Bronopol

Carcinogenicity

Description of key information

In an oral chronic toxicity study (Huntigdon Research Centre, 1976), systemic toxicity was observed at the mid and high dose level (32 and 142 mg/kg bw/day) but not at the low dose level (7 mg/kg bw/day). A carcinogenic potential for Bronopol could not be evidenced in this study.

This is confirmed by the results of a dermal chronic toxicity study in mice (Huntigdon Research Centre, 1975). In this study, an increased incidence of skin papilloma was reported for the highest tested concentration of Bronopol (0.5%); however, these tumours rather resulted from the irritant potential of Bronopol than from a carcinogenic potential of Bronopol. Therefore, a carcinogenic potential could not be evidenced for Bronopol under the test conditions used.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: oral

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 test substance bronopol was determined in terms of batch numbers (batch No: CT 92495T used for the test period ranging from week 1 to week 51, and batch No: CT 95274W used for the test period ranging from week 52 to week 104), but no further details were provided.The study was conducted prior to the implementation of guideline and GLP was compulsory at this time. The stability of the test substance in aqueous solution was monitored at a later time point (see addendum). However, all data taken together are of scientific acceptability.

Principles of method if other than guideline:

Method: the study was conducted prior to the implementation of guideline

GLP compliance:

no

Species:

rat

Strain:

other: CD, caesarian-derived strain of Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Age: 28 +/- 1 day.
Acclimatization: 4 days; Predosing period: 1 week.
Housing: 5 animals a cage.
Animal room temperature and relative humidity were controlled at 21 +/- 2°C and 50 +/- 5% respectively, and lighting was controlled to give 12 hours light (8 a.m. to 8 p.m. B.S.T.) and 12 hours dark per 24 hours.
Diet: All rats had free access to standard laboratory rat food, Spratt's Laboratory Diet 1.
Source: Charles River Laboratories, St. Aubin-les-Elbeuf, France.

Route of administration:

oral: drinking water

Vehicle:

water

Duration of treatment / exposure:

104 weeks

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
10, 40, 160 mg/kg bw/day
Basis:
nominal in water

No. of animals per sex per dose:

Each test group of the main test series comprised 45 animals/sex/group whereas each test group of the satellite
series comprised 15 animals/sex/group.

Control animals:

yes, concurrent no treatment

Details on study design:

Post-exposure period: None

Observations and examinations performed and frequency:

The animals of the main test series were used for evaluation of the carcinogenic potential of the test substance.
The animals of the satellite series were used for the evaluation of haematological and clinical-chemical
parameters, and for urinalysis. These animals were not considered for carcinogenicity evaluation.
Blood samples were removed at defined time points (Week 26, 52, 78 and 102) from the orbital sinus of fasted animals for the purpose of examination of haematological (Packed cell volume, haemoglobin, red cell count, mean corpuscular haemoglobin concentration, mean cell volume, total white cell count, differential count (neutrophils, lymphocytes, eosinophils, basophils, monocytes), platelet count, and thrombotest) and clinical-chemical parameters (Plasma urea, plasma glucose, total serum proteins, serum protein electrophoresis and albumin/globulin (AG) ratio, serum alkaline phosphatase (SAP), serum glutamic-pyruvic transaminase (SGPT), sodium and potassium).
Urinalysis:
Time points: Test groups 1 and 4: Week 26, 52, 77 and 103. Test groups 2 and 3: Week 77 and 103.
Parameters: Test groups 1 and 4: Specific gravity, pH, protein, reducing substances, glucose, ketones, bile pigments, urobilin, haemoglobin.
Test groups 2 and 3: Haemoglobin, microscopy of spun deposits (examination was repeated for verification in case of animals showing haemoglobinuria).

Body weight: The body weight of each rat was recorded at test initiation and weekly thereafter.
Food consumption: The quantity of food consumed was recorded for each cage of rats (each cage contained 5 animals) and the mean weekly intake was calculated.
Food efficiency: Food efficiency was assessed by calculation of the mean food conversion ratios (FCR values) during the period of fastest growth (week 1 to 24) as weights of food consumed per unit gains in body weight.
Water consumption: Water consumption was measured daily and the mean weekly intake was calculated.
Clinical signs: The animals were regularly observed for clinical symptoms of toxicity, changes in behaviour, and mortalities.
Ophthalmoscopic examination: Ophthalmoscopic examinations were conducted on the animals of the control and the high dose groups (i.e. 0 and 160 mg/kg bw/day) of the main test series only. Examination time points were: prior test initiation, week 0 (pre-dosing period), week 26, week 52, week 78 and week 103.

Sacrifice and pathology:

At test ending, all surviving animals of both test series (main and satellite) were sacrificed for the purpose of necropsy and were subjected to organ weighing and to gross pathological and histopathological examination. Animals that died or were sacrificed in extremis during the test period also were subjected to gross pathological and histopathological examination for determination of the cause of death.

Gross pathology: All superficial tissues including urogenital orifices, tails, auricles, auditory meatus, and painted skin areas were checked for abnormalities such as swelling, distortion or other evidence of tumor formation. The nares, mouth, tongue, pharynx and auditory region were examined. The cranial roof was removed for examination of the brain, the pituitary gland and the cranial nerves. The subcutaneous tissues including regional lymph nodes, mammary glands and salivary glands were examined. The abdominal and thoracic contents were examined. All gross abnormalities were recorded.
Organ weights: All surviving animals of both test series, at terminal sacrifice
Absolute and relative weights were determined for liver, kidneys, adrenals, testes, uterus, ovaries, spleen, lungs, brain, heart, seminal vesicles, prostate, pituitary and thyroid.
Histopathology: All surviving animals of both test series, at terminal sacrifice.
Samples of following organs/tissues were taken, fixed in buffered 10% formalin and processed for histological examination:
All abnormal tissues, brain, pituitary, thymus, salivary glands, stomach, caecum, ileum, liver, kidneys, spleen, heart, lungs, gonads, uterus, adrenals, urinary bladder, lymph nodes, pancreas, bone marrow, eye (fixed in Davidson´s fixative), blood smears.
For microscopical examination, the tissue samples were embedded in paraffin; the embedded samples were then sectioned, and the sections were Haematoxylin/Eosin stained. Liver and kidney samples were frozen and frozen sections were stained for fat with Oil Red O and for glycogen (liver) or basement membranes (kidney) with periodic acid Schiff reagent. Additional sections from tissues in which colonies of microorganisms were seen were prepared and stained with Gram stains.
Selected samples for microscopical examination:
All tissue listed above and collected from 10 animals/sex from the control group and from 12 males and 15 females of the high dose group (main test series) were subjected to microscopical examination.
Samples of following organs/tissues were taken, fixed in buffered 10% formalin and were not processed for histological examination in the first instance:
Oesophagus, spinal cord, tongue, jejunum, trachea, aorta, prostate, seminal vesicles, female mammary gland, sciatic nerve, bone, skeletal muscle, skin, second eye (fixed in Davidson´s fixative).



