Bronopol

Endpoint summary

Description of key information

Repeated dose toxicity: oral

Repeated oral application of Bronopol to rats was performed at doses of 10, 40 and 160 mg/kg bw/day via the drinking water over a period of 2 years. Systemic toxicity, non neoplastic and neoplastic findings were noticed. The chronic toxicity and carcinogenicity study 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 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. At the lowest tested dose of 10 mg/kg bw/day, no conspicious differences from control could be evidenced, and the only clear effect observed was 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 10 mg/kg bw/day. Taking into account the re-evaluation of test concentrations, the actual NOAEL was 7 mg/kg bw/day; the corresponding LOAEL was 32 mg/kg bw/day.

Repeated dose toxicity: dermal

Repeated dermal application of Bronopol at 0.2 and 0.5 % in methylcellulose suspension over a period of 21 days was not associated with signs of systemic toxicity or deaths. Severe skin irritations were observed after application of 0.5 % Bronopol, whereas 0.2 % Bronopol caused skin reactions comparable to, but slightly more persistent than those elicited by the vehicle alone. The NOAEL for systemic effects was 5 mg/kg bw/day (5 %). The NOAEL for local effects was established at 2 mg/kg bw/day (2 %).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

chronic toxicity: 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 . However, all data taken together are of scientific acceptability.

Principles of method if other than guideline:

The study was conducted prior to the implementation of guideline and GLP was compulsory at this time.

GLP compliance:

not specified

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

A total of 480, 28 +/- 1 day old CD rats, a caesarian-derived strain of Sprague-Dawley origin, was obtained from
Charles River Laboratories, St. Aubin-les-Elbeuf, France, on 14 May 1973.
Following an initial quarantine and acclimatisation period of four days to accustom the rats to the environmental conditions existing in our laboratories, each animal was weighed and allocated to one of several arbitrary weight ranges, each containing rats with bodyweights differing from one another by no more than +/- 2.5 g. Equal numbers of animals from within each weight range were randomly allocated to each of the four treatment groups, and all animals then identified by earmark. In this way we ensured that each group contained a similar population of rats arid initial.
mean bodyweights were also approximately equalised. Treatment conmmenced after a predosing period of one week during which time the water intake, food consumption and bodyweights were measured.
The rats were housed five to a cage (unless the number was reduced by mortality) in suspended cages with wiremesh floors. Animal room temperature and relative humidity were controlled at 21 +/- 20C 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.
The cages constituting each group were dispersed in batteries so that possible environmental influences arising from their spatial distribution were equilibrated, as far as possible, for all treatments.
All rats had free access to standard laboratory rat food, Spratt's Laboratory Diet 1.

Route of administration:

oral: drinking water

Vehicle:

water

Duration of treatment / exposure:

104 wk

Frequency of treatment:

7days/week

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

re-evaluated dose: 7 mg/kg bw/day

Dose / conc.:

40 mg/kg bw/day (nominal)

Remarks:

re-evaluated dose: 32 mg/kg bw/day

Dose / conc.:

160 mg/kg bw/day (nominal)

Remarks:

re-evaluated dose: 142 mg/kg bw/day

No. of animals per sex per dose:

45 male and 45 female rats per dose in main groups.
15 male and 15 female rats per dose in satellite groups.

Control animals:

yes

Details on study design:

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.
Main test series: The animals of the main test series were used for evaluation of the carcinogenic potential of the test substance.
Satellite test series: 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.

Observations and examinations performed and frequency:

The body weight of each rat was recorded at test initiation and weekly thereafter.
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 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 was measured daily and the mean weekly intake was calculated.
The animals were regularly observed for clinical symptoms of toxicity, changes in behaviour, and mortalities.
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.
Haematology: Test groups 1 and 4 (i.e. control and 160 mg/kg bw/day), 10 animals /sex /group, blood samples removal from the orbital sinus of fasted animals, Time points: Week 26, 52, 78 and 102.
Parameters: 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.
Clinical chemistry:
Time points: Week 26, 52, 78 and 102.
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.
Urinanalyses:
Number of animals: Satellite test series
Test groups 1 and 4 (i.e. control and 160 mg/kg bw/day), and additionally test groups 2 and 3 (i.e. 10 and 40 mg/kg bw/day), 10 animals /sex /group, Urine samples were collected overnight from fasted animals. 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).

