Registration Dossier

Administrative data

Description of key information

The LD50/LC50 values derived from the key or WoE studies were:
- LD50 (oral, rat) = 193 mg/kg bw
- LC50 (inhalation, rat, 4 hour) > 0.588 mg/l; LC50 (inhalation, rat, 4 hour) > 0.120 mg/l - < 1.140 mg/l
- LD50 (dermal, rat) > 2000 mg/kg bw

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records
Reference
Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 July 2001 - 25 September 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 401 (Acute Oral Toxicity)
Qualifier:
according to
Guideline:
EPA OPPTS 870.1100 (Acute Oral Toxicity)
Qualifier:
according to
Guideline:
other: JMAFF Acute Oral Toxicity Study, 2000
Qualifier:
according to
Guideline:
EU Method B.1 (Acute Toxicity (Oral))
GLP compliance:
yes
Test type:
standard acute method
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
Fischer 344 rats obtained from Charles River Laboratories Inc. (Raleigh, North Carolina) and weighing 102-245 grams at study start were used for this study. Rats were born on May 7, 2001 or June 11, 2001. The rats were dosed on July 17, 18, 24, 31, or August 2, 8, 2001 and were sent to necropsy on August 1, 7, 14, 16, or 22, 2001. Animal care facilities are fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. Upon arrival at the laboratory, the rats were examined for health status by the laboratory veterinarian. Animals were housed two or three per cage in stainless steel cages in rooms that were designed to maintain adequate environmental conditions concerning temperature, humidity, photocycle, and air exchanges. The relative humidity and room temperature were maintained within a range of 40-70% and 22 ± 3 °C, respectively. A 12-hour light/dark photocycle was maintained for all animal rooms with lights on at 6:00 a.m. and off at 6:00 p.m. Room air was exchanged approximately 12-15 times/hour, and the water lines automatically bled every six hours.
Animals were provided LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in pelleted form. Feed and municipal water were provided ad libitum. Analysis of the feed was performed by PMI Nutrition International to confirm the nutritional adequacy of the diet and to quantify the levels of selected contaminants. Drinking water obtained from the municipal water source was periodically analyzed for chemical parameters and biological contaminants by the municipal water department. In addition, specific analyses for chemical contaminants were conducted at periodic intervals by an independent testing facility. Copies of these analyses are maintained at Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland Michigan. Animals were acclimated to the laboratory environment for at least one-week prior to study start. Animals were identified via a code number transmitted by a subcutaneously implanted transponder.
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
Five male rats per dose level received 100, 200, or 300 mg of Bronopol per kg body weight as a 2%, 4%, or 6% mixture in distilled water by single dose gavage. Five female rats per dose level also received 100, 150, or 200 mg of the test material per kg body weight as a 2%, 3%, or 4% mixture in distilled water by single dose gavage. Rats were fasted the night prior to treatment. Feed was provided to all rats immediately following administration of the test material.
Doses:
Males: 100, 200, 300 mg/kg bw
Females: 100, 150, 200 mg/kg bw
No. of animals per sex per dose:
5
Control animals:
no
Details on study design:
A Detailed Clinical Observation (DCO) was conducted for all rats prior to test material administration for comparison with the observations recorded throughout the study. Animals were observed a minimum of two times on the day of treatment. A DCO was done each day (including weekends and holidays) during the study. Hand-held and open-field observations included a careful physical examination according to an established format. For scored DCOs only observations other than typically expected were recorded. Observations were dictionary based, and the dictionary contained most of the common physical and neurologic abnormalities seen in toxicity studies. Since not all potential observations were contained in the dictionary, free-field descriptions also were allowed. Each animal was weighed, pre-study, the day of treatment, and on test days 2, 8, and 15. A necropsy was performed on all animals.

