

As medium, Eagle's minimal essential medium (MEM, Earle) with the following sup­plements was used:
nonessential amino acids
L-glutamine (2 mM)
MEM-vitamins
NaHC03-solution (final concentration: 0.225 %) penicillin (50 units/ml)
streptomycin (50 µg/ml)
heat-inactivated fetal calf serum

The karyotype of the V79 cells (modal number of chromosomes: 22) was confirmed on August 11, 2000.
A routine check for mycoplasma was performed on October 29, 1998. There was no evidence of mycoplasma contamination.

Cytokinesis block (if used):

colcemid

Metabolic activation:

with and without

Metabolic activation system:

The S9 fraction was isolated in house from the livers of Aroclor 1254 induced male Sprague Dawley rats; co-factors were added.

Test concentrations with justification for top dose:

Pretest:
-S9: 0, 0.1, 0.5, 1, 5, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50 µg/mL
+S9: 0, 15, 20, 25, 30, 35, 40, 45, 50 µg/mL

Aberration test:
-S9: 0, 3, 5, 7, 9, 11 µg/mL
+S9: 0, 10, 15, 20, 23, 26 µg/mL

Vehicle / solvent:

deionized water

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Cytotoxic effects of the test substance were assessed in the pre-test as well as in the main-study. Cell survival as well as mitotic index were determined in the presence and absence of S9 mix.
At the end of the respective incubation period cells of all cultures of the respective period were trypsinized, and an appropriate dilution was counted using a hemocy­tometer (improved Neubauer) to determine cell survival.
The mitotic index was determined for all cultures. The number of mitotic cells among a total of 1000 cells per culture was determined. All cells which were not in inter­phase were defined as mitotic.
In the main study, cultures with a total incubation period of 8 hours were additionally and exclusively used to determine the cytotoxicity of Bronopol Bayer.

The general protocol of the test system was similar to published procedures (e.g. Dean and Danford, 1984). Chinese hamster V79 cells were passaged on the day prior to treatment. Approximately 1E6 cells were seeded in 20 ml of medium per 75 cm2 flasks and incubated. All cultures were set up in duplicate.
Immediately before treatment, the medium was removed from the cultures. For treatment, fresh solutions of medium containing 2% FCS (20 ml), S9 mix (+/-; 1 ml), and test substance (0.2 ml) were added to each flask. Cells were incubated for 4 hours. After this treatment period, the medium was removed, the cells were washed with pre-warmed PBS (about 37°C), 20 ml of fresh medium containing 10 % FCS was added to the flasks and the flasks were placed in a CO2-incubator for the remaining incubation time.
0.2 ml Colcemid-solution (40 µg/ml water) were added to each flask two hours prior to the end of the incubation period to arrest the cells in a metaphase-like stage of mitosis (c-metaphase).

Positive controls (see section 4. 1.2) and solvent controls (0.2 ml solvent per culture), and, if indicated, untreated controls (no addition of solvent) were set up in parallel and handled as described for Bronopol Bayer-treated cultures. Untreated controls and solvent controls are used as negative controls.

After harvesting the cells, hypotonic treatment, fixation and staining 200 metaphases (100 metaphases from two parallel cultures per concentration) were counted for aberrations.
In addition at least 1000 cells per culture were evaluated to determine the mitotic index. The highest concentration for metaphase evaluation should lead to a relevant reduction of survival indices.

Evaluation criteria:

An increased incidence of gaps of both types without concomitant increase of other aberration types was not considered as indication of a clastogenic effect.
A test was considered positive, if there was a relevant and statistically significant in­crease in the aberration rate.
A test was considered negative, if there was no such increase at any time interval.
A test was also considered negative, if there were statistical significant values, which were, however, within the range of historical negative controls.
A test was considered equivocal, if there was an increase above the range of histori­cal negative controls which was statistically significant but not considered relevant, or if an increase occurred, which was considered relevant, but which was not statisti­cally significant.

Statistics:

The statistical analysis was performed by pair-wise comparison of Bronopol Bayer­treated and positive control groups to the respective solvent control group. The mitotic index was statistically analyzed (provided that it was reduced compared to the mean of the respective solvent control) using the one-sided chi2-test. The numbers of metaphases with aberrations (including and excluding gaps) and of metaphases with exchanges were compared (provided that these data superceded the respective solvent control). The statistical analysis followed the recommenda­tions outlined by Richardson et al. (1989). Fisher's exact test was used for the statis­tical evaluation. A difference was considered to be significant if the probability of error was below 5%.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

pH and Osmolality: Concentrations of up to 50 µg/ml Bronopol Bayer did not change the pH in the me­dium in the pre-test. The osmolality in the medium of the pre-test was not changed by concentrations of up to 50 µg/ml Bronopol Bayer.

Cytotoxicity: In the pre-test the survival indices were reduced at >/=5 µg/mL and >/=15 µg/mL in absence and presence of S9, respectively. Marked cytotoxicity (survival indices <10% of control) was seen at >/=15 µg/mL and >/=30 µg/mL in absence and presence of S9, respectively.
In the chromosome aberration test in absence of S9 relevant cytotoxicity was seen at Bronopol concentrations >/=5 µg/mL and >/=7 µg/mL after 18 and 30 hours of incubation, respectively. In presence of S9 relevant cytotoxicity was seen at >/=20 µg/mL and >/=23 µg/mL after 18 and 30 hours of incubation, respectively

Genotoxicity:
without metabolic activation: After 18 hours of incubation there was no relevant increase in the number of aberrant metaphases +/- gaps. While not attaining statistical significance the number of metaphases with exchanges exceeded the historical control range at the highest concentration of 7 µg/mL. After 30 hours of incubation there was a relevant and statistically significant increase in aberrant metaphases also at 7 µg/mL. A slight increase of polyploid metaphases was observed at 7 µg/mL after 30 hours incubation. The positive control compound also revealed a positive response in aberrant metaphases.

with metabolic activation: After 18 hours of incubation there was a relevant, statistically significant increase in the number of aberrant metaphases +/- gaps. While not attaining statistical significance the number of metaphases with exchanges exceeded the historical control range at the highest concentration of 23 µg/mL. After 30 hours of incubation there was a relevant and statistically significant increase in aberrant metaphases also at 23 µg/mL. The positive control compound also revealed a positive response in aberrant metaphases.