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.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

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:

mortality observed, treatment-related

Description (incidence):

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).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

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 till 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 and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

For the males of the high dose group, food consumption was significantly reduced in comparison to control starting from week 14 of treatment till 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 and compound intake (if drinking water study):

effects observed, non-treatment-related

Description (incidence and severity):

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

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Examination of the eyes revealed no treatment-related effects.

Haematological findings:

no effects observed

Description (incidence and severity):

No treatment-related and/or biologically relevant differences between treated and control animals could be evidenced.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No treatment-related and/or biologically relevant differences between treated and control animals could be evidenced.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

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.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Kidney: Histopathology of the kidney revealed no treatment-related changes, which would explain the increase in relative kidney weight reported. 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: 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.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

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.
Carcenogenicity: negative

Details on results:

Body weigth gain:
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:
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:
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: 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: 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.

Key result

Dose descriptor:

NOAEL

Effect level:

7 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Systemic toxicology

Dose descriptor:

LOAEL

Effect level:

32 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Systemic toxicology

Critical effects observed:

not specified

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) and achieved dose of bronopol, 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

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

Table 4: Mortality in the main and satellite test series after treatment with bronopol.

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

*, p<0.05; ***, p<0.001

Table 5: The incidences and severity of histopathological findings in the stomach.

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
No of rats affected/No 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)
No of rats affected/No 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

Dose descriptor:

NOAEL

7 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

The database for repeated dose oral toxicity (systemic effects) is complete and is considered to meet the relevant data requirements of REACH.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was conducted in the seventies, i.e. prior to implementation of guidelines, and GLP was not compulsory at that time. However, the study was well-documented and the reported data are of scientific accetability.

Principles of method if other than guideline:

Method: other: no data

GLP compliance:

no

Species:

rabbit

Strain:

New Zealand White

Sex:

male/female

Details on test animals or test system and environmental conditions:

Source: Hylyne Commercial Rabbits, Cheshire
They were housed individually in metal cages.

Type of coverage:

other: non-occlusive

Vehicle:

other: methylcellulose

Details on exposure:

Route of Administration: dermal
At a dose level of 1 ml/kg b.w. the daily application of Bronopol at a concentration of 0.2 and 0.5% corresponds to a dose of 2 and 5 mg/kg bw/day
respectively.
Vehicle: 2,5% methylcellulose.
The residual test substance was removed after each treatment period of 6 hours by gentle washing of the skin application site with a warm dilute soap solution, followed by rinsing with clean water; thereafter the skin was blotted dry with absorbent paper.

Duration of treatment / exposure:

21 days, 6 hours/day

Frequency of treatment:

7 d/week

Dose / conc.:

0.2 other: %

Remarks:

bronopol in 2.5% methyl cellulose, 1 ml/kg bw, corresponding to 2 mg/kg bw/day

Dose / conc.:

0.5 other: %

Remarks:

bronopol in 2.5% methyl cellulose, 1 ml/kg bw, corresponding to 5 mg/kg bw/day

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

Post-exposure period: none

Observations and examinations performed and frequency:

Observations and examinations included local skin reaction (erythema and oedema), behavioural changes and
signs of toxicity, food consumption, body weight, haematology (packed cell volume, haemoglobin, red cell count,
total and differential white cell count), clinical chemistry (plasma urea, total serum proteins, serum protein,
serum alkaline phosphatase, serum transaminase (SGPT), ophthalmoscopic examination.
Body weights were recorded at test initiation and weekly thereafter; the group mean value (g/rabbit) was calculated. Food consumption for each rabbit was recorded weekly and the group mean food intake (g/rabbit/week) was calculated. The eyes of the animals were examined prior test start and again after 3 weeks of treatment (Keeler indirect ophthalmoscope).

Sacrifice and pathology:

Terminal studies after sacrifice of the animals included macroscopic examination and weighing of inner organs
(thyroid, heart, liver, kidneys, adrenals, and gonads) and microscopic examination of a series of tissues
(adrenal, brain, duodenum, eye, gall-bladder, gonads, heart, ileum, kidney, liver, lymph nodes, marrow smear,
mid-colon, pancreas, pituitary, salivary gland, skin (treated and untreated), spleen, stomach
(glandular and non-glandular), thymus, thyroid, urinary bladder.