Animals submitted alive for necropsy were anesthetized by inhalation of carbon dioxide and were euthanized by decapitation after clamping of the trachea. A complete necropsy was conducted on all animals by a veterinary pathologist assisted by a team of trained individuals. The eyes were examined in situ using a moistened glass microscope slide applied to the corneal surface. Following inspection of the externum and body orifices, the nasal, cranial, oral, thoracic, and abdominal cavities were opened and the visceral organs were examined both in situ and following dissection, and tissues were not saved.
Statistics:
Means and standard deviations were calculated for body weights. The data were evaluated for statistical outliers by a sequential test (Grubbs, 1969); however, outliers were not routinely excluded from statistical analysis. An approximate LD50 was calculated by linear interpolation of the arc sine transformed data (Stephan, 1977).
Sex:
male
Dose descriptor:
LD50
Effect level:
211 mg/kg bw
Based on:
test mat.
Sex:
female
Dose descriptor:
LD50
Effect level:
193 mg/kg bw
Based on:
test mat.
Mortality:
Two male rats given 200 mg/kg and all males given 300 mg/kg died on test day 1 or 2. One female given 100 mg/kg and three females given 200 mg/kg died on test day 1.
Clinical signs:
Clinical observations in rats that died were consistent with their moribund condition. Three males and two females given 200 mg/kg, all females given 150 mg/kg, and all males and four females given 100 mg/kg survived the two-week observation period. Clinical observations for surviving rats given 100 or 200 mg/kg consisted of various combinations of: lacrimation, noisy respiration, decreased responsiveness to touch, decreased activity, decreased reactivity to handling, soft feces, vocalization, and soiling of the perioral, perinasal, perineal, and/or periocular regions. Clinical signs for surviving animals given 100 or 200 mg/kg diminished over time, and these animals were normal at study termination. Females given 150 mg/kg had no remarkable clinical observations.
Body weight:
Two of the surviving female rats given 200 mg/kg gained weight over the duration of the study. Two surviving male rats given 200 mg/kg lost weight between test days 1 and 2 or 8, but gained weight over the remainder of the study. All of the female rats given 150 mg/kg gained weight over the duration of the study. Three male rats and three female rats given 100 mg/kg gained weight over the duration of the study. One female rat and two male rats given 100 mg/kg lost weight between test days 1 and 2, but gained weight over the remainder of the study.
Gross pathology:
Male rats given 200 or 300 mg/kg and females given 100 or 200 mg/kg that died had treatment-related gross findings consisting of various combinations of: dark glandular mucosa of the stomach, dilatation of the stomach with cloudy fluid, perineal soiling, and hemolyzed blood in the gastrointestinal tract. Dark foci in the lungs were also noted in two males given 300 mg/kg that died. Surviving animals from all dose groups had no treatment-related gross pathologic observations.

Table 1. Mortality

Dose
[mg/kg bw]

Number of dead /
number of investigated

Time of death (range)

100

0/5 males
1/5 females

-
day 1

150

0/5 females

-

200

2/5 males
3/5 females

day 1
day 1

300

5/5 males

day 1 (4x), day 2 (1x)

Interpretation of results:
Category 3 based on GHS criteria
Conclusions:
Under the conditions of this study, the acute oral LD50 of Bronopol in Fischer 344 rats was approximately 211 mg/kg in males and approximately 193 mg/kg in females.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LD50
Value:
193 mg/kg bw
Quality of whole database:
The key study was selected.