Table 1. Cytotoxicity pre-test

Bronopol [µg/mL]	-S9		+S9
	Survival index [% control]	Mitotic index [%control]	Survival index [% control]	Mitotic index [% control]
0	100	100	100	100
0.1	95	86	nd	nd
0.5	96	86	nd	nd
1	111	94	nd	nd
5	80	80	nd	nd
10	52	45	nd	nd
12	33	25	nd	nd
15	3.5	55	80	77
20	0	0	56	61
25	0	nd	34	64
30	0	nd	2.2	32
35	0	nd	0	0
40	0	nd	0	0
45	0	nd	0	nd
50	0	nd	0	nd

nd = not determined

Table 2. Chromosome aberrations in the absence of S9

Bronopol [µg/mL]	Survival index	Mitotic index	% Metaphases with aberrations			Polyploid metaphases
	[% control]	[% control]	+ gaps	- gaps	exchanges
4 h treatment + 4 h recovery
0  7  9 11	100 84 89 48°	100 52** 22** 16**	not evaluated
4 h treatment + 14 h recovery
0  3  5  7  9 11 pos. control	100 90 72° 53° 47° 30° 46°	100 87* 80** 65** 56** 36** 83**	1.0 1.0 1.5 3.0 - - 36.0^++	1.0 1.0 1.5 3.0 - - 36.0^++	0.0 0.0 0.5 2.0 - - 20.5^++	25 23 23 30 - - 19
4 h treatment + 26 h recovery
0  7  9 11	100 49° 20° 10°	100 66** 42** 44**	0.0 11.0^++ - -	0.0 11.0^++ - -	0.0 8.5^++ - -	18 40 -

° relevant reduction of survival index

* p<0.05, ** p <0.01 (one-sidedc²-test)

⁺p <0.05, ⁺⁺p <0.01 (Fisher’s exact test)

Table 3. Chromosome aberrations in the presence of S9

Bronopol [µg/mL]	Survival index	Mitotic index	% Metaphases with aberrations			Polyploid metaphases
	[% control]	[% control]	+ gaps	- gaps	exchanges
4 h treatment + 4 h recovery
0 20 23 26	100 75° 59° 37°	100 74** 51** 31**	not evaluated
4 h treatment + 14 h recovery
0 10 15 20 23 26 pos. control	100 84 93 67° 54° 44° 76°	100 127 124 125 81* 41** 78**	3.5 - 2.0 5.5 10.0^++ - 42.0^++	3.5 - 2.0 5.5 9.5^+ - 40.5^++	2.0 - 0.0 2.5 4.0 - 22.5^++	18 - 28 12 20 - 17
4 h treatment + 26 h recovery
0 23 23 26	100 56° 24° 11°	100 101 58** 48**	1.0 16.5^++ - -	1.0 16.5^++ - -	0.5 14.0^++ - -	32 41 - -

° relevant reduction of survival index

* p<0.05, ** p <0.01 (one-sidedc²-test)

Conclusions:

Bronopol is considered to be clastogenic in this in vitro study both in presence and absence of S9.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 April - 7 Dec 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

other: histidine deficient

Metabolic activation:

with and without

Metabolic activation system:

Prepared from livers of male Sprague-Dawley rats induced with a single i.p. injection of 500 mg/kg bw Arochlor 1254 in corn oil five days prior t sacrifice; co-factors were added.

Test concentrations with justification for top dose:

Cytotoxicity tests: 50, 158, 500, 1581, 5000 µg/plate in absence and presence of S9
Mutagenicity tests: 1-64 µg/plate in absence and presence of S9

Vehicle / solvent:

deionized water

Untreated negative controls:

other: not applicable

Negative solvent / vehicle controls:

yes

True negative controls:

other: not applicable

Positive controls:

yes

Positive control substance:

sodium azide

other: 4-nitro-1,2,-phenylene diamine, nitrofurantoin, cumene hydroperoxide, 2-aminoantracene

Details on test system and experimental conditions:

Plate incorporation test: The bacterial suspensions (0.1 mL) were mixed with soft agar (2.0 mL), 0.1 mL of Bronopol stock solutions or vehicle control, 0.5 mL S9 mix (in presence of metabolic activation) or buffer (in absence of S9) before being poured onto minimal agar plates.
Preincubation test: The bacterial suspensions (0.1 mL) were mixed with 0.1 mL of Bronopol stock solutions or vehicle control, 0.5 mL S9 mix (in presence of metabolic activation) or buffer (in absence of S9) and incubated for 20 min at 37°C. Soft agar (2.0 mL) was then added to the mixture before being poured onto minimal agar plates
The plates (three per concentration, negative and positive controls in each test) were then incubated for 48 hours at 37°C.
Total bacterial counts were also determined (soft agar with 5x higher histidine concentration) using two plates each in absence and presence of S9.
Examination for toxicity was (reduction of) background growth and number of mutant colonies and examination for mutagenicity was frequency of revertant colonies

Evaluation criteria:

The test was considered to be positive if a reproducible and dose-related increase in mutant counts of at least one strain was seen. The increase should be at least:
• twice that of negative controls for strains TA 1535, TA 100, TA 98
• threefold that of negative controls for strain TA 1537
• about 100 mutant colonies higher than negative controls for strain TA 102

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

In the cytotoxicity test marked toxicity was seen at >/=50 µg/plate for TA 98, TA 1537, TA 100 in presence or absence of S9 and for TA 1535 ( S9), at >/=158 µg/plate for TA 1535 (+S9) and TA 102 (+/- S9).
In the plate incorporation assay, cytotoxicity was seen in absence of S9 at 64 µg/plate for TA 1535 and TA 100. The same applied to TA 100, TA 1537, TA 98 and TA 102 in absence of S9 in the preincubation test. Also in the preincubation assay cytotoxicity was seen at >/=32 µg/plate in absence of S9 for TA 1535.

In the plate incorporation test mutant counts about twice the concurrent control level were seen in several concentrations in strain TA 1535 without metabolic activation. This was not considered to be relevant as this was in absence of a dose relationship, as there were no increases in the plate incorporation test and as the mutant counts observed with Bronopol were within the range of the normal fluctuation of negative controls.
In comparison to historical controls (ranging from median 8, semi-Q range 2 to median 10 semi-Q range 2), the concurrent control of TA 1535 in the plate incorporation test was relatively low (6).
Positive control compounds gave a clear positive result.