Neoplastic findings: All rats of the main test series were examined for neoplastic changes.
Gross abnormalities and lesions suggestive of neoplasia were recorded. Following series of organs was
considered for tumorigenicity screening: Adrenals, thyroids, ovaries, liver, spleen, lymph nodes and pituitary
gland. Blood smears also were examined for abnormalities indicative of neoplastic change, and when such
abnormalities were seen, an examination of bone marrow was added.

Statistics:

Original study: The statistical assessment of intergroup differences was based on Student´s t-test. The incidence of mortality and of progressive glomerulonephrosis respectively was compared by means of a 2x2 one-tailed contingency test.
Statistical re-assessment of mortality, incidence of skin papilloma tumour, and incidence nodular hyperplasia of the liver (Huntingdon Life Sciences Ltd (1998), Report No: TXA93007):
In an additional statistical study conducted in 1998, the incidences of mortality and forestomach papilloma tumours were assessed by means of life-table analysis and log-rank analysis, according to:
Kaplan EL, Meier P (1958) Nonparametric estimation from incomplete observations. J. Am. Statist. Assoc., 53, 457-481
Mantel N (1966 ): "Evaluation of survival data and two new rank order statistics arising in its consideration". Cancer Treat Rep, 50 :163-170
Peto R (1974) Guidelines on the analysis of tumor rates and death rates in experimental animals. Br J. Cancer 29:101 – 105
Peto R et al. (1980) Guidelines for simple, sensitive significance tests for carcinogenic effects in long-term animal experiments. In: Long term and Short term Screen Assays for Carcinogens: A critical Appraisal. IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Supplement 2, pp. 311-426. International Agency for Research on Cancer, Lyon.

Details on results:

Body weigth gain (see table 1):
For the males of the high dose group, body weight gain was decreased in comparison to control starting from week 3 of treatment: The differences against control values were of statistical significance throughout the whole test period. From week 78 til test ending, a significant loss in mean body weight could be evidenced, which however also was related to the high mortality observed towards test ending. For the males of the 40 mg/kg bw group, body weight gain was found to be significantly below control for the period ranging from week 26 to week 78 of treatment. Body weight gain of the males treated with the lowest test dose of 10 mg/kg bw/day was similar to control and therefore inconspicuous. For the females of the high dose group, no differences in body weight gain were seen during the first weeks of treatment. Thereafter and throughout the remaining time of treatment, body weight gain almost was significantly lowered compared to control, and from week 78 until test ending, a significant loss in mean body weight could be evidenced. Body weight gains for the females of the low and mid dose groups almost were similar to control and inconspicuous.
Food consumption (see table 2):
For the males of the high dose group, food consumption was significantly reduced in comparison to control starting from week 14 of treatment til test ending. A slight reduction in food consumption also was reported for the males of the 40 mg/kg bw group for the period between week 53 and 78 of treatment. For the females of the high dose group, food consumption was similar to control during the first 78 weeks of treatment; thereafter, a slight reduction in food consumption was observed.
Water consumption (see table 3):
Prior initiation of treatment, water consumption was similar for all groups of animals. Starting from week 1 of dosage and throughout the whole test period, all treated animals showed reduced water consumption compared to control, which furthermore was dose-related. The effect was more pronounced for treated males than for treated females. The reduction in water consumption rather was due to a palatability problem that to the treatment as such.
Test substance intake: The group mean bronopol intakes throughout the whole treatment period of 104 weeks were calculated on the basis of water consumption and body weight and were as follows:
Group Expected dosage Achieved dosage (mg/kg bw/day)
Males Females
2 10 mg/kg bw/day 10.5 mg/kg bw/day 10.4 mg/kg bw/day
3 40 mg/kg bw/day 40.2 mg/kg bw/day 40.7 mg/kg bw/day
4 160 mg/kg bw/day 152.2 mg/kg bw/day 158.4 mg/kg bw/day



Result (carcinogenicity): negative

Clinical symptoms of toxicity: During the last year of treatment, a reduction in grooming activity was noted in the groups treated with 160 mg/kg bw/day of test substance. No further treatment-related effects were reported.
From week 9 to 10 of treatment all animals suffered from a viral disease, which occasionally affects rats of the used strain. The viral disease was identified as sialodacryoadenitis and persisted for 2 to 3 weeks; during this time, the rats showed reduced appetite and decrease in body weight gain and body weight. Thereafter, the rats recovered and by week 12, they appeared normal. This disease did not affect the test conduct.
Mortality (see table 4): An increase in mortality was observed in the 160 mg/kg bw groups when compared to controls; mortality affected more male animals than females. In the high dose group of the main test series the difference to control was statistically significant. For the remaining groups, recorded mortalities were within control range (see Table 4).