Acute toxicity: via inhalation route

Link to relevant study records

Referenceopen allclose all

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1986
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: No guideline was mentioned. It was not specified whether the study did follow GLP. The study was however well-documented and of scientific acceptability.
Principles of method if other than guideline:
No guideline given.
GLP compliance:
not specified
Remarks:
not specified
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Animals source: Charles River (UK) Ltd., Margate
Weight range for both, males and females: 180 – 200 g
Age: about 6 to 8 weeks
The chamber temperature and the relative humidity for the control and the treated groups ranged between 15 and 19 °C and between 30 to 89%. The air flow rate was 15 litres/min. for the control group and between 13 to 18 litres/min. for the treated groups. The oxygen concentrations were within the range of 21 to 22 % for all groups.
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose/head only
Vehicle:
other: filtered air
Duration of exposure:
4 h
Concentrations:
Analytical concentrations: 0, 0.038, 0.089, 0.588 mg/l
Nominal concentrations: 0, 1.80, 2.59, 23.23 mg/l
No. of animals per sex per dose:
5
Control animals:
yes
Details on study design:
The test was conducted with 3 test groups and one control group. Each test group (defined as group 2, 3 and 4) comprised 5 male and 5 female Sprague-Dawley rats. For test atmosphere preparation and in order to get an adequate preparation in terms of particle size and respirability, the test substance was milled by means of an ultracentrifugal mill prior to use. The test atmospheres were then prepared in a Perspex chamber (capacity: ca. 10 litres) by means of a Wright duct feed generator. The test substance was conducted continuously into the chamber with filtered air at flow rate of 13 - 18 litres/min; the air flow rate was monitored and recorded at 30 minute-intervals. The nominal test concentrations were 0, 1.80, 2.59, 23.23 mg/l; the corresponding measured concentrations were 0, 0.038, 0.089 and 0.588 mg/l. The animals were nose-head exposed to the test atmosphere for a period of 4 hours; the control animals received filtered air without test substance. The rats were observed hourly during the exposure and once a day over the observation period of 14 days for mortality and clinical signs of toxicity. Body weight was assessed prior test initiation, at the end of the 4 hour-exposure, once daily between day 1 and 7 of observation, on day 14 and prior sacrifice. Animals that died were subjected to gross pathological examination as well as histopathology. The surviving rats were sacrificed at the end of the observation period and were also subjected to gross pathology and histopathology. Particular attention was given to the nasal passages, the cranial cavity and the respiratory tract. Lungs, bronchi, trachea and nasal passages were examined; lungs, liver, kidney and skin samples from 2 animals were fixed in 10% neutral buffered formalin for the purpose of histopathological examination.
Sex:
male/female
Dose descriptor:
LC50
Effect level:
>= 0.588 mg/L air
Exp. duration:
4 h
Mortality:
Of the high dose group (0.588 mg/l) one male animal was found dead on the day following exposure; and 2 more animals (one male and one female) were killed for humane reasons because they suffered from inflammation of the eyes. The authors attribute the deaths of 3 animals at this level only to the local irritancy of bronopol. However, symptoms of local irritation only occured at concentrations causing lethality. Thus, acute inhalation toxicity is the predominant effect.No deaths occurred in the control groups or at concentrations of 0.038 or 0.089 mg/l.
Clinical signs:
other: High dose group (0.588 mg/l): nasal discharge, red staining from the eyes, staining of the head and inflammation of the eyes was observed on the day of exposure in most animals, sometimes accompanied by swelling of the head, throat and/or forepaws. Thes
Body weight:
Small body weight losses were observed for both control and treated animals. Almost all animals had returned to their pre-exposure body weight by day 4 of the observation period, and body weight gains were normal thereafter. The initial body weight losses were similar in treated and control groups and were attributed to the restraint procedure and not to the exposure to bronopol.
Gross pathology:
There were no treatment-related gross or histopathological findings in the kidneys, livers or lungs of the high dose level group animals. Local dermatitis and ulceration were seen in 2 high dose animals and were attributed to dermal exposure, but not to inhalation.
Other findings:
All the mean mass median diameters indicated that the test atmosphere was respirable. In fact, the reported mean values were 1.31, 1.99 and 6.66 µm respectively for the 0.038, the 0.089 and the 0.588 mg/L groups.

The NOEC was about 0.038 mg/L.

The ratio between measured and nominal test concentrations indicated a low efficiency in the generation of the test aerosol. However, the authors indicated that such a low efficiency is not unusual for aerosol generation from powder using the Wright dust feed generator and mainly is due to losses within the chamber.