Strain/concen-tration [µg/plate]	Number of mutant cells
	Plate incorporation test		Preincubation test
	— S9	+ S9	— S9	+ S9
TA 1535:  0                    1                    2                    4                    8                   16                   32                   64 Positive control	6 10 13 15 11 13 13 0 580	10 10 9 11 9 11 10 9 183	11 10 12 11 10 5 0 0 542	12 10 11 12 17 11 9 13 123
TA 100:     0                    1                    2                    4                    8                   16                   32                   64 Positive control	127 121 116 118 113 97 58 0 277	151 137 146 152 154 160 160 133 1719	114 112 104 114 118 137 66 0 272	108 104 86 104 107 111 118 120 1311
TA 1537:  0                    1                    2                    4                    8                   16                   32                   64 Positive control	10 7 8 10 5 9 11 2 114	8 7 10 8 9 9 9 9 298	12 13 12 9 10 11 8 0 115	13 17 14 13 18 15 12 19 248
TA 98:       0                    1                    2                    4                    8                   16                   32                   64 Positive control	36 35 30 42 40 31 36 13 221	37 38 46 45 46 38 45 54 1767	32 37 31 26 28 25 23 0 192	31 31 34 25 25 29 36 32 1458
TA 102:     0                    1                    2                    4                    8                   16                   32                   64 Positive control	251 255 285 272 276 268 247 229 413	240 245 245 253 269 251 262 264 704	213 208 228 220 211 209 189 43 401	199 207 162 190 204 244 214 216 456

Conclusions:

No mutagenic potential of Bronopol was detected in S. typhimurium strains in presence and absence of metabolic activation.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 5 2000 - January 29 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5300 (Detection of Gene Mutations in Somatic Cells in Culture)

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian cell transformation assay

Target gene:

Chinese hamster lung fibroblasts (V79), hypoxanthine-guanin phosphoribosyl transferase (HPRT) proficient

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

The V79 cell line was originally derived from the lung of a male Chinese hamster (Chu and Malling, 1968). The V79 cells used in this study ( designated V79 in th is report) were a kind gift from Prof. G. Speit, University of Ulm, Germany. These cells have since been recloned to maintain karyotypic stability. They have a modal chro­mosome number of 22 and a rapid population doubling time (10 to 14 hours).

V79 cell stocks are stored in liquid nitrogen. Laboratory cultures were maintained in plastic tissue culture vessels at 37°C in a humidified atmosphere containing ap­proximately 5% CO2 Exponential growth of cell cultures was maintained by subculturing at least twice a week. For cell detachment in order to subculture, a solution consisting of 0.1% trypsin and 0.04% EDTA (ethylenediamine-N,N,N',N'-tetraacetic acid) in phosphate buffered saline (PBS) has been employed.

The V79 cells were checked on August 3, 2000 for karyotype stability utilising a modified protocol of the method of Moorhead et al. (1960) and the karyotype (modal chromosomes number: 22) was confirmed. A routine check for mycoplasma was performed on August 10, 2000 using a DNA-Staining Kit (Biochrom) according to the method provided by the supplier. There was no evidence of rnycoplasma contamination.

To keep the number of spontaneous 6-TG resistant mutants at a low level, cell cultures were subcloned by plating about 1,000 cells per culture vessel at least every two weeks. If necessary, the spontaneous frequency of HPRT-mutants was additionally reduced by supplementing the culture medium with thymidine (9 µg/ml), hypoxanthine (10 µg/ml), glycine (22.5 µg/ml) and methotrexate (0.3 µg/ml). A 6-TG sensitive subclone was then used for the HPRT-test.

Media
Cells were maintained in hypoxanthine-free Eagle's Minimal Essential Medium (MEM, Gibco). The hypoxanthine-free MEM has been proven suitable for the growth of V79 cells (Abbondandolo et al., 1977). The MEM was supplemented with nones­sential amino acids, L-glutamine (2 mM), MEM-vitamins, NaHCO3 , penicillin (100 units/ml), streptomycin (100 µg/ml) and heat-inactivated fetal calf serum (final concentration: 10%) (Seromed). This medium is referred to as culture medium. During treatment with Bronopol Bayer, the serum content was reduced to 2%.
For selection of mutants, a hypoxanthine-free culture medium was used, containing 10 µg/ml of 6-thioguanine (6-TG).

Additional strain / cell type characteristics:

other: HPRT proficient

Metabolic activation:

with and without

Metabolic activation system:

S9 mix, Prepared from livers of male Sprague-Dawley rats induced with Arochlor 1254; co-factors were added.

Test concentrations with justification for top dose:

Preliminary cytotoxicity test:
0, 5, 5, 10 and 15 µg/mL (-S9)
0, 5, 5, 10, 15 and 30 µg/mL (+S9)
Mutagenicity test (1st experiment):
0, 1, 2, 4, 8, 10 and 12 µg/mL ( S9)
0, 3, 6, 12, 18, 21, 24 and 27 µg/mL (+S9)
Mutagenicity test (2nd experiment):
0, 3, 6, 9, 12, 15, 18 and 21 µg/mL ( S9)
0, 6, 9, 12, 18 and 21 µg/mL (+S9)

Vehicle / solvent:

deionized water

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9,10-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

Determination of Cytotoxicity:
Exponentially growing V79 cells were plated in 20 ml culture medium in a 250 ml flask (4x10E6cells per flask). For each concentration one culture was available. After attachment (16-24 hours later), cells were exposed without S9 mix to vehicle alone and to a range of concentrations of the test substance for 5 hours in 20 ml medium containing 2% FCS. In experiments with metabolic activation 1 ml of medium was replaced by 1 ml S9 mix. Thereafter, cell monolayers were washed with PBS, tryp­sinized and replated in 5 ml culture medium at a density of 200 cells into each of 3 Petri dishes (0 60 mm). These dishes were incubated for 6 to 8 days to allow colony development.
Thereafter, colonies were fixed with 95% methanol, stained with Giemsa (Merck; stock solution diluted 1 :5 with deionized water) and counted automatically, if auto­matic counting was not interfered by precipitation etc. Cytotoxicity was expressed by comparison of colonies in treated cultures versus vehicle control cultures (relative cloning efficiency).