Ophthalmoscopic examination: Examination of the eyes revealed no treatment-related effects.
Haematology: No treatment-related and/or biologically relevant differences between treated and control animals could be evidenced.
Clinical Chemistry: No treatment-related and/or biologically relevant differences between treated and control animals could be evidenced.
Urinanalysis: In the 160 mg/kg bw group a decrease in urine volume was seen during weeks 26 and 52, which was related to the decreased water consumption. During week 77, urine volumes of the high dose animals were similar to those of control animals; however, towards test ending (i.e. week 103), the urine volume produced by the high dose animals again was lower than control.
Neoplastic findings:
Treatment-related macroscopical findings were reported for the high dose animals and affected the stomach (table 5).
Lesions in the stomach included thickening of the non-glandular region, raised area with central depressions, warty excrecences and ulceration; these lesions were seen in 9 males and 10 females treated with 160 mg/kg bw of bronopol.
Incidental/spontaneous findings were reported, which occurred in both treated and untreated animals, and/or showed no dose-response relationship. Especially in the stomach of high dose rats that died during the experiment, histopathological examination revealed squamous cell papillomata associated with epithelial hyperplasia and ulceration. These findings were rather related to the irritant potential of bronopol than indicative of a tumorigenic potential for this substance. Re-evaluation of the findings: The more recent re-evaluation of the histopathological findings confirmed the findings of the older evaluations, and treatment-related papillomas were reported for the forestomachs of the rats treated with bronopol. The fact to the papillomas were mainly seen in the high dose group and were associated to ulcerations indicates that the tumours rather were a consequence of the irritant potential of bronopol than of a carcinogenic potential of the test substance as such.

Histopathology:
Kidney: Histopathology of the kidney revealed no treatment-related changes, which would explain the increase in relative kidney weight reported above. Moreover, progressive glomerulonephrosis of varying degree was reported for animals of both treated and untreated groups; however, the incidence of this lesion in rats treated with 160 mg/kg bw group was statistically significantly increased compared to control. With regard to histopathology, glomerulonephrosis was characterized by glomerular abnormalities, atrophic or cystically dilated nephrons, which often were lined by basophilic epithelium, and interstitial fibrosis with foci of lymphocytic infiltration. Glomerulonephrosis is not untypical for the rat strain used. The treatment with bronopol at a test dose of 160 mg/kg bw/day probably exacerbed the occurrence of this spontaneous lesion as a consequence of the reduced renal output observed at the high dosing level.
Salivary glands (table 6): Squamous metaplasia was seen in the ducts of the salivary glands, and was often associated with minimal mixed or chronic inflammatory cell infiltration, and with groups of atrophic acini. The affected glands often were dilated and displayed minimal epithelial hyperplasia. The incidence and gravity of these effects were increased in the males of the 40 and the 160 mg/kg bw groups and in the females of the 160 mg/kg bw group when compared to control. Squamous metaplasia in the salivary glands is regarded as a spontaneous changes occurring in the rat strain used; the treatment with bronopol probably exacerbed the occurrence of this spontaneous lesion.
Lymph nodes: A treatment-related dilatation of the sinusoids in the gastric lymph nodes was reported for 4/12 males and 5/22 females of the 160 mg/kg bw group. The finding generally was associated with hyperplasia and ulceration of the non-glandular epithelium of the stomach. No such changes were seen in the control and the low and mid doses groups.

Stomach (see table 7): With regard to histopathology, ulceration in the non-glandular epithelium of the stomach often was accompanied by epithelial hyperplasia of varying degree and hyperkeratosis. Occasional squamous cell papillomata also were seen. The increased incidence and severity of the hyperplasia and areas of ulceration seen in the high dose group compared to control indicated that the findings were treatment-related.

In none of the remaining examined organs and tissues, treatment-related changes were seen. In fact, changes such as foci of myocarditis in the heart, small areas of interstitial pseumonitis in the lung, cytoplasmic vacuolisation in hepatocytes of the liver, changes in endocrine glands (e.g. vacuolisation and distention of cells) and changes in the reproductive systems (e.g. testicular atrophy in males or follicular cysts in ovaries) were reported, which occurred in both, treated and untreated animals and therefore were of no toxicological relevance.

Dose descriptor:

other: No effect level

Effect level:

7 mg/kg bw/day

Remarks on result:

other: Effect type: carcinogenicity (migrated information)

Table 1: Group mean body weight gains (g), data summary (extract from tables presented in the study report):

Period (week)	Males				Females
	G1	G2	G3	G4	G1	G2	G3	G4
0 – 3	166	168	171	166	83	86	84	83
3 – 6	105	102	98	90	53	57	52	55
0 – 6	271	270	269	256**	136	137	136	138
6 – 13	100	98	95	73***	45	47	47	40**
0 - 13	371	368	364	329***	181	184	183	178
13 - 26	103	108	98	52***	45	47	57*	38
26 – 52	110	107	88**	18***	60	58	59	28***
52 – 78	98	74	47***	36***	69	80	83	41**
78 – 104	40	7	23	-104***	35	39	0	-7***

G1, control group, G2, 10 mg/kg bw/day group, G3, 40 mg/kg bw/day group, G4, 160 mg/kg bw/day group; *, p<0.05; **, p<0.01;***, p<0.001

Table 2: Group mean food consumption (g/rat/week), data summary (extract from tables presented in the study report).

Period (week)	Males				Females
	G1	G2	G3	G4	G1	G2	G3	G4
1 – 13 (T)	2221	2208	2193	2169	1649	1678	1671	1657
1 – 13 (%)	-	99	99	98	-	102	101	100
14 – 26 (T)	2186	2164	2133	2000	1649	1648	1636	1589
14 – 26 (%)	-	99	98	91***	-	100	99	96
27 – 52 (T)	4233	4260	4171	3954***	3239	3185	3136	3116
27 – 52 (%)	-	101	99	93	-	98	97	96
53 – 78 (T)	4820	4687	4477*	4065***	3702	3670	3637	3640
53 – 78 (%)	-	97	93	84	-	99	98	98
79 – 104 (T)	4797	4659	4649	4021	3936	3909	3737	3659
79 – 104 (%)	-	97	97	84***	-	99	95	93

G1, control group, G2, 10 mg/kg bw/day group, G3, 40 mg/kg bw/day group, G4, 160 mg/kg bw/day group; T, Total (g); %, percentage of control; *, p<0.05; **, p<0.01;***, p<0.001.