Statistics:

Student's t-test and analysis of variance were employed to assess the significance of intergroup differences.

Clinical signs:

no effects observed

Description (incidence and severity):

No mortality and no signs of toxicity were observed.

Dermal irritation:

effects observed, non-treatment-related

Description (incidence and severity):

Local skin reaction (slight to well-defined erythema in 9/10 animals, and slight edema in all animals) was observed in the control rabbits. Similar irritation was seen in rabbits treated with Bronopol 0.2% although possibly with slightly more edema formation and slightly more persistent.Bronopol 0.5% treatment caused progression of slight to well-defined erythema to moderate erythema in 9/10 animals. Edema occurred on the third day and became well defined in all rabbits. Moderate edema developed in 3/5 males.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Initial loss of body weight was seen in all groups including controls; weight gain was comparable and no treatment related trend could be observed which would indicate a specific effect of Bronopol on growth.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption of all male and female animals was normal except for 2 animals in group 2 (0.2%) and one animal in group 3 (0.5%) where food consumption was depressed.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Ophthalmoscopic examination revealed no abnormalities.

Haematological findings:

no effects observed

Description (incidence and severity):

Haematology and clinical chemistry were inconspicious.

Clinical biochemistry findings:

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The macroscopical examination of the treated skin areas at necropsy revealed a varying degree of inflammatory reaction in control (2 cases) and in the 0.5% bronopol group (one case).One animal treated with 0.2% bronopol showed an isolated area of hyperkeratosis. One animals of the 0.2% bronopol group and 4 animals of the 0.5% bronopol group showed areas of dermal scarring (1 to 8 mm in length). All these reactions were considered to be non-specific and without toxicological significance. Areas of scabbing with associated dermal inflammation were seen in 2 animals treated with 0.5% bronopol. A similar effect also was seen in an animal of the 0.2% bronopol group, however within an untreated skin area. Therefore, this effect was considered to rather be related to the scarification of the skin than to the treatment with bronopol.
Neither gross- nor histopathological treatment-related changes were seen; organ weights were inconspicious.

Histopathological findings: non-neoplastic:

no effects observed

Details on results:

Local skin reaction (slight to well-defined erythema in 9/10 animals, and slight edema in all animals)was observed in the control rabbits. Similar irritation was seen in rabbits treated with Bronopol 0.2% although possibly with slightly more edema formation and slightly more persistent.Bronopol 0.5% treatment caused progression of slight to well-defined erythema to moderate erythema in 9/10 animals. Edema occurred on the third day and became well defined in all rabbits. Moderate edema developed in 3/5 males. The macroscopical examination of the treated skin areas at necropsy revealed a varying degree of inflammatory reaction in control (2 cases) and in the 0.5% bronopol group (one case).One animal treated with 0.2% bronopol showed an isolated area of hyperkeratosis.One animals of the 0.2% bronopol group and 4 animals of the 0.5% bronopol group showed areas of dermal scarring(1 to 8 mm in length). All these reactions were considered to be non-specific and without toxicological significance. Areas of scabbing with associated dermal inflammation were seen in 2 animals treated with 0.5% bronopol. A similar effect also was seen in an animal of the 0.2% bronopol group, however within an untreated skin area. Therefore, this effect was considered to rather be related to the scarification of the skin than to the treatment with bronopol.
No mortality and no signs of toxicity were observed.
Food consumption of all male and female animals was normal except for 2 animals in group 2 (0.2%) and one animal in group 3 (0.5%) where food consumption was depressed.Initial loss of body weight was seen in all groups including controls; weight gain was comparable and no treatment related trend could be observed which would indicate a specific effect of Bronopol on growth.
Haematology and clinical chemistry were inconspicious.
Ophthalmoscopic examination revealed no abnormalities.
Neither gross- nor histopathological treatment-related changes were seen; organ weights were inconspicious.

Key result

Dose descriptor:

NOAEL

Effect level:

0.2 other: %

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: skin irritation

Remarks on result:

other: 2 mg/kg bw/day

Dose descriptor:

LOAEL

Effect level:

0.5 other: %

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: skin irritation

Remarks on result:

other: 5 mg/kg bw/day

Critical effects observed:

not specified

Table 1: Numerical scores awarded to dermal reactions elicited by Bronopol

(group 1: vehicle control; 2: Bronopol 0.2%; 3: Bronopol 0.5%).