Nonactivation Assay:
The method is based on the publication of Myhr and DiPaolo (1978). Exponentially growing V79 cells were plated in culture medium at a final volume of 20 ml in two 250 ml flasks per concentration (4x106 per flask) including all control groups. After attachment (16-24 hours later), the cells were exposed for 5 hours in 20 ml culture medium with reduced serum content (2%). The corresponding controls were incubated under the same conditions. Thereafter, cell monolayers were washed with PBS, trypsinized and rep lated in 20 ml culture medium using 1.5x10E6 cells per 250 ml flask and in 5 ml culture medium using 200 cells per Petri dish (0 60 mm). Per culture one flask and 3 Petri dishes were used. The Petri dishes were incubated (normally 6 days) to allow colony development and to determine the cytotoxicity associated with each test substance directly after treatment ("Survival to Treatment").
Cells in 250 ml flasks were incubated to permit growth and expression of induced mutations. Cells were subcultured (= count 1, normally after 3 days) by reseeding 1.5E6 cells into 20 ml medium in 250 ml flasks. At the end of the expression period (= count 2, normally a total of 6 days), cultures were reseeded in Petri dishes (100 mm) at 3E5 cells per dish (8 dishes per culture) in 20 ml culture medium with­out hypoxanthine but containing 10 µg/ml 6-TG for selection of mutants. In addition, 200 cells per dish (60 mm, 3 dishes per culture) were seeded in 5 ml culture me­dium to determine the absolute cloning efficiency for each concentration. After incubation for 6 to 8 days, the colonies were fixed, stained with Giemsa and counted to determine the number of 6-TG resistant colonies in the mutation assay dishes and the number of colonies in the cloning efficiency dishes.
At least two trials will be performed. Mutant frequencies for at least four concentrations should be determined in each trial.

Activation Assay
The activation assay was performed independently. The procedure was identical to the nonactivation assay except for the addition of S9 mix. In these experiments 19 instead of 20 ml culture medium and additionally 1 ml of S9 mix were added to the flasks for the treatment period, resulting in a concentration of 5% S9 mix in the cul­tures. The number of 6-TG resistant mutants and viability were determined as in the nonactivation assay.

Evaluation criteria:

Only concentration with a relative survival, relative population growth and absolute cloning efficiency >/=10% are evaluated. A result is interpreted as positive if the following criteria are met:
a dose-related and reproducible increase in the mutant frequency by at least factor 2-3 over the negative controls (+/- vehicle)
A positive result is considered irrelevant, if the test compound leads to relevant changes in osmolarity of the culture medium in comparison to negative controls.

Statistics:

The statistical analysis relies on the mutant frequencies which are submitted to a weighted analysis of variance as well as to a weighted recursive regression, both with Poisson derived weights (Hsie et al., 1981; Arlett et al., 1989).

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Cytotoxicity:
Pre-test: Slight cytotoxicity was seen at >/=10 µg/mL in absence of S9 and marked cytotoxicity at 30 µg/mL in presence of S9. Accordingly 21 and 27 µg/mL were selected as highest concentrations for the 1st mutagenicity experiment.
Mutagenicity test: Overall, cytotoxicity was seen at concentrations >/=15 µg/mL and >/=18 µg/mL in absence and presence of S9, respectively.

Genotoxicity:
without metabolic activation: Even in absence of a completely clear dose-relationship, there was a reproducible relevant increase in mutant frequency in Bronopol treated cultures reaching occasionally statistical significance in the 2nd experiment.
The positive control induced markedly increased mutant frequencies.

with metabolic activation: Statistically significant increases in mutant frequencies were observed for the 1st experiment, which was not reproducible in the 2nd experiment. Due to the positive results in the experiments in absence of S9, the somewhat equivocal results in presence of S9 were not further evaluated.
The positive control induced markedly increased mutant frequencies.

Table 1. Cytotoxicity Pre-Test

Concentration [µg/mL]	Survival after treatment
	-S9		+S9
	absolute	relative	absolute	relative
Preliminary cytotoxicity test
0  3  5 10 15 30	58.3 59.0 71.0 61.3 77.5 -	100 101 122 105 133 -	57.7 62.3 62.3 43.0 40.7 1.7	100 108 108 75 71 3

Table 2. Mutagenicity

	Survival after treatment [% of control]	Relative population growth [% of control]	Relative cloning efficiency° [% of control]	Mutation frequency [mutants/10^6cells]
1^stexperiment, - S9
Negative control  0 (vehicle)  1  2  4  6  8 10 12 Positive control	123 100 130 140 159 106 134 139 94 58	72 100 92 93 94 103 116 93 86 76	139 100 119 82 97 98 53 52 65 53	13.3 4.6 9.1 14.9 8.0 13.0 9.7 21.9 16.7 6785.5*
2^ndexperiment, -S9
Negative control  0 (vehicle)  3  6  9 12 15 18 21 Positive control	108 100 148 114 72 54 44 45 t 38	131 100 31 34 49 76 25 18 - 33	119 100 90 117 101 91 126 115 - 67	3.3 1.2 14.0 21.2* 15.0* 24.3* 14.5* 17.6 - 1094.6*
1^stexperiment, + S9
Negative control  0 (vehicle)  3  6 12 18 21 24 27 Positive control	87 100 146 131 94 18 t t t 54	85 100 33 47 64 12 - - - 46	82 100 105 93 80 75 - - - 90	0.7 0.6 0.9 2.8* 8.6* 16.1* - - - 159.9
2^ndexperiment, +S9
Negative control  0 (vehicle)  6  9 12 15 18 21 Positive control	120 100 131 128 131 115 97 99 97	125 100 110 88 103 90 94 49 136	57 100 75 73 86 94 95 62 55	5.4 2.9 6.2 4.9 3.5 4.2 8.1 11.3 52.3*

° after expression

* p <0.05 (one-sided Dunnett test)

t = not cloned as cytotoxic

Conclusions:

Bronopol Bayer was tested in the V79/HPRT-test in concentrations ranging from 1 µg/ml to 21 µg/ml without S9 mix and from 3 µg/ml to 27 µg/ml with S9 mix. Under both activation conditions, clear cytotoxic effects were induced. Bronopol Bayer induced at least without S9 mix biologically relevant increases in mutant frequencies. The positive controls EMS and DMBA induced a distinct mutagenic effect and thus demonstrated the sensitivity of the test system and the activity of the used S9 mix. Due to this sensitivity, Bronopol Bayer is considered to be mutagenic in the V79/HPRT forward mutation assay.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Cytogenicity in vivo

CD1 mouse (micronucleus test), up to 120 mg/kg bw p.o.: negative (GLP, OECD 474; Boots Company PLC Research Department, 1986/TX86049)

Mouse (dominant lethal assay), up to 100 mg/kg bw/day for 6 days; p.o or 10 mg/kg bw i.p.:negative (no GLP, no guideline, Boots Company Limited Research Department, 1974/TX74034)

DNA Repair

Wistar rat (UDS assay), up to 150 mg/kg bw p.o.: negative (GLP, OECD 486; Covance Laboratories Limited, 1998/TXO98007)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The whole report summarizes the data of three mutagenic testing approaches, which were conducted in 1974, prior to the implementation of guidelines. Furthermore, no data referring to purity and stability of the tested substance were provided. However, the results obtained can be considered as scientifically acceptable.

Principles of method if other than guideline:

Method: other: according to the method of Bateman AJ (Heredity 12: 213-232, 1958) and Bateman AJ and Epstein SS (in Chemical Mutagens, Vol. 2, ed. Hollaender, Plenum Press, 1971)

GLP compliance:

no

Type of assay:

rodent dominant lethal assay

Species:

mouse

Strain:

other: OLAC

Sex:

male

Details on test animals or test system and environmental conditions:

Animals: OLIC ex-breeding stock males of proven fertility. OLIC virgin females, 12 weeks old when mated.
Temperature range 22-24 °C; daylight supplemented by fluorescent tubes on a controlled li.ght/dark cycle, 16 hours on/8 hours off.
Diet: Oxoid cubes and water ad libitum.
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.

Route of administration:

other: gavage and i.p., respectively

Details on exposure:

Application volume: 0.1 ml/10 g bw

Duration of treatment / exposure:

Oral dosage: once daily for 6 consecutive days.
Intraperitoneal dosage: single i.p. injection

Frequency of treatment:

Substance: once daily
Positive control: once (last day of substance dosing)

Post exposure period:

4 hours (i.e. 4 hours after the last dosing, the males were housed together with the females for mating.)

Dose / conc.:

20 mg/kg bw/day (actual dose received)

Remarks:

oral application

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

oral application

Dose / conc.:

10 mg/kg bw (total dose)

Remarks:

intraperitoneal application, (0.9% saline)

No. of animals per sex per dose:

10 for test group and positive control
20 for negative control

Control animals:

other: Twenty 20 males served as negative control. They were treated orally with water.

Positive control(s):

Ten males mice were used for positive control.
Positive controls received a single i.p. injection of 25mg/kg bw tris(2-methyl-1-aziridinyl)-phosphine oxide (METEPA) in 0.1 ml/10 g bw of 0.9% physiological saline.

Tissues and cell types examined:

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.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Positive controls validity:

valid

Additional information on results:

Summary of the main findings:

Oral treatment with 100 and 20 mg/kg bw bronopol:
Four of the 10 males treated with 100 mg/kg bw bronopol died within 2 weeks (Table 1). The implantation rate was significantly reduced in weeks 2 and 3; this effect was further accompanied by a reduction in fertility as indicated by a decreased pregnancy rate observed in this group (Table 2, 3). No such effects were seen at 20 mg/kg bw. Moreover, for both test doses, no increase in the frequency of dead implants were seen. In fact, for 100 mg/kg bw, the number of dead implants/pregnant female ranged was 0.5, 0.4, 0.2 and 0.5 respectively after 1, 2, 3 and 4 weeks; the corresponding control values respectivley were 0.6, 0.5, 1.0 and 0.7. For 20 mg/kg bw, the values also were within control range.

Injection (i.p.) of 10 mg/kg bw bronopol:
One male from the group receiving 10 mg/kg i.p. died during the third week (Table 1). The implantation rate was significantly reduced in week 4; this effect was further accompanied by a reduction in fertility as indicated by a decreased pregnancy rate observed in this group (Table 2, 3). In this group, a decrease in the frequency of dead implants was seen after 4 weeks (0.2 dead implant per pregnant female versus 0.7 for control).

Positive control (METEPA, 25 mg/kg bw, i.p. injection):
In this group, the number of dead implants per pregnant female was significantly increased compared to untreated control. Mean values of 3.5, 3.9 and 3.1 were reported respectively after week 1, 2 and 3, versus 0.6, 0.5 and 1.0 for controls.

For the highest orally applied dose of bronopol (i.e. 100 mg/kg bw, 6 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 (Table 2).

Table 1: Cumulative mortalities of Bronopol-treated animals.

Dose	Cumulative deaths per week (W)
	W1	W2	W3	W4
0 mg/kg bw (negative control; oral)	0	0	0	0
Bronopol, 6 x 100 mg/kg bw(oral)	1	4	4	4
Bronopol, 6 x  20 mg/kg bw(oral)	0	0	0	0
Bronopol, 1 x 10 mg/kg bw(i.p.)	0	0	1	1
Positive control (METEPA 1x 25 mg/kg bw (i.p.)	0	0	0	0

Table 2: Number of pregnant females per week (per cent).

Dose	Pregnant females per week (W)
	W1	W2	W3	W4
0 mg/kg bw (negative control; oral)	42 (70%)	34 (57%)	33 (55%)	34 (56%)
Bronopol, 6 x 100 mg/kg bw(oral)	8 (44%)	7 (39%)	9 (50%)	10 (56%)
Bronopol, 6 x  20 mg/kg bw(oral)	20 (67%)	22 (73%)	24 (80%)	23 (76%)
Bronopol, 1 x 10 mg/kg bw(i.p.)	9 (33%)	13 (45%)	14 (48%)	12 (44%)
Positive control (METEPA 1x 25 mg/kg bw (i.p.)	24 (83%)	20 (67%)	21 (70%)	17 (56%)

Table 3: Implants per pregnant female (live, dead, total).