Interpretation of results:
Category 3 based on GHS criteria
Conclusions:
The acute inhalation LC50 in rats was >0.588 mg/L. The classification is based on a weight of evidence approach additionally considering the LC50 > 0.120 - < 1.140 mg/m3 determined in the study by Dow (2003).
Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
5 December 2002 - 30 May 2003
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
As only two dose levels are available pinpointing the exact LC50 for classification is difficult.
Qualifier:
according to
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Qualifier:
according to
Guideline:
EPA OPPTS 870.1300 (Acute inhalation toxicity)
Qualifier:
according to
Guideline:
other: EEC (1992) and MAFF (1985)
GLP compliance:
yes
Test type:
traditional method
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
Source: Charles River Laboratories Inc., Raleigh, North Carolina, USA
Age/weight at study initiation: 102-245 g at start of study; 7 weeks old at arrival, 8 weeks old at exposure
Physical/Acclimation: Upon arrival at the laboratory, each animal was evaluated by a laboratory veterinarian or a trained animal/toxicology technician, under the direct supervision of a laboratory veterinarian, to determine the general health status and acceptability for study purposes. The animals were housed 2 - 3 per cage in stainless steel cages, in rooms designed to maintain adequate conditions (temperature, humidity, and photocycle), and acclimated to the laboratory for approximately seven days prior to the start of the study.
Animals were acclimated to the nose cones and chamber for at least two hours on the day preceding exposure to the test material.
Housing: Prior to and after exposure, the rooms in which animals were housed had a 12-hour photocycle (on at 6:00 a.m., off at 6:00 p.m.), and are designed to maintain adequate environmental conditions concerning temperature and relative humidity and regulated for the specific species under study (22 ± 3 °C, 40 - 70%, respectively for rats). The relative humidity and minimum/maximum temperature of the animal room was recorded daily.
Randomization/Identification: All animals were randomized by weight into groups of five using a computerized randomization program. Animals were singly housed following randomization. All animals used for exposure were individually identified with alphanumeric metal ear tags.
Feed and Water: Animals were provided LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in pelleted form. Feed and municipal water was provided ad libitum except during exposure. Analysis of the feed was performed by LabDiet to confirm the diet provides adequate nutrition and to quantify the levels of selected contaminants. Drinking water obtained from the municipal water source was periodically analyzed for chemical parameters and biological contaminants by the municipal water department. In addition, specific analyses for chemical contaminants were conducted at periodic intervals by an independent testing facility.
Randomization and Identification: All animals were randomized by weight into groups of five using a computer randomization program designed to ensure homogeneous distribution of rats based on body weight. For each exposure, one group of male and female rats was selected for use on this study from those that were randomized. The study animals were individually identified with alphanumeric metal ear tags.
Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
>= 3.29 - <= 9.34 µm
Geometric standard deviation (GSD):
>= 1.64 - <= 4.67
Remark on MMAD/GSD:
1.14 mg/L exposure: MMAD of 9.34 microns with average GSD of 4.67 microns
0.12 mg/L exposure: MMAD of 3.29 microns with average GSD of 1.64 microns
Details on inhalation exposure:
Exposures occurred under dynamic airflow conditions. Milled material was aerosolized with a Model 00 Jet-O-Mizer jet mill (Fluid Energy Processing & Equipment Company, Hatfield, PA).
Due to test material agglomeration, a respirable aerosol with a MMAD between 1 and 4 microns could not be reliably maintained at a chamber concentration greater than 1 mg/L.
Mass concentration determined gravimetrically at least 3 times during each exposure period.
Particle size was determined at least twice during each exposure period through a multi-stage cascade impactor.
Analytical verification of test atmosphere concentrations:
yes
Remarks:
gravimetric
Duration of exposure:
4 h
Concentrations:
time-weighted average concentrations of 1.14 and 0.12 mg/L
No. of animals per sex per dose:
5
Control animals:
no
Details on study design:
Clinical signs, mortality: prior to exposure and every 30 minutes during exposure, at least once daily thereafter
Detailed clinical observations: prior and after application, daily thereafter
Body weights: prior to exposure and on days 2, 4, 8, 11 and 15
Decedents, animals killed in a moribund condition, and all survivors were subjected to necropsy and post mortem examination.
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 0.12 - < 1.14 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Mortality:
At 1140 mg/m3 4/5 males and 3/5 females died during exposure and the remaining animals died until end of day 3. At 120 mg/m3 one male rat died during exposure.
Clinical signs:
other: Clinical signs during exposure included bloody nose and mouth breathing (at 1140 mg/m3 only) as well as labored breathing, and soiling of the hair coat (both concentrations). At the high concentration the observed gross nasal lesions and impaired respirat
Body weight:
A slight loss of body weight was noted on day 2 but rats gained body weight afterwards.
Gross pathology:
Necropsy findings in the animals that died during the study: congestion of the brain, kidneys, liver, pharynx, and lung; edema of the lung; froth in the tracheas well as gas in the gastrointestinal tract and stomach.
No visible lesions were noted in animals that survived until the scheduled sacrifice.