Group	Sex	Animal No.	Reaction	Day 0	1	2	3	14	21
1	Male	324	Erythema	0	1	1	1	1	1
			Oedema	0	0	0	0	1	0
		469	Erythema	0	1	1	1	2	1
			Oedema	0	0	0	0	1	1
		478	Erythema	1	1	1	1	1	1
			Oedema	0	0	0	0	1	0
		2438	Erythema	0	1	1	1	1	1
			Oedema	0	0	0	0	0	0
		327	Erythema	0	1	1	1	1	1
			Oedema	0	0	0	0	1	0
	Female	350	Erythema	0	1	1	1	1	1
			Oedema	0	0	0	0	1	2
		2081	Erythema	1	1	1	1	1	1
			Oedema	0	0	0	0	1	0
		2082	Erythema	0	0	0	1	2	1
			Oedema	0	0	0	0	1	0
		332	Erythema	0	1	1	2	2	1
			Oedema	0	0	0	0	2	2
		344	Erythema	0	1	1	1	2	1
			Oedema	0	0	0	0	1	2

Group	Sex	Animal No.	Reaction	Day 0	1	2	3	14	21
2	Male	326	Erythema	1	1	1	1	2	1
			Oedema	0	0	0	0	1	1
		330	Erythema	1	1	1	1	2	1
			Oedema	0	0	0	0	1	0
		476	Erythema	0	1	1	1	2	2
			Oedema	0	0	0	0	1	1
		2054	Erythema	1	1	1	1	2	1
			Oedema	0	0	0	0	1	0
		2473	Erythema	0	0	1	1	1	1
			Oedema	0	0	0	0	1	0
	Female	342	Erythema	0	1	1	2	1	2
			Oedema	0	0	0	0	1	2
		2089	Erythema	0	1	1	1	1	1
			Oedema	0	0	0	0	0	0
		2097	Erythema	0	1	1	1	1	1
			Oedema	0	0	0	0	1	1
		2092	Erythema	0	1	1	1	1	1
			Oedema	0	0	0	0	1	1
		2406	Erythema	0	1	1	1	2	1
			Oedema	0	0	0	0	1	1

Group	Sex	Animal No.	Reaction	Day 0	1	2	3	14	21
3	Male	316	Erythema	0	2	2	2	3	3
			Oedema	0	0	0	1	3	2
		331	Erythema	0	2	2	2	3	3
			Oedema	0	0	0	1	3	2
		2057	Erythema	0	1	2	2	3	3
			Oedema	0	0	0	1	2	1
		2439	Erythema	0	1	1	1	3	3
			Oedema	0	0	0	1	3	3
		2055	Erythema	0	1	1	1	3	3
			Oedema	0	0	0	1	2	2
	Female	336	Erythema	0	1	1	1	3	3
			Oedema	0	0	0	1	2	2
		354	Erythema	0	1	1	2	3	3
			Oedema	0	0	0	1	2	2
		2083	Erythema	0	1	1	2	3	1
			Oedema	0	0	0	1	2	1
		335	Erythema	0	1	1	2	2	1
			Oedema	0	0	0	1	1	1
		337	Erythema	0	1	1	2	3	1
			Oedema	0	0	0	1	2	1

Conclusions:

CL-Freetext:
Repeated dermal application of Bronopol at 0.2 and 0.5% in methyl cellulose suspension over a period of 21 days was not associated with signs of systemic toxicity or deaths. Severe skin irritation was observed after application of 0.5% Bronopol, whereas 0.2% Bronopol caused skin reaction comparable to, but slightly more persistent than that elicited by the vehicle alone. Correspondingly, NOAEL- and LOAEL-values of 2 mg/kg bw/day and 5 mg/kg bw/day were estimated on the basis of the skin findings.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

5 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Quality of whole database:

The database for repeated dose dermal toxicity is complete and is considered to meet the relevant data requirements of REACH. No concentration of Bronopol with clear effects of systemic toxicity were noted in a well-documented study in rabbits up to and including the high-dose level of 5 mg/kg bw/day.