Dose	Live implants per pregnant female and week (W)
	W1	W2	W3	W4
0 mg/kg bw (negative control; oral)	9.4	9.6	9.4	10.4
Bronopol, 6 x 100 mg/kg bw(oral)	8.5	6.7	6.0*	9.6
Bronopol, 6 x  20 mg/kg bw(oral)	9.8	8.4	9.3	10.6
Bronopol, 1 x 10 mg/kg bw(i.p.)	9.2	8.1	9.9	8.7*
Positive control (METEPA 1x 25 mg/kg bw (i.p.)	7.4***	4.2***	6.9**	8.7*
Dose	Dead implants per pregnant female and week (W)
	W1	W2	W3	W4
0 mg/kg bw (negative control; oral)	0.6	0.5	1.0	0.7
Bronopol, 6 x 100 mg/kg bw(oral)	0.5	0.4	0.2	0.5
Bronopol, 6 x  20 mg/kg bw(oral)	1.0	0.9	0.5	0.7
Bronopol, 1 x 10 mg/kg bw(i.p.)	0.7	1.2	0.3	0.2*
Positive control (METEPA 1x 25 mg/kg bw (i.p.)	3.5***	3.9***	3.1***	1.2
Dose	Total Number of implants per female and week (W)
	W1	W2	W3	W4
0 mg/kg bw (negative control; oral)	10.0	10.1	10.4	11.1
Bronopol, 6 x 100 mg/kg bw(oral)	9.0	7.1*	6.2*	10.1
Bronopol, 6 x  20 mg/kg bw(oral)	10.8	9.3	9.8	11.2
Bronopol, 1 x 10 mg/kg bw(i.p.)	9.9	9.3	10.2	8.9*
Positive control (METEPA 1x 25 mg/kg bw (i.p.)	10.9	8.1*	10.0	9.9

*, p=<0.05; **, p=<0.01; ***, p=<0.001

Conclusions:

CL-Freetext:
Overt signs of toxicity were seen after 6 daily oral doses
of 100 mg/kg Bronopol in male mice, but not after 6 daily oral doses of 20 mg/kg, nor after a single i.p. injection of 10 mg/kg. Reduced fertility was seen in the high dose group, but not in the others. This effect was considered to be related to toxicity rather than dominant lethality. No increase in dead implants was observed after Bronopol treatment. From these results it was concluded that Bronopol lacks cytotoxicity for mouse meiotic and post-meiotic sperm stages.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study conducted in accordance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Principles of method if other than guideline:

OECD repeated sampling micronucleus assay:
Report of the UKEMS Sub-Committee on Guidelines for Mutagenicity Testing :Part I (February, 1983), Ed :Brian J. Dean p 134
(The United Kingdom Environmental Mutagen Society).

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

Bodyweight range : 30-35 g (males) and 25-30 g (female) at collection
Age : 9 weeks at collection
Normally in the range: 22+/- 3°C. Humidity was measured daily.
Light cycle: A 12/12 hour light/dark cycle will be maintained, switching an at 0600 h.
Air change: The ventilation system provides 15 air changes per hour in the vented cabinets housing the animals.
Diet: Labsure Animal Diet (CRM Nuts) and tap water will be available at all times.

Route of administration:

oral: gavage

Vehicle:

sterile double distilled water

Details on exposure:

The mice received single oral application of the test substance by gavage.
Bronopol was tested at following doses: 80 mg/kg bw (12 animals per sex) and
160 mg/kg bw (24 animals/sex), the latter dose being the maximum dose tolerated by the mice.
8 males and 8 females per dose group per sampling time (high dose), 4 males and 4 females per dose group per sampling time (low dose, positive and negative controls).

Frequency of treatment:

single application

Post exposure period:

24, 48, 72 hours after treatment

Dose / conc.:

80 mg/kg bw (total dose)

Remarks:

actual ingested

Dose / conc.:

160 mg/kg bw (total dose)

Remarks:

actual ingested

No. of animals per sex per dose:

80 mg/kg bw (12 animals per sex)
160 mg/kg bw (24 animals/sex)
Positive control group (Cyclophosphamide, 75 mg/kg bw): 12 males and 12 females
Negative control group (purified water B.P.): 12 males and 12 females

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide (75 mg/kg bw)

Tissues and cell types examined:

After 24, 48 and 72 hours following treatment, 4 animals/sex and 8 animals/sex, respectively from the 80 and the 160 mg/kg bw group were sacrificed, and the femoral bone marrow was extracted, prepared and examined.

Details of tissue and slide preparation:

The polychromatic/normochromatic erythrocytes ratio and the number of micronuclei in 1000 polychromatic erythrocytes per animal.

Statistics:

The statistical assessment of the findings was based on the Analysis of variance, the Freeman-Tukey transformation and the use of F-distribution tables.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Additional information on results:

The main findings can be summarized as follows:
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 hours, 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 withoutbeing statistically significant.

Table 1: Ratio of polychromatic to normochromatic erythrocytes in the femoral bone marrow of bronopol treated CD1-mice:

Test group	Dose  (mg/kg bw)	Ratio of polychromatic/normochromatic erythrocytes
		24 h		48 h		72 h
		M	F	M	F	M	F
Neg. Cont.*	-	1:0.92	1:1.02	1:0.67	1:0.79	1: 0.95	1: 1.04
BN	80	1:1.05	1:0.87	1:1.72	1:1.40	1:6.57	1:4.06
	160	1:0.82	1:0.85	1:0.99	1:0.73	1:3.52	1:0.81
Pos. Cont.**	75	1:2.31	1:0.87	1:7.65	1:2.18	1:15.77	1:2.37

BN, Bronopol; *, purified water; **, Cyclophosphamide

Table 2: Incidence of micronuclei (MN) in the polychromatic erythrocytes (PCE) of bronopol treated CD1-mice at time point 24 h.