Table 3: Group mean water consumption (ml/rat/week), data summary (extract from tables presented in the study report):

Period (week)	Males				Females
	G1	G2	G3	G4	G1	G2	G3	G4
1 – 13 (T)	3769	2838	2435	1840	2574	2273	2014	1589
1 – 13 (%)	-	75	65	49	-	88	78	62
14 – 26 (T)	3493	2595***	2196***	1657***	2454	2181***	1925***	1560***
14 – 26 (%)	-	74	63	47	-	89	78	64
27 – 52 (T)	5829	5010***	4338***	3264***	5225	4687***	4250***	3457***
27 – 52 (%)	-	86	74	56	-	90	81	66
53 – 78 (T)	5997	5418	4692***	4076***	5661	5270	4836**	4279***
53 – 78 (%)	-	90	78	68	-	93	85	76
79 – 104 (T)	6747	6397	6306	3652***	6695	6268	5601*	4021***
79 – 104 (%)	-	95	93	54	-	94	84	60

G1, control group, G2, 10 mg/kg bw/day group, G3, 40 mg/kg bw/day group, G4, 160 mg/kg bw/day group; T, Total (g); %, percentage of control; *, p<0.05; **, p<0.01;***, p<0.001

Table 4: Mortality occuring during the test.

Dose level	Main test series		Satellite test series
	Males	Females	Males	Females
0 mg/kg bw/day	21/45	19/45	8/15	9/15
10 mg/kg bw/day	20/45	19/45	5/15	10/15
40 mg/ kg bw/day	20/45	22/45	7/15	8/15
160 mg/kg bw/day	36/45***	28/45*	12/15	10/15

*, p0.05; ***, p0.001

Table 5: Numbers of rats with treatment-related macroscopic lesions in the stomach (extract from table 12 of the study):

Main treatment-related effects in the stomach:	Control group				160 mg/kg bw/day Bronopol
	Males		Females		Males		Females
	D	S	D	S	D	S	D	S
Thickening	0	0	0	0	2	3	1	2
Ulceration	1	0	1	0	0	1	0	4
Raised areas/excrescences	0	0	0	0	8	9	3	7

D, rats that died or were sacrificed in extremis; S, rats that were sacrificed at test ending

Table 6: Number of rats showing abnormalities of the salivary glands.

Lesion	Male				Female
	G1	G2	G3	G4	G1	G2	G3	G4
Moderate squamous metaplasia in ducts:								4
Minimal squamous metaplasia in ducts:		1	4	4				1
Focal squamous metaplasia in ducts:	2	1	4	5	1	1	2	3
Dilatation of the ducts				7				11
Acinar atrophy	3	4	6	11			1	8
Inflammation	2	5	12	9 (1)	2	1	3	9
Minimal epithelial hyperplasia				2
Nr of rats affected/Nr of rats in which salivary glands were examined	3/24	5/24	12/25	11 (1)/ 12 (1)	2/26	1/26	2/23	12/20

G1, control group, G2, 10 mg/kg bw/day group, G3, 40 mg/kg bw/day group, G4, 160 mg/kg bw/day group; (), rat died during the treatment period

Table 7: The incidences and severity of histopathological findings in the stomach of the animals.

Lesion	Male
	G1		G2		G3		G4
	D	S	D	S	D	S	D	S
Squamous cell papilloma							2 (1)
Papillomatous/marked epithelial hyperplasia							5 (3)	3
Moderate epithelial hyperplasia	1				1		3 (1)	4 (1)
Minimal focal epithelial hyperplasia
Ulceration	1						5 (3)	5
Nr of rats affected/Nr of rats in which salivary glands were examined	1/4	0/24	1/1	0/25	1/1	0/21	8 (4)/ 8 (4)	7 (1)/ 8 (3)

Lesion	Females
	G1		G2		G3		G4
	D	S	D	S	D	S	D	S
Squamous cell papilloma							(1)
Papillomatous/marked epithelial hyperplasia	2						0	(2)
Moderate epithelial hyperplasia	1				1		2	3 (2)
Minimal focal epithelial hyperplasia								5
Ulceration	1						(2)	6 (4)
Nr of rats affected/Nr of rats examined	2/2	0/26	1/1	0/26	0/0	0/23	(3)/(3)	8 (4)/ 17 (5)

G1, control group, G2, 10 mg/kg bw/day group, G3, 40 mg/kg bw/day group, G4, 160 mg/kg bw/day group; (), satellite group animals

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Quality of whole database:

Findings related to the irritant potential of Bronopol were observed. A carcinogenic potential for Bronopol could not be evidenced.

Carcinogenicity: via dermal route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: dermal

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 study was conducted in the seventies, i.e. prior to implementation of guideline; furthermore at this time GLP was not compulsory. However, the methods and results were well documented and reported and can therefore considered to be of scientific acceptability. The stability of the test substance in aqueous solution was monitored at a later time point (see addendum).

Principles of method if other than guideline:

Method: other: no data; the study was conducted prior to the implementation of guideline

GLP compliance:

no

Species:

mouse

Strain:

other: CFLP mice (hysterectomy-derived strain from Swiss origin)

Sex:

male/female

Details on test animals or test system and environmental conditions:

The CFLP mice were obtained from Carworth Europe (UK) and were 28 days old at arrival in the testing laboratory;
they were subjected to an acclimatisation period of 14 days prior test initiation.
Animal-room temperature was controlled at 21 ± 2°C, and lighting was controlled to give 12 hours light ( 8 a.m. to 8 p.m. B.S.T.) and 12 hours dark
per 24 hours.
Housing: 4 animals a cage.
All mice had free access to tap water and to quantities of Oxoid Pasteurised Breeding Diet for Rats and Mice.

Route of administration:

dermal

Vehicle:

other: 90% acetone in water

Details on exposure:

Bronopol batch No: CT 89075N was used for the first 13 weeks of treatment; treatment was then continued with batch No: CT 92495T.
The dorsum of each animal was closely shaved by means of electric clippers prior to the initial treatment; thereafter, shaving was repeated at weekly intervals until test ending. The test solution was applied on the shaved area of the dorsal skin. The test solution was applied on the shaved area of the dorsal skin at a volume of 0.3 ml/mouse.