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was conducted in the seventies, i.e. prior to implementation of guidelines, and GLP was not compulsory at that time. However, the study was well-documented and the reported data are of scientific accetability.

Principles of method if other than guideline:

Method: other: no data

GLP compliance:

no

Species:

rabbit

Strain:

New Zealand White

Sex:

male/female

Details on test animals or test system and environmental conditions:

Source: Hylyne Commercial Rabbits, Cheshire
They were housed individually in metal cages.

Type of coverage:

other: non-occlusive

Vehicle:

other: methylcellulose

Details on exposure:

Route of Administration: dermal
At a dose level of 1 ml/kg b.w. the daily application of Bronopol at a concentration of 0.2 and 0.5% corresponds to a dose of 2 and 5 mg/kg bw/day
respectively.
Vehicle: 2,5% methylcellulose.
The residual test substance was removed after each treatment period of 6 hours by gentle washing of the skin application site with a warm dilute soap solution, followed by rinsing with clean water; thereafter the skin was blotted dry with absorbent paper.

Duration of treatment / exposure:

21 days, 6 hours/day

Frequency of treatment:

7 d/week

Dose / conc.:

0.2 other: %

Remarks:

bronopol in 2.5% methyl cellulose, 1 ml/kg bw, corresponding to 2 mg/kg bw/day

Dose / conc.:

0.5 other: %

Remarks:

bronopol in 2.5% methyl cellulose, 1 ml/kg bw, corresponding to 5 mg/kg bw/day

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

Post-exposure period: none

Observations and examinations performed and frequency:

Observations and examinations included local skin reaction (erythema and oedema), behavioural changes and
signs of toxicity, food consumption, body weight, haematology (packed cell volume, haemoglobin, red cell count,
total and differential white cell count), clinical chemistry (plasma urea, total serum proteins, serum protein,
serum alkaline phosphatase, serum transaminase (SGPT), ophthalmoscopic examination.
Body weights were recorded at test initiation and weekly thereafter; the group mean value (g/rabbit) was calculated. Food consumption for each rabbit was recorded weekly and the group mean food intake (g/rabbit/week) was calculated. The eyes of the animals were examined prior test start and again after 3 weeks of treatment (Keeler indirect ophthalmoscope).

Sacrifice and pathology:

Terminal studies after sacrifice of the animals included macroscopic examination and weighing of inner organs
(thyroid, heart, liver, kidneys, adrenals, and gonads) and microscopic examination of a series of tissues
(adrenal, brain, duodenum, eye, gall-bladder, gonads, heart, ileum, kidney, liver, lymph nodes, marrow smear,
mid-colon, pancreas, pituitary, salivary gland, skin (treated and untreated), spleen, stomach
(glandular and non-glandular), thymus, thyroid, urinary bladder.

Statistics:

Student's t-test and analysis of variance were employed to assess the significance of intergroup differences.

Clinical signs:

no effects observed

Description (incidence and severity):

No mortality and no signs of toxicity were observed.

Dermal irritation:

effects observed, non-treatment-related

Description (incidence and severity):

Local skin reaction (slight to well-defined erythema in 9/10 animals, and slight edema in all animals) was observed in the control rabbits. Similar irritation was seen in rabbits treated with Bronopol 0.2% although possibly with slightly more edema formation and slightly more persistent.Bronopol 0.5% treatment caused progression of slight to well-defined erythema to moderate erythema in 9/10 animals. Edema occurred on the third day and became well defined in all rabbits. Moderate edema developed in 3/5 males.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Initial loss of body weight was seen in all groups including controls; weight gain was comparable and no treatment related trend could be observed which would indicate a specific effect of Bronopol on growth.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption of all male and female animals was normal except for 2 animals in group 2 (0.2%) and one animal in group 3 (0.5%) where food consumption was depressed.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Ophthalmoscopic examination revealed no abnormalities.

Haematological findings:

no effects observed

Description (incidence and severity):

Haematology and clinical chemistry were inconspicious.