Test group		Incidence of micronuclei (MN) in the polychromatic erythrocytes (PCE) of bronopol treated CD1 Time point 24 hours
		Males		Females
		MN/1000 PCE/mouse	Mean (%)	MN/1000 PCE/mouse	Mean (%)
Negative control  (Purified water)	1	2	0.23	3	0.23
	2	1		1
	3	2		3
	4	4		2
Bronopol 80 mg/kg bw	1	2	0.13	0	0.05
	2	2		1
	3	1		0
	4	0		1
Bronopol 160 mg/kg bw	1	- (Mouse died)	0.10	2	0.10
	2	0		0
	3	0		0
	4	0		3
	5	1		- (Mouse died)
	6	3		0
	7	2		1
	8	1		1
Positive control (Cyclophosphamide, 75 mg/kg bw)	1	39	2.73**	42	2.25**
	2	13		7
	3	34		24
	4	23		17

**, Significantly different from negative control (p<0.01)

Table 3: Incidence of micronuclei (MN) in the polychromatic erythrocytes (PCE) of bronopol treated CD1-mice at time point 48 h.

Test group		Incidence of micronuclei (MN) in the polychromatic erythrocytes (PCE) of bronopol treated CD1 Time point 48 hours
		Males		Females
		MN/1000 PCE/mouse	Mean (%)	MN/1000 PCE/mouse	Mean (%)
Negative control  (Purified water)	1	0	0.13	0	0.15
	2	1		0
	3	2		2
	4	2		4
Bronopol 80 mg/kg bw	1	2	0.23	0	0.20
	2	3		5
	3	4		1
	4	0		2
Bronopol 160 mg/kg bw	1	- (Mouse died)	0.18	2	0.24
	2	2		5
	3	0		3
	4	- (Mouse died)		- (Mouse died)
	5	- (Mouse died)		2
	6	3		0
	7	2		- (Mouse died)
	8	2		- (Mouse died)
Positive control (Cyclophosphamide, 75 mg/kg bw)	1	6	0.78**	11	1.25**
	2	5		9
	3	7		9
	4	13		21

**, Significantly different from negative control (p0.01)

Table 4: Incidence of micronuclei (MN) in the polychromatic erythrocytes (PCE) of bronopol treated CD1-mice at time point 72 h.

Test group		Incidence of micronuclei (MN) in the polychromatic erythrocytes (PCE) of bronopol treated CD1 Time point 72 hours
		Males		Females
		MN/1000 PCE/mouse	Mean (%)	MN/1000 PCE/mouse	Mean (%)
Negative control  (Purified water)	1	1	0.13	2	0.28
	2	0		2
	3	0		3
	4	4		4
Bronopol 80 mg/kg bw	1	1	0.18	1	0.17
	2	1		1
	3	3		3
	4	2		- (Mouse died)
Bronopol 160 mg/kg bw	1	1	0.26	0	0.33
	2	1		2
	3	1		3
	4	2		3
	5	4		3
	6	3		8
	7	6		4
	8	3		3
Positive control (Cyclophosphamide, 75 mg/kg bw)	1	2	0.30	5	0.25
	2	3		2
	3	1		0
	4	6		3

**, Significantly different from negative control (p0.01)

Conclusions:

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.

Reference 3

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study conducted in accordance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

GLP compliance:

yes

Type of assay:

unscheduled DNA synthesis

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

Source: Charles River Ltd (UK)
Age/weight:
Range-finding pre-test:
The rats were about 44 to 58 days old and weighed between 160 and 233 g.
Main assay:
The rats were about 56 to 59 days old and weighed between 204 and 286 g.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Vehicle: Acidified water (pH 4)
Total volume applied: 10 ml/kg bw

Frequency of treatment:

single application

Post exposure period:

Range-finding pre-test: 4 days
Main assay:
Experiment 1: 12 –14 h
Experiment 2: 2-4 h

Dose / conc.:

100 mg/kg bw (total dose)

Remarks:

pre-test

Dose / conc.:

125 mg/kg bw (total dose)

Remarks:

pre-test

Dose / conc.:

150 mg/kg bw (total dose)

Remarks:

pre-test

Dose / conc.:

200 mg/kg bw (total dose)

Remarks:

pre-test

Dose / conc.:

60 mg/kg bw (total dose)

Remarks:

main test

Dose / conc.:

150 mg/kg bw (total dose)

Remarks:

main test

No. of animals per sex per dose:

Range-finding pre-test: 4 test groups comprising 3 rats each (total: 12 rats).
Main assay: 2 test groups comprising 25 rats each (total: 50 rats).
Negative (vehicle) control group: total of 10 rats (5 per experiment)
Positive controls: 2-Acetamidofluorene (2-AAF) in corn oil, 75 mg/kg bw (5 rats)
Dimethylnitrosamine (DMN) in purified water, 10 mg/kg bw (5 rats)

Control animals:

yes, concurrent vehicle

Positive control(s):

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.

Tissues and cell types examined:

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.
Parameters:
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)
Cytoxicity (i.e. cell viability) and conspicuous changes in cell morphology (trypan blue exclusion technique)
technique. DNA damage and repair was measured by incorporation of 3H-thymidine using autoradiography technique.

Details of tissue and slide preparation:

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).

Evaluation criteria:

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.

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Clinical signs:
Range-finding pre-test:
No clinical signs of toxicity were seen at the both lowest tested doses of 100 and 125 mg/kg bw. At 150 and 200 mg/kg bw, clinical signs of toxicity were seen, which included abnormal gait and abnormal breathing. All 3 animals of the 150 mg/kg bw group as well as 2/3 animals of the 200 mg/kg bw group died or were sacrificed in extremis within day 1 to 3 of observation. On the basis of the findings of this pre-test, 150 mg/kg bw was selected as maximum tolerable dose for the main assay.
Main assay:
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 animals showed similar symptoms. No mortality was seen. Body weights were inconspicuous

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.

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.

Table 1: Group mean net grain count values of experiment 1 (sacrifice time 12 -14 h) and experiment 2 (2 -4 h).