Duration of treatment / exposure:

80 weeks

Frequency of treatment:

One application/day on 3 days/week (Monday, Wednesday and Friday)

Dose / conc.:

0.2 other: %

Remarks:

in 90% acetone in water
Corresponds to approx 20 mg/kg bw/day (assuming a body weight of 30 g/mouse)

Dose / conc.:

0.5 other: %

Remarks:

in 90% acetone in water
Corresponds to approx 50 mg/kg bw/day (assuming a body weight of 30 g/mouse)

No. of animals per sex per dose:

52

Control animals:

yes, concurrent vehicle

Details on study design:

Post-exposure period: None

Observations and examinations performed and frequency:

Body weight: The body weights of the mice were recorded prior test initiation, and thereafter, at weekly intervals during the 12 first weeks of treatment. During the remaining treatment period, body weights were recorded in 2 weeks-intervals until test ending.
Food consumption: Food consumption was recorded for each cage of mice (four mice/cage) and the mean weekly food intake was calculated. The efficiency of food utilization was assessed by calculation of the mean food conversion ratios (FCR values) during successive four-weekly periods for the first 24 weeks, as weights of food consumed per unit gains in body weight.
Water consumption: Water consumption only was assessed by inspection of the water bottle. As no treatment-related effect was expected, no more detailed measurement of water consumption was considered.
Clinical signs: The animals were daily observed for clinical symptoms of toxicity, changes in behaviour, mortalities and skin reaction.
Macroscopic investigations: Particular attention was given to skin lesions, cataracts and/or palpable masses as well as to the progression or regression of such findings.

Sacrifice and pathology:

Animals that died or were sacrificed in extremis during the test period were subjected to gross pathological
examination for determination of the cause of death. Where possible, tissue samples were preserved in buffered
10% formalin for further histopathological examination. Animals that survived the treatment were sacrificed at
test ending and were subjected to gross pathology and histopathology, Particular attention was given to the
development of tumours, and standard criteria were used for the classification of certain tumour-types in the
liver, the lung and the lympho-reticular system
(classification according to Walters, Brit. J. Cancer 20: 148-160 and to Dunn, J. ntn.Cancer. Inst. 14:1281-1432,
1954).
Organs:
All superficial tissues including urogenital orifices, tails, auricles, auditory meatus, mammary tracts and cervical subcutaneous structures were checked for abnormalities such as swelling or distortion or other evidence of tumour formation. The nares, mouth, tongue, pharynx and auditory region were examined. The cranial roof was removed for examination of the brain, the pituitary gland and the cranial nerves. The subcutaneous tissues including regional lymph nodes, mammary glands and salivary glands were examined. The abdominal and thoracic contents were examined.
The urinary bladder from all mice that died after week 52 of treatment was distended with fixative in situ, removed, opened and examined under low-power magnification. In the thorax, particular attention was given to the thymus, the lymph nodes and the heart. The mucosa of the esophagus, intestines and uterus were examined. The lungs were removed and all pleural surfaces were examined. Liver and kidney were sectioned and any lesions indicative of neoplasia were noted. Gonads, adrenals, thyroid, intra-abdominal lymph nodes and accessory reproductive organs were checked for abnormalities in appearance and size. Evidences of adhesion, invasion or other interaction between presumptive neoplasia and the adjacent structures also were noted. All gross abnormalities were recorded.

Histopathology: All surviving animals, at terminal sacrifice. All animals that died or were sacrificed in extremis during the test period.
Samples of following organs/tissues were taken, fixed in buffered 10% formalin and processed for histological examination: All abnormal tissues, aorta, brain, mid-colon, pituitary, thymus, salivary glands, stomach, jejunum, liver, kidneys, spleen, heart, lungs, gonads, uterus, adrenals, seminal vesicles, skeletal muscle, skin (untreated), treated skin areas, tongue, urinary bladder, gall bladder, lymph nodes, mammary gland, sciatic nerve, thyroid, pancreas, bone, bone marrow, eye (fixed in Davidson´s fixative), blood smears.
For microscopic examination, the tissue samples were embedded in paraffin; the embedded samples were then sectioned, and the sections were Haematoxylin/Eosin stained. Selected samples for microscopic examination:
All macroscopically abnormal tissues from animals that died during treatment, all macroscopically lesions suggestive of neoplasia (all animals), and following series of tissues: adrenals, thyroids, ovaries, liver, spleen, lymph nodes, pituitary gland, treated skin areas, bone marrow and blood smears.

The histopathological findings were re-evaluated and completed in 1992 by Robinson AJ.

Statistics:

For statistical assessment, differences in tumour incidence were compared by a 2 x 2 contingency test used as a
one-tailed test. The main findings were re-assessed statistically (cf. Huntingdon Life Sciences Ltd, 1998).

Details on results:

Range finding study:
Mortality:
A female of the 4% bronopol group was found dead on day 4; because of the autolytic state, no cause of death could be determined. The dead animal showed no skin abnormalities.
Clinical signs of toxicity:
2% and 4% bronopol: After the 3 first applications, the treated skin areas of mice of the 2 and the 4% bronopol groups were dark and dehydrated, with small areas of epidermal loss and ulceration. Because of the increasing severity of the skin findings, all the mice of the 2 and the 4% groups were sacrificed on day 8. Necropsy of the 4% treated mice revealed extensive intradermal haemorrhage and scab formation, associated with several small areas of epidermal loss, ulceration, slight thickening of the skin and slight edema. Similar but less severe pathological findings were also found in 7 mice of the 2% test group; further 7 mice of this group showed areas of epidermal loss or edema. The remaining two mice of the 2% group (one male and one female) showed no abnormalities.
1% bronopol: After the first 3 to 5 applications, the treated skin areas of one male and 5 females of the 1% bronopol group showed small areas of scab formation and epidermal loss; no further progression or increase in severity of these findings was seen over the remaining treatment period.
0.5% bronopol: Some mice treated with 0.5% bronopol showed minimal hair loss during the initial phase of treatment; thereafter, hair growth turned back to normal.
0.2% bronopol: No signs of toxicity were seen in mice treated with 0.2% bronopol.
No Bronopol-related effect on body weight was evident in the groups treated with up to 1% bronopol.
No Bronopol-related effect on food consumption was evident in the groups treated with up to 1% bronopol.
Gross and histopathology:
In 3 males and 4 females of the 1% bronopol group necropsy revealed small areas of epidermal loss; 6 females showed slight thickening of the dorsalskin and one of these showed scab formation in the dorso-cervical region. No further treatment-related abnormalities were evident.
Necropsy of mice treated with 0.2 and 0.5% bronopol revealed no treatment-related abnormalities.