Clinical biochemistry findings:

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The macroscopical examination of the treated skin areas at necropsy revealed a varying degree of inflammatory reaction in control (2 cases) and in the 0.5% bronopol group (one case).One animal treated with 0.2% bronopol showed an isolated area of hyperkeratosis. One animals of the 0.2% bronopol group and 4 animals of the 0.5% bronopol group showed areas of dermal scarring (1 to 8 mm in length). All these reactions were considered to be non-specific and without toxicological significance. Areas of scabbing with associated dermal inflammation were seen in 2 animals treated with 0.5% bronopol. A similar effect also was seen in an animal of the 0.2% bronopol group, however within an untreated skin area. Therefore, this effect was considered to rather be related to the scarification of the skin than to the treatment with bronopol.
Neither gross- nor histopathological treatment-related changes were seen; organ weights were inconspicious.

Histopathological findings: non-neoplastic:

no effects observed

Details on results:

Local skin reaction (slight to well-defined erythema in 9/10 animals, and slight edema in all animals)was observed in the control rabbits. Similar irritation was seen in rabbits treated with Bronopol 0.2% although possibly with slightly more edema formation and slightly more persistent.Bronopol 0.5% treatment caused progression of slight to well-defined erythema to moderate erythema in 9/10 animals. Edema occurred on the third day and became well defined in all rabbits. Moderate edema developed in 3/5 males. The macroscopical examination of the treated skin areas at necropsy revealed a varying degree of inflammatory reaction in control (2 cases) and in the 0.5% bronopol group (one case).One animal treated with 0.2% bronopol showed an isolated area of hyperkeratosis.One animals of the 0.2% bronopol group and 4 animals of the 0.5% bronopol group showed areas of dermal scarring(1 to 8 mm in length). All these reactions were considered to be non-specific and without toxicological significance. Areas of scabbing with associated dermal inflammation were seen in 2 animals treated with 0.5% bronopol. A similar effect also was seen in an animal of the 0.2% bronopol group, however within an untreated skin area. Therefore, this effect was considered to rather be related to the scarification of the skin than to the treatment with bronopol.
No mortality and no signs of toxicity were observed.
Food consumption of all male and female animals was normal except for 2 animals in group 2 (0.2%) and one animal in group 3 (0.5%) where food consumption was depressed.Initial loss of body weight was seen in all groups including controls; weight gain was comparable and no treatment related trend could be observed which would indicate a specific effect of Bronopol on growth.
Haematology and clinical chemistry were inconspicious.
Ophthalmoscopic examination revealed no abnormalities.
Neither gross- nor histopathological treatment-related changes were seen; organ weights were inconspicious.

Key result

Dose descriptor:

NOAEL

Effect level:

0.2 other: %

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: skin irritation

Remarks on result:

other: 2 mg/kg bw/day

Dose descriptor:

LOAEL

Effect level:

0.5 other: %

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: skin irritation

Remarks on result:

other: 5 mg/kg bw/day

Critical effects observed:

not specified

Table 1: Numerical scores awarded to dermal reactions elicited by Bronopol

(group 1: vehicle control; 2: Bronopol 0.2%; 3: Bronopol 0.5%).

Group	Sex	Animal No.	Reaction	Day 0	1	2	3	14	21
1	Male	324	Erythema	0	1	1	1	1	1
			Oedema	0	0	0	0	1	0
		469	Erythema	0	1	1	1	2	1
			Oedema	0	0	0	0	1	1
		478	Erythema	1	1	1	1	1	1
			Oedema	0	0	0	0	1	0
		2438	Erythema	0	1	1	1	1	1
			Oedema	0	0	0	0	0	0
		327	Erythema	0	1	1	1	1	1
			Oedema	0	0	0	0	1	0
	Female	350	Erythema	0	1	1	1	1	1
			Oedema	0	0	0	0	1	2
		2081	Erythema	1	1	1	1	1	1
			Oedema	0	0	0	0	1	0
		2082	Erythema	0	0	0	1	2	1
			Oedema	0	0	0	0	1	0
		332	Erythema	0	1	1	2	2	1
			Oedema	0	0	0	0	2	2
		344	Erythema	0	1	1	1	2	1
			Oedema	0	0	0	0	1	2