Table 1. Mortality

Dose [mg/m3]

Number of dead /
number of investigated

Time of death (range)

 120

1/5 males
0/5 females

day 1
-

1140

5/5 males

5/5 females

day 1 (4x), day 3
day 1 (4x), day 2

Interpretation of results:
Category 3 based on GHS criteria
Conclusions:
The acute inhalation LC50 in rats is >120 mg/m3 but <1140 mg/m3. As only two dose levels are available pinpointing the exact LC50 for classification is difficult. The classification is based on a weight of evidence approach additionally considering the LC50 > 580 mg/m3 determined in the study by Hazleton Laboratories Europe Ltd (1986).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
discriminating conc.
Value:
588 mg/m³
Quality of whole database:
No exact LC50 could be determined. Therefore, the classification is based on a weight of evidence approach considering the LC50 (4h) > 580 mg/m3 determined in the study by Hazleton Laboratories Europe Ltd (1986) and the LC50 (4h) > 120 - < 1140 mg/m3 determined in the study by Dow (2003). An aerosol was tested in both studies.

Acute toxicity: via dermal route

Link to relevant study records
Reference
Endpoint:
acute toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted in accordance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
GLP compliance:
yes
Test type:
fixed dose procedure
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Young adult animals
(male animals approx. 8 - 16 weeks
female animals approx. 9 - 16 weeks)
Animal weights at start of Animals of comparable weight (200 g - 300 g ± 20% of the the study: mean weight)
Source: Charles River Laboratories, Sulzfeld, Germany
Type of coverage:
semiocclusive
Vehicle:
other: Tylose CB 30.000
Details on dermal exposure:
The test substance was prepared in 0.5% aqueous Tylose® and was applied to a skin area of about 50 cm2 (10% of body surface) under semi occlusive conditions for 24 hours.The test substance concentration in the vehicle was 80g/100 ml (w/v) and the application volume was 2.5 ml/kg
Duration of exposure:
24 hours
Doses:
2000 mg/kg bw
No. of animals per sex per dose:
5
Control animals:
no
Details on study design:
Five male and five female Wistar rats were treated with 2000 mg/kg bw test substance.The test substance was prepared in 0.5% aqueous Tylose® and was applied to a skin area of about 50 cm2 under semi-occlusive conditions for 24 hours.The test substance concentration in the vehicle was 80g/100 ml (w/v) and the application volume was 2.5 ml/kg. After 24 hours of exposure the treated skin was cleaned with water. The animals were checked for mortality twice each working day and once on Saturday, Sundays or holidays. They were observed for clinical symptoms of toxicity several times on the day of treatment and once daily thereafter. Body weights were recorded prior test start, weekly thereafter, and at the end of the observation period. The skin was examined 30 to 60 minutes following removal of the dressing, weekly thereafter, and at the end of the observation period. All rats that died and the surviving rats at the end of the study (18 days observation period) were subjected to necropsy and examined for gross pathology.Analytical determinations of the test substance preparation with respect to stability in the vehicle were performed prior to starting the toxicological study.
The assessment of skin findings was based on Draize JH (Appraisal of the safety of chemicals in food, drugs and cosmetics. The association of food and drug officials of the United States Austin, Texas, 1959).
Statistics:
Binomial test according to SNEDECOR GW (“Statistical methods”, 8th ed., Iowa State University Press/Ames, 1989).
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Remarks on result:
other: No mortality occurred
Mortality:
No mortality occurred
Clinical signs:
The animals of both sexes displayed poor general state, dyspnoea and apathy as clinical symptoms; these symptoms lasted until day 1 of observation.Local effects at the application sites were seen in both, males and females and included white discoloration of the skin, erythema, edema, eczematoid skin change, scaling and crust formation.These effects lasted until the end of the observation period.
Body weight:
Body weights were inconspicuous.
Sex BW on Day 0 BW on Day 7 BW on Day 13
Males 246 g 247 g 263 g
Females 220 g 219 g 221 g
Gross pathology:
Excepted for incrustation and full thickness necrosis seen at the application site in 4/5 males and 5/5 females, pathological examination at necropsy revealed no further abnormalities.
Interpretation of results:
GHS criteria not met
Conclusions:
The LD50 (males+females) was determined as >2000 mg/kg bw (no mortlity occured).
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
discriminating dose
Value:
2 000 mg/kg bw
Quality of whole database:
The key study was selected. As LD50 value >2000 mg/kg was determined.