Main study:
Body weight: A slight but statistically significant decrease in mean body weight gain was reported for the males treated with 0.5% Bronopol for the period ranging from week 26 to week 52 (see table 1).
Food consumption: Both, food intake and food efficiency were similar in all groups.
Clinical signs: Some mice of the group treated with 0.5% bronopol showed slight loss in hair around the treated skin area of mice during the first 3 weeks of treatment. No further treatment-related effects were seen.
Mortality for the treated females (both test groups) was within control range and therefore inconspicuous (after 80 days:35% mortality in the 0.2% test group and 40% mortality in the 0.5% group, versus 46% mortality in the control group). In contrast, mortality of the treated males was slightly increased compared to control males (after 80 days: 50% mortality in the 0.2% test group and 48% mortality in the 0.5% group, versus 36% mortality in the control group). However, the causes of death of the males were common for the strain used and the age of the animals, and no relationship to treatment could be evidenced.
The statistical assessment of mortality findings (Freemantle MR, 1998) revealed no statistical significance for the results

Pathology (see Table 1 and 2):
Macroscopical tumour findings:
Suspected skin tumours in the treated area were recorded for 1 male of the control group and for 3 females and 1 male of the 0.5% bronopol test group. The tumours were first noted between week 39 and week 79 of the treatment period. However, in vivo palpation of the underlying skin revealed no evidence of malignancy for these tumours. Skin tumours also were seen in one control female, two females of the 0.2% bronopol test group and one female of the 0.5% test group. These tumours were first noted between week 66 and week 79 of treatment but then regressed (week 79 to 80). Further skin tumours were recorded in untreated skin areas of one control female (ventral thoracic region) and one female of the 0.2% test group (right forelimb); both tumours were first noted on week 79 of treatment.

Histological neoplastic findings: Animals that died during the experiment, summary of main findings:
Respiratory system: incidental findings such as e.g.. perivascular and peribronchiolar lymphoid aggregations were seen in both, treated and control mice; these findings were common to the strain used and were not treatment related
Liver: Some incidental changes seen in control and treated mice, such as e.g. vacuolization of hepatocytes and foci of degeneration; these changes were not treatment-related.
Lympho-reticular system: changes known to occur commonly in the strain used, such as e.g. foci of hyaline degeneration were reported for both, control and treated mice. These changed were not treatment-related.
Reproductive system: Testicular atrophy was seen in some males of the control and the treated groups whereas changes in the uterus were reported for some females. These findings were incidental and not treatment-related.
Skin: In control and treated mice, epidermal hyperplasia often associated with hyperkeratosis or parakeratosis and inflammatory cell response was reported. Further incidental findings included ulceration of the untreated skin areas, which also often was associated with hyperkeratosis or parakeratosis and inflammatory cell response. Such findings are commonly seen in laboratory mice and were therefore not treatment-related.
All reported findings were incidental and without relationship to the treatment with Bronopol.
Animals that were sacrificed at test ending:
Incidental findings similar to those reported above also were seen in the animals that were sacrificed at the end of the test period.

Re-evaluation of the histopathological findings: Robinson AJ (1992) confirmed the increased incidence of papillomas on the treated skin site of one male and three females of the 0.5% bronopol test dose; the examination of these papillomas revealed that these tumours rather were related to the irritant potential of bronopol than to a carcinogenic potential of the test substance. As non-neoplastic lesion. Robinson AJ reported an increased incidence of nodular hyperplasia in the livers of male mice treated with 0.5% bronopol; no such lesions were seen in the males of the 0.2% bronopol test group. However, there was no effect on the incidence of liver-cell tumours in either of the treated groups.

Table 1: Group mean body weight gains (g), data summary (extract from tables presented in the study report).

Period (week)	Males			Females
	Control	0.2% Bronopol	0.5% Bronopol	Control	0.2% Bronopol	0.5% Bronopol
0 – 26	20 g	19.8 g	19.1 g	14.7 g	14.1 g	13.2 g
26 – 52	6.8 g	6.8 g	3.6 g**	5.7	6.8	6.3
52 – 80	- 2.5 g	- 3.2 g	0.3 g	0 g	- 0.1 g	- 0.2 g
0 - 80	24.9 g	24.2 g	22.4 g	19.6 g	21.6 g	19.3 g

**, p<0.01

Table 2: Summary of tumour incidence after microscopic classification, male mice

Number of mice with:	MALES
	Control (acetone)		0.2% Bronopol		0.5% Bronopol
	D	S	D	S	D	S
Lymphoreticular system:
Lymphoma	2	2	3	-	3	3
Reticulum cell sarcoma	-	-	-	-	1	-
Lymph node squamous cell carcinoma	-	1^b	-	-	-	-
Lymph node haemangioma	-	1	-	1	-	3
Splenic haemangioma	-	-	-	-	-	1
Liver:
Liver cell tumour	-	1	-	-	-	-
Haemangioma	-	-	-	1	-	-
Heart
Fibrosarcoma	1^a	-	-	-	-	-
Lungs
Lung tumour grade 1	1	5(6)	3	6(7)	3	6
Lung tumour grade 2	3	3	-	1	-	3
Lung tumour grade 3	-	1	2	1	-	1
Testes
Interstitial cell adenoma	-	3	-	-	-	2
Endocrine glands
Pituitary adenoma	-	1	-	1	-	-
Thyroid papillary cystadenoma	-	1	-	1	-	-
Pancreatic islet cell adenoma	-	-	-	1	-	-
Adrenal phaeochromocytoma	-	1	-	-	-	-
Mesentery
Haemangiosarcoma	-	-	-	1^c	-	-
Subcutaneous tissues
Anaplastic sarcoma	-	1	-	-	-	-
Fibrosarcoma	-	-	-	-	1^d	-
Harderian gland
Papillary adenoma	-	-	-	-	-	1
Cutaneous tissues
Treated areas, squamous cell papilloma	1^e	-	-	-	-	1
Tumour bearing mice (N)	6	18	8	13	8	15
Examined mice (N)	17	33	24	26	23	27
Not examined mice (N; because of cannibalism or autolysis)	2	-	2	-	2	-