Group	Sex	Animal No.	Reaction	Day 0	1	2	3	14	21
2	Male	326	Erythema	1	1	1	1	2	1
			Oedema	0	0	0	0	1	1
		330	Erythema	1	1	1	1	2	1
			Oedema	0	0	0	0	1	0
		476	Erythema	0	1	1	1	2	2
			Oedema	0	0	0	0	1	1
		2054	Erythema	1	1	1	1	2	1
			Oedema	0	0	0	0	1	0
		2473	Erythema	0	0	1	1	1	1
			Oedema	0	0	0	0	1	0
	Female	342	Erythema	0	1	1	2	1	2
			Oedema	0	0	0	0	1	2
		2089	Erythema	0	1	1	1	1	1
			Oedema	0	0	0	0	0	0
		2097	Erythema	0	1	1	1	1	1
			Oedema	0	0	0	0	1	1
		2092	Erythema	0	1	1	1	1	1
			Oedema	0	0	0	0	1	1
		2406	Erythema	0	1	1	1	2	1
			Oedema	0	0	0	0	1	1

Group	Sex	Animal No.	Reaction	Day 0	1	2	3	14	21
3	Male	316	Erythema	0	2	2	2	3	3
			Oedema	0	0	0	1	3	2
		331	Erythema	0	2	2	2	3	3
			Oedema	0	0	0	1	3	2
		2057	Erythema	0	1	2	2	3	3
			Oedema	0	0	0	1	2	1
		2439	Erythema	0	1	1	1	3	3
			Oedema	0	0	0	1	3	3
		2055	Erythema	0	1	1	1	3	3
			Oedema	0	0	0	1	2	2
	Female	336	Erythema	0	1	1	1	3	3
			Oedema	0	0	0	1	2	2
		354	Erythema	0	1	1	2	3	3
			Oedema	0	0	0	1	2	2
		2083	Erythema	0	1	1	2	3	1
			Oedema	0	0	0	1	2	1
		335	Erythema	0	1	1	2	2	1
			Oedema	0	0	0	1	1	1
		337	Erythema	0	1	1	2	3	1
			Oedema	0	0	0	1	2	1

Conclusions:

CL-Freetext:
Repeated dermal application of Bronopol at 0.2 and 0.5% in methyl cellulose suspension over a period of 21 days was not associated with signs of systemic toxicity or deaths. Severe skin irritation was observed after application of 0.5% Bronopol, whereas 0.2% Bronopol caused skin reaction comparable to, but slightly more persistent than that elicited by the vehicle alone. Correspondingly, NOAEL- and LOAEL-values of 2 mg/kg bw/day and 5 mg/kg bw/day were estimated on the basis of the skin findings.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

2

Study duration:

subacute

Species:

rabbit

Quality of whole database:

The database for repeated dose dermal toxicity is complete and is considered to meet the relevant data requirements of REACH. No concentration of Bronopol with clear toxic effects was tested in this well-documented study on rabbits. NOAEL: 2 mg/kg bw/day; LOAEL: 5 mg/kg bw/day (referring to skin findings).

Additional information

Repeated dose toxicity: oral

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 whereas each test group of the satellite series comprised 15 animals/sex. 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; the concentration of test substance in the drinking water was changed as necessary to preserve the required dosage levels. The animals were checked for mortality and clinical symptoms of toxicity; food and water consumption as well as body weight were determined. Ophthalmoscopic examinations were done. Blood samples were removed at defined time points from the orbital sinus of fasted animals for the purpose of examination of haematological and clinical-chemical parameters. Urine samples were collected for the assessment of urinalysis parameters. 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. All rats of the main test series were examined for neoplastic changes also. 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. Moreover, the histopathological findings were re-evaluated and completed.

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.

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 a spontaneously 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 spontaneously 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 dose groups.

Neoplastic findings:

With regard to neoplastic changes, 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 the findings of the older evaluations, 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 10 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 10 mg/kg bw/day. Taking into account the performed re-evaluation of test concentrations, the actual NOAEL 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.

Furthermore, the sub-chronic toxicity of Bronopol in rats and dogs was investigated after daily oral gavage with the test substance over a period of 13 weeks (Huntingdon Research Centre, 1973; Rivett, 1973). For rats, as LOAEL 20 mg/kg bw /day and NOAEL <20 mg/kg bw/day was determined, respectively.In case of dogs, as LOAEL 20 mg/kg bw /day and a of NOAEL 8 mg/kg bw/day was determined, respectively.