Additional information

Acute toxicity: oral

In an GLP compliant acute oral toxicity study according to OECD test guideline 401 groups of 5 male and 5 female Fischer 344 rats were given single oral doses of Bronopol at 100, 200 or 300 mg/kg bw (males) and 100, 150, 200 mg/kg bw (females), as a suspension in water (The Dow Chemical Company, K-081547-002, 2001). The rats were observed for up to 14 days after treatment. A necropsy was performed on all animals. Two male rats given 200 mg/kg and all males given 300 mg/kg died on test day 1 or 2. One female given 100 mg/kg and three females given 200 mg/kg died on test day 1. Clinical observations in rats that died were consistent with their moribund condition. Three males and two females given 200 mg/kg, all females given 150 mg/kg, and all males and four females given 100 mg/kg survived the two-week observation period. Clinical observations for surviving rats given 100 or 200 mg/kg consisted of various combinations of: lacrimation, noisy respiration, decreased responsiveness to touch, decreased activity, decreased reactivity to handling, soft feces, vocalization, and soiling of the perioral, perinasal, perineal, and/or periocular regions. Clinical signs for surviving animals given 100 or 200 mg/kg diminished over time, and these animals were normal at study termination. Females given 150 mg/kg had no remarkable clinical observations. Two of the surviving female rats given 200 mg/kg gained weight over the duration of the study. Two surviving male rats given 200 mg/kg lost weight between test days 1 and 2 or 8, but gained weight over the remainder of the study. All of the female rats given 150 mg/kg gained weight over the duration of the study. Three male rats and three female rats given 100 mg/kg gained weight over the duration of the study. One female rat and two male rats given 100 mg/kg lost weight between test days 1 and 2, but gained weight over the remainder of the study. Male rats given 200 or 300 mg/kg and females given 100 or 200 mg/kg that died had treatment-related gross findings consisting of various combinations of: dark glandular mucosa of the stomach, dilatation of the stomach with cloudy fluid, perineal soiling, and hemolyzed blood in the gastrointestinal tract. Dark foci in the lungs were also noted in two males given 300 mg/kg that died. Surviving animals from all dose groups had no treatment-related gross pathologic observations.

The oral administration of Bronopol to male rats resulted in a LD50 value of 211 mg/kg bw in males 193 mg/kg bw in females.

The study was conducted under GLP and the study method used followed that described in the OECD Guideline No. 401. Thus, this study is classified as acceptable (key study).