N, Number; D, animals that died or were sacrificed in extremis; S, animals that were sacrificed at test ending; (), total number of tumours recorded;

a, tumour with metastasis to lung and kidney; b, tumour of unknown origin invading lymph nodes; c, tumour with metastasis to liver;

d, tumour with metastasis to liver, kidney, and epididymis; e, histological diagnosis preclude by autolytic change.

Table 3: Summary of tumour incidence after microscopic classification, female mice.

Number of mice with:	FEMALES
	Control (acetone)		0.2% Bronopol		0.5% Bronopol
	D	S	D	S	D	S
Lymphoreticular system:
Lymphoma	4	1	3	5	5	5
Thymic lymphosarcoma	1	-	-	-	-	-
Reticulum cell sarcoma	-	1	-	-	-	-
Liver:
Haemangioma	-	2	-	-	-	-
Lungs
Lung tumour grade 1	2	2	1	6	2	4
Lung tumour grade 2	4	2	-	2	-	2
Lung tumour grade 3	-	-	-	-	-	2
Lung tumour grade 4	-	-	-	-	-	1
Kidney
Adenocarcinoma	-	1	-	-	-	-
Ovary
Papillary cystadenoma	1	-	-	-	-	-
Arrhenoblastoma	-	-	-	1	-	-
Uterus/Vagina
Leiomyoma	-	1	-	-	-	-
Leiomyosarcoma	2^f	-	-	-	-	-
Endocrine glands
Pituitary adenoma	2	1	1	2	-	1
Subcutaneous tissues
Fibrosarcoma	2	-	-	-	-	-
Haemangioma	1	-	-	-	-	-
Cutaneous tissues
Treated areas, squamous cell papilloma	-	-	-	-	1	2
Untreated areas, squamous cell papilloma	-	1	-	1	-	-
Tumour bearing mice (N)	16	9	4	14	8	14
Examined mice (N)	23	28	16	34	18	31
Not examined mice (N; because of cannibalism or autolysis)	1	-	2	-	3	-

N, Number; D, animals that died or were sacrificed in extremis; S, animals that were sacrificed at test ending; f,

one vaginal tumour with metastasis to ovaries, liver and lymph node, one uterine and ovarian tumour with metastasis to pancreas and liver.

Conclusions:

An increased incidence of skin papilloma was reported for the highest tested concentration of bronopol (0.5%); however, these tumours rather resulted from the irritant potential of bronopol than from a carcinogenic potential of bronopol. Therefore, a carcinogenic potential could not be evidenced for bronopol under the test conditions used.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Quality of whole database:

Findings related to the irritant potential of Bronopol were observed. A carcinogenic potential for Bronopol could not be evidenced.

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 carcinogenicity under Regulation (EC) No 1272/2008, as amended for thetenth time in Regulation (EU) No 2017/776.

Additional information

In a chronic toxicity (and carcinogenicity) study (Huntingdon Research Centre, 1976) Bronopol was tested for its carcinogenic potential in Sprague-Dawley rats of the CD strain following chronic administration in the drinking water over a period of 104 weeks. The test consisted of a main test series and a satellite test series. Each test series consisted of 4 test groups. Each test group of the main test series comprised 45 animals/sex/group whereas each test group of the satellite series comprised 15 animals/sex/group. The animals of the main test series were used for evaluation of the carcinogenic potential of the test substance. The animals of the satellite series were used for the evaluation of haematological and clinical-chemical parameters, and for urinalysis. These animals were not considered for carcinogenicity evaluation. Bronopol was offered to the animals via the drinking water, and following doses were tested: 10, 40 and 160 mg/kg bw/day. As no monitoring of the stability of Bronopol in solution was done at the time the study was performed, the selected test doses were re-evaluated in 1985/1986, resulting in following dosages: 7, 32 and 142 mg/kg bw/day corresponding to respectively 69%, 81% and 89% of the intended doses of 10, 40 and 160 mg/kg bw /day. Systemic toxicity and non neoplastic findings: Treatment- and dose-related effects seen at 32 and 142 mg/kg bw/day included reduction in food consumption and in body weight gain; additional findings in the high dose group included increased mortality and reduction in grooming activity. The incidence of glomerulonephrosis, which is regarded as a spontaneous occurring lesion in the used rat strain, appeared to be exacerbated by the treatment with 142 mg/kg bw/day of Bronopol, as a consequence of reduced renal output, which again was related to reduced water uptake. Reduced water uptake, which was seen in all groups but was more pronounced at the high dose, was rather due to a palatability problem.Thus, the series of effects ranging from reduced water uptake to increased incidence of glomerulonephrosis was not indicative of Bronopol toxicity. Lesions in the stomach were seen at the high dose and were attributed to the irritant potential of Bronopol.Ulceration in the non-glandular epithelium of the stomach was seen, often associated to epithelial hyperplasia of varying degree and hyperkeratosis. Occasional squamous cell papillomata also was seen. The increased incidence and severity of the hyperplasia and areas of ulceration seen at the high dose compared to control indicated that the findings were treatment-related. An exacerbated incidence of squamous metaplasia in the salivary glands of the Bronopol-treated rats was reported, which often was associated with inflammation and atrophic acini; the occurrence of squamous metaplasia in the salivary glands is known to be a spontaneous occurring change in the used rat strain. A treatment-related dilatation of the sinusoids in the gastric lymph nodes was reported for males and females of the 142 mg/kg bw/day group. The finding generally was associated with hyperplasia and ulceration of the non-glandular epithelium of the stomach. No such changes were seen in the control and the low and mid doses groups. Neoplastic findings: Incidental/spontaneous findings were reported, which occurred in both treated and untreated animals, and/or showed no dose-response relationship. Especially in the stomach of high dose rats that died during the experiment, histopathological examination revealed squamous cell papillomata associated with epithelial hyperplasia and ulceration. These findings were rather related to the irritant potential of Bronopol than indicative of a tumorigenic potential for this substance. The more recent re-evaluation of the histopathological findings confirmed this evaluation, and treatment-related papillomas were reported for the forestomachs of the rats treated with Bronopol. The fact that papilloma were mainly seen in the high dose group and were associated to ulcerations indicates that the tumours rather were a consequence of the irritant potential of Bronopol than of a carcinogenic potential of the test substance as such. The neoplastic (stomach papillomas) findings were due to the irritant potential of Bronopol. A carcinogenic potential for Bronopol could not be evidenced in the present study. Conclusion: At the lowest tested dose of 7 mg/kg bw/day, no conspicuous differences from control could be evidenced, and the only clear effect, which was reported, consisted of a slight decrease in water uptake and was due to a palatability problem. Therefore, the NOAEL for the systemic toxicity of Bronopol in the present study was 7 mg/kg bw/day and the LOAEL was 32 mg/kg bw/day. The study is classified as acceptable (key study). Investigations were conducted prior to the implementation of guideline and GLP was compulsory at this time. With regard to the test substance, batch No: CT 92495T was used for the test period ranging from week 1 to week 51, and batch No: CT 95274W was used for the test period ranging from week 52 to week 104; no further details were provided. The stability of the test substance in aqueous solution was monitored at a later time point. However, all data taken together are of scientific acceptability.