Experiment 1:

Group mean net grain count values, sacrifice time 12 – 14 h
Test group	Net nuclear grain count (NNG)		Net grain count of cells in repair		% of cells in repair (NNG >= 5)
	Mean	SD	Mean	SD	Mean	SD
Neg. Cont.	-2.5	1.8	7.3	0.0	1.0	1.7
Bronopol 60 mg/kg bw	-1.9	0.7	6.1	1.0	1.7	1.2
Bronopol 150 mg/kg bw	-1.6	0.8	6.6	0.2	2.3	0.6
2-AFF 75 mg/kg bw	8.8	2.6	11.8	2.2	67.7	16.3

2-AFF: 2-Acetamidofluorene, positive control

Experiment 2:

Group mean net grain count values, sacrifice time 2 –  4 h
Test group	Net nuclear grain count (NNG)		Net grain count of cells in repair		% of cells in repair (NNG >= 5)
	Mean	SD	Mean	SD	Mean	SD
Neg. Cont.	-2.6	0.3	5.8	1.2	1.3	0.6
Bronopol 60 mg/kg bw	-1.8	0.5	5.9	0.0	1.3	2.3
Bronopol 150 mg/kg bw	-2.0	0.2	8.0	2.1	3.0	3.6
DMN 10 mg/kg bw	15.2	5.9	19.0	4.9	78.3	12.5

DMN: Dimethylnitrosamine, positive control

Conclusions:

The test substance bronopol (Myacide AS) showed no genotoxic potential within the in vivo UDS assay performed with rats.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro study: Bacterial systems

In a GLP-conform study, S. typhimurium TA1535, TA1537, TA98, TA100 and TA102 were exposed to Bronopol in the preincubation test and plate incorporation test with and without metabolic activation according to OECD TG 471 (Lanxess, 2000/PH 30497). Concentrations of 50, 158, 500, 1581, 5000 µg/plate and 1-64 µg/plate were used for the Cytotoxicity tests and Mutagenicity tests, respectively. Concurrent vehicle and positive controls were performed. No increase in revertants was recorded up to cytotoxic concentrations. The controls were valid. Bronopol showed no mutagenic potential in the Ames test.

This GLP-conform guideline study is classified as acceptable (key study) and was used for classification.

The test substance Bronopol (purity 99.7 %) was tested negative for mutagenicity in another 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 (supporting study). However, this study was not used for classification since no strains like S. typhimutium TA102 or E.coli WP2 uvrA were testedwhich have an AT base pair at the primary reversion sitecapable of detecting certain oxidizing mutagens, cross-linking agents and hydrazines. Moreover, no guideline was 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

Bronopol was assessed for its potency to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster lung fibroblasts (V79) cells in vitro in a GLP-conform study according to OECD TG 476 (Lanxess, 2001/PH 30670). Two independent experiments were carried out, both with and without the addition of liver S9 mix from Arochlor 1254 induced rats (exogenous metabolic activation). According to an initial range-finding cytotoxicity test for the determination of the experimental doses, the following concentrations were tested and evaluated for gene mutations:

Mutagenicity test (1st experiment):

           0, 1, 2, 4, 8, 10 and 12 µg/mL (-S9)

           0, 3, 6, 12, 18, 21, 24 and 27 µg/mL (+S9)

Mutagenicity test (2nd experiment):

           0, 3, 6, 9, 12, 15, 18 and 21 µg/mL (-S9)

           0, 6, 9, 12, 18 and 21 µg/mL (+S9)

Following attachment of the cells for 16 - 24 hours, cells were treated with the test substance for 5 hours in the absence and presence of metabolic activation. Subsequently, cells were cultured for 6 days and then selected in 6-thioguanine-containing medium for another 6-8 days. Finally, the colonies of each test group were fixed, stained with Giemsa and counted. The vehicle controls gave mutant frequencies within the range expected for the V79 cell line. Both positive control substances, ethyl methanesulfonate (EMS) and 7,12-dimethylbenz[a]-anthracene (DMBA), led to the expected statistically significant increase in the frequencies of forward mutations. Under both activation conditions, clear cytotoxic effects were induced. Bronopol induced at least without S9 mix biologically relevant increases in mutant frequencies. The positive controls EMS and DMBA induced a distinct mutagenic effect and thus demonstrated the sensitivity of the test system and the activity of the used S9 mix. Due to this sensitivity, Bronopol is considered to be mutagenic in the V79/HPRT forward mutation assay.

This GLP-conform guideline study is classified as acceptable (key study) and was used for classification.

Another 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 (supporting 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). This study was not used for classification since an GLP-conform guideline study is avaliable.

 

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 a GLP conform assay according to OECD guideline 473, V79 CHL fibroblasts were tested for chromosome aberrations in doses up to cytotoxic concentrations with and without metabolic activation (S-9 mix from Aroclor 1254 induced rat liver) (Lanxess, 2000/PH 30397). Results without metabolic activation: After 18 hours of incubation there was no relevant increase in the number of aberrant metaphases +/- gaps. While not attaining statistical significance the number of metaphases with exchanges exceeded the historical control range at the highest concentration of 7 µg/mL. After 30 hours of incubation there was a relevant and statistically significant increase in aberrant metaphases also at 7 µg/mL. A slight increase of polyploid metaphases was observed at 7 µg/mL after 30 hours incubation. The positive control compound also revealed a positive response in aberrant metaphases.Results with metabolic activation: After 18 hours of incubation there was a relevant, statistically significant increase in the number of aberrant metaphases +/- gaps. While not attaining statistical significance the number of metaphases with exchanges exceeded the historical control range at the highest concentration of 23 µg/mL. After 30 hours of incubation there was a relevant and statistically significant increase in aberrant metaphases also at 23 µg/mL. The positive control compound also revealed a positive response in aberrant metaphases. Therefore, Bronopol is considered to be clastogenic in this in vitro study both in presence and absence of S9.

This GLP-conform guideline study is classified as acceptable (key study) and was used for classification.

In line with the results mentioned above, Bronopol was tested positive in another 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 (supporting study). No guideline mentioned; however the study to a large extent was in accordance with the requirements of OECD guideline 473. This study was not used for classification since an GLP-conform guideline study is avaliable.

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 (supporting 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.

The negative result was confirmed in two other independent GLP-conform in vivo micronucleus tests according to OECD TG 474 (Dow, 2001/DR-0365 -7736 -001; Lanxess, 2001/PH-31370).

 

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/TXO98007). 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 (Macide 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.

The negative result was confirmed in another GLP-conform in vivo UDS assay according to OECD TG 486 (Dow, 2001/K-081547 -001).

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. In a gene mutation test in cultured mammalian cells (HPRT assay) Bronopol was tested positive in absence of metabolic activation. In an in vitro chromosomal aberration test in cultured V79 cells, positive effects were noted 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

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on the available data, the substance is not considered to be classified for genetic toxicity under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.