In a single dose study (Safepharm Laboratories Limited, 1987) the acute oral median lethal dose (LD50) of Bronopol was estimated by oral gavage in rat: Five male and five female Sprague-Dawley rats per test group were administered following doses of Bronopol: 250, 500, 1000 and 2000 mg/kg bw; the doses were selected on the basis of the results of a range-finding study conducted prior to the main test.The 2000 mg/kg bw was tested alone within a limit test, prior to the range-finding test.The test substance was applied as single dose by gavage, using distilled water as vehicle; the administration volume was 10 mL/kg bw.

Following treatment, the animals were regularly examined for mortality and clinical signs of toxicity over an observation period of 14 days; body weights were recorded at test starting and during the observation period. All rats that died during the observation period as well as the surviving rats, which were sacrificed at the end of the observation period, were subjected to necropsy. The LD50 and the 95% confidence limits were calculated by means of the method of Weil (1952). Mortality in the 250 and the 500 mg/kg bw group was 30 and 70%, respectively. At both highest tested doses of 1000 and 2000 mg/kg bw, mortality was 100%. Clinical symptoms indicative of toxicity were seen in all groups during the first hours following treatment and mainly included hunched posture, piloerection, lethargy and decrease in respiratory rate. Excepted for one male suffering from hunched posture, piloerection, lethargy and decreased respiratory rate from day 4 to day 9 of observation, all survivors of the 250 mg/kg bw group were free from symptoms from day 2 of observation. In the 500 mg/kg bw group, one surviving female displayed hunched posture and piloerection up to day 11 of observation; this animal furthermore showed uncontrolled head movements from day 3 to day 5. A small number of surviving animals showed reduced body weight gain over the first week; body weight gain over the second week of observation was as expected. Necropsy of the animals that died during the observation period revealed dark liver and kidneys, severe haemorrhage or ulceration of the gastric mucosa and haemorrhage of the small and large intestines. The dead animals of the 500, 1000 and 2000 mg/kg bw groups additionally showed abnormally red lungs, and those of the 2000 mg/kg be group had dark spleens and sloughing of the non-glandular stomach region. Oral administration of Bronopol to male rats resulted in a LD50 value of 273 mg/kg bw and to female rats in a LD50 value of 374 mg/kg bw. A LD50 value for males and females was 305 mg/kg bw.

The study method used followed that described in the OECD Guidelines for Testing of Chemicals (1981) No. 401 "Acute Oral Toxicity".

Furthermore, in a study (Boots Pharmaceuticals, 1992/TX92163) groups of ten male and ten female Boots-Wistar rats were given single oral doses of Bronopol at 200, 280, 390, 550 or 770 mg/kg/bw, as a solution in distilled water. The rats were observed for a seven-day period. Most of the decedents and, at the end of the study, the survivors given 280 mg/kg/bw or more and subsequently killed, were dissected and examined macroscopically. No gross abnormalities were observed at necropsy of decendents or animals sacrificed at the end of the study. Oral administration of Bronopol to male rats resulted in a LD50 value of 307 mg/kg bw and to female rats in a LD50 value of 342 mg/kg bw. At the time the study was conducted (1972) GLP was not compulsory.

In an acute oral toxicity study groups of ten male Boots-Wistar rats were given single oral doses of Bronopol at 36, 54, 80, 120, 180, 270, 400 or 600 mg/kg bw, as a suspension in 0.4 % aqueous cellosize solution (Boots Company, 1988). The rats were observed for up to ten days after treatment. Decedents, and all rats given 80 mg/kg or more and killed at the end of the study, were dissected and examined post mortem. The oral administration of Bronopol to male rats resulted in a LD50 value of 254 mg/kg bw bw with a 95 % confidence interval of 177 to 431 mg/kg bw. At the time the study was conducted (1970) GLP was not compulsory. The study is classifed as acceptable (key study). This study is reported also in another study report (Boots Pharmaceuticals, 1992/TX92160) and represents an addendum of a further study (Sutton, 1970).