In a chronic toxicity (and carcinogenicity) study (Huntingdon Research Centre, 1975) Bronopol was tested for its carcinogenic potential in mice following chronic dermal administration at doses of 0, 0.2 and 0.5 % over a period of 80 weeks. Mortality, clinical signs, body weight and food and water consumption were assessed at regular intervals. At the end of the treatment period, all survivors were necropsied and examined macroscopically and histopathologically. Following results were obtained: Body weight: A slight but statistically significant decrease in mean body weight gain was reported for the males treated with 0.5% Bronopol for the period ranging from week 26 to week 52. Food consumption: Both, food intake and food efficiency were similar in all groups. Clinical signs: Some mice of the group treated with 0.5% Bronopol showed slight loss in hair around the treated skin area during the first 3 weeks of treatment. No further treatment-related effects were seen. Mortality for the treated females (both test groups) was within control range and therefore inconspicuous (after 80 days: 35% mortality in the 0.2% test group and 40% mortality in the 0.5% group, versus 46% mortality in the control group). In contrast, mortality of the treated males was slightly increased compared to control males (after 80 days: 50% mortality in the 0.2% test group and 48% mortality in the 0.5% group, versus 36% mortality in the control group). However, the causes of death of the males were common for the strain used and the age of the animals, and no relationship to treatment could be evidenced. The statistical assessment of mortality findings (Huntingdon Life Sciences Ltd, 1998) revealed no statistical significance for the results. Macroscopical tumour findings: Suspected skin tumours in the treated area were recorded for 1 male of the control group and for 3 females and 1 male of the 0.5% Bronopol test group. The tumours were first noted between week 39 and week 79 of the treatment period. However, in vivo palpation of the underlying skin revealed no evidence of malignancy for these tumours. Skin tumours also were seen in one control female, two females of the 0.2% Bronopol test group and one female of the 0.5% test group. These tumours were first noted between week 66 and week 79 of treatment but then regressed (week 79 to 80). Further skin tumours were recorded in untreated skin areas of one control female (ventral thoracic region) and one female of the 0.2% test group (right forelimb); both tumours were first noted on week 79 of treatment. Histological neoplastic findings: Animals that died during the experiment, summary of main findings: Respiratory system: incidental findings such as e.g. perivascular and peribronchiolar lymphoid aggregations were seen in both, treated and control mice; these findings were common to the strain used and were not treatment related. Liver: Some incidental changes seen in control and treated mice, such as e.g. vacuolization of hepatocytes and foci of degeneration; these changes were not treatment-related. Lympho-reticular system: changes known to occur commonly in the strain used, such as e.g. foci of hyaline degeneration were reported for both, control and treated mice. These changed were not treatment-related. Reproductive system: Testicular atrophy was seen in some males of the control and the treated groups whereas changes in the uterus were reported for some females. These findings were incidental and not treatment-related. Skin: In control and treated mice, epidermal hyperplasia often associated with hyperkeratosis or parakeratosis and inflammatory cell response was reported. Further incidental findings included ulceration of the untreated skin areas, which also often was associated with hyperkeratosis or parakeratosis and inflammatory cell response. Such findings are commonly seen in laboratory mice and were therefore not treatment-related. All reported findings were incidental and without relationship to the treatment with Bronopol.

Incidental findings similar to those reported above also were seen in the animals that were sacrificed at the end of the test period. Re-evaluation of the histopathological findings: Boots Pharmaceuticals Research Department (1992) confirmed the increased incidence of papillomas on the treated skin site of one male and three females of the 0.5% Bronopol test dose; the examination of these papillomas revealed that these tumours rather were related to the irritant potential of Bronopol than to a carcinogenic potential of the test substance. Boots Pharmaceuticals Research Department (1992) reported an increased incidence of nodular hyperplasia in the livers of male mice treated with 0.5% Bronopol; no such lesions were seen in the males of the 0.2% Bronopol test group. However, there was no effect on the incidence of liver-cell tumours in either of the treated groups. The study is classified as acceptable (key study). Investigations were conducted prior to the implementation of guideline and GLP was compulsory at this time.