 

Repeated dose toxicity: dermal

In a sub-acute dermal toxicity study (HuntingdonResearchCentre, 1973) the effects of repeated administration of Bronopol to the skin of rabbit for three weeks were examined.

Bronopol, suspended at 0.2 % and 0.5 % in 2.5 % methylcellulose was applied to the abraded skin of rabbits, at a dose volume of 1 mL/kg bw, daily, 7 days a week, for a period of 3 weeks resulting in dose levels of 2 and 5 mg/kg bw/day. At the end of the daily 6 hour exposure period the skin was washed and blotted dry. During the experimental period abrading was repeated weekly and hair clipping as necessary. The rabbits were equipped with a collar to prevent licking the treated area. They were housed individually in metal cages. A total of 30 rabbits (5 animals/sex/group) were used for the test. Observations and examinations included: behavioural changes and signs of toxicity, local skin reaction, body weight gain, food consumption and ophthalmologic examinations. Prior test initiation and at termination, blood samples were collected for analysis of a series of haematological and clinical-chemical parameters (packed cell volume, haemoglobin, red cell count, total white cell count and differential count, plasma urea, total serum proteins, serum alkaline phosphatase and serum glutamic-pyruvic transaminase). At the end of the treatment period, the animals were sacrificed for the purpose of necropsy, and were subjected to gross pathology, organ weighing and histopathology. The statistical assessment of significant inter-group differences was based on the Student's t-test and the analysis of variance.

No clinical effects were seen and no mortality was reported.

The treatment of the skin with the vehicle 2.5% methylcellulose resulted in slight to well-defined erythema in the control rabbits. Similar but slightly more persistent reactions were reported for the rabbits treated with 0.2% Bronopol. Treatment with 0.5% Bronopol resulted in stronger dermal reactions, including moderate erythema and edema, with extensive scabbing within the application site.Scabbing was considered to result from a non-specific effect on the normal healing of scarified skin and was therefore of no toxicological relevance.

No Bronopol-related effect on body weight was evident.In fact, all groups including the control showed loss in body weight or poor body weight gain. Only one female treated with 0.2 % Bronopol showed heavy loss in body weight. Food consumption for males was similar for all groups including control and therefore was inconspicuous.For the females, two animals treated with 0.2% Bronopol and one treated with 0.5 % Bronopol showed depressed food consumption within week 2 and 3 of treatment. One of the 0.2 % Bronopol treated females showed heavy loss in body weight. Food consumption of the remaining females was inconspicuous. No treatment-related ophthalmologic effects were seen. Neither the haematological nor the clinical chemical parameters showed treatment-related changes. At necropsy, the organ weights of all animals were within normal range and no treatment-related trends were seen. Neither gross- nor histopathological examination revealed treatment-related abnormalities.

Repeated dermal application of Bronopol at 0.2 and 0.5 % in methylcellulose suspension over a period of 21 days was not associated with signs of systemic toxicity or deaths. Severe skin irritations were observed after application of 0.5 % Bronopol, whereas 0.2 % Bronopol caused skin reactions comparable to, but slightly more persistent than those elicited by the vehicle alone. Correspondingly, NOAEL- and LOAEL-values were estimated for skin irritation reactions as endpoint: LOAEL: 5 mg/kg bw/day (0.5%), NOAEL: 2 mg/kg bw/day (0.2%). The NOAEL for systemic effects was established at 5 mg/kg bw/day.

The study is classified as acceptable (key study). Investigations were conducted in 1973, prior to implementation of guideline; GLP was not compulsory at that time. The conduct of the study was guideline-like, excepted for the fact, that no concentration with clear toxic effects was tested. However, the study was well-documented. No further data on the test substance was provided.

Furthermore, 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 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. Furthermore, observed tumors rather were related to the irritant potential of Bronopol than to a carcinogenic potential of the test substance. The study is classified as acceptable (supporting study). Investigations were conducted prior to the implementation of guideline and GLP was compulsory at this time. However, these results emphasizes the results of the dermal toxicity study in rabbits.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
As relevant study the key study was selected which covers a time period of 2 years.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
The key study was selected.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:

The key study was selected.

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 1272/2008. As a result the substance is not considered to be classified for repeated dose toxicity under Regulation (EC) No. 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.