 

Based on a supporting study in mice (Shaw, 1972), after oral administration of Bronopol signs of toxicity appeared soon after dosing and were encountered at all doses (250, 300, 360, 430 or 520 mg/kg bw) in both males and female. The majority of mice died within 19 hours of dosing but there were some deaths up to 6 days later. At autopsy, ulceration of the stomach or duodenum, thickening of the intestinal wall, adhesions of the stomach to the liver and lesions on the liver surface were observed. The liver lesions appeared to be secondary reactions to the gastrointestinal damage. The oral LD50 to male mice was 374 mg/kg bw and to female mice 327 mg/kg bw.

 

In an acute oral toxicity study groups of ten male Boots-Wistar rats were given single oral doses of Bronopol at 36, 54, 80, 120, 180, 270, 400 or 600 mg/kg bw, as a suspension in 0.4 % aqueous cellosize solution (Boots Company, 1988). The rats were observed for up to ten days after treatment. Decedents, and all rats given 80 mg/kg or more and killed at the end of the study, were dissected and examined post mortem. The oral administration of Bronopol to male rats resulted in a LD50 value of 254 mg/kg bw bw with a 95 % confidence interval of 177 to 431 mg/kg bw. At the time the study was conducted (1970) GLP was not compulsory. The study is classifed as acceptable (key study). This study is reported also in another study report (Boots Pharmaceuticals, 1992/TX92160) and represents an addendum of a further study (Sutton, 1970).

Reference: Weil CS (1952) Tables for convenient calculation of median-effective dose (LD50 or ED50) and instructions in their use Biometrics 8: 249-265.

 

Acute toxicity: inhalation

Bronopol was tested for its acute inhalation toxicity in rats (Hazleton Laboratories Europe Ltd, 1986). The test was conducted with 3 test groups and one control group. Each test group comprised 5 male and 5 female Sprague-Dawley rats. The test substance was milled before the test atmospheres were prepared. The test substance was conducted continuously into the chamber with filtered air at flow rate of 13–18 L/min; the air flow rate was monitored and recorded at 30 minute-intervals. The nominal test concentrations were 0, 1.80, 2.59, 23.23 mg/L; the corresponding measured concentrations were 0, 0.038, 0.089 and 0.588 mg/L.

The animals were nose-head exposed to the test atmosphere for a period of 4 hours; the control animals received filtered air without test substance. The rats were observed hourly during the exposure and once a day over the observation period of 14 days for mortality and clinical signs of toxicity. Body weight was assessed prior test initiation, at the end of the 4-hour-exposure, once daily between day 1 and 7 of observation, on day 14 and prior sacrifice. Animals that died were subjected to gross pathological examination as well as histopathology. The surviving rats were sacrificed at the end of the observation period and were also subjected to gross pathology and histopathology. Particular attention was given to the nasal passages, the cranial cavity and the respiratory tract. Lungs, bronchi, trachea and nasal passages were examined; lungs, liver, kidney and skin samples from 2 animals were fixed in 10% neutral buffered formalin for the purpose of histopathological examination. The chamber temperature and the relative humidity for the control and the treated groups ranged between 15 to 19 °C and between 30 to 89 %. The air flow rate was 15 L/min for the control group and between 13 to 18 L/min for the treated groups. The oxygen concentrations were within the range of 21 to 22 % for all groups. One male rat of the 0.588 mg/L group died whereas two further animals of the same test group (one male and one female) were sacrificed for humane reasons as they suffered from inflammation of the eyes. All animals of the remaining groups survived.Nearly all animals of the 0.588 mg/L group suffered from nasal discharge, red staining and inflammation of the eyes and staining of the head; sometimes accompanied by a swelling of the head, throat and/or the forepaws.These effects were consistent with local irritation of those areas, which came in direct contact with the test substance. These symptoms mainly were seen on the day of exposure and disappeared within 3 days; in some case, staining of the head reappeared at the end of the observation period. In one case (female of the 0.588 mg/L group) the marked staining persisted throughout the whole observation period and was accompanied by sores, fissuring and desquamation of the head skin during the second week of observation. In the 0.089 mg/L group, symptoms of toxicity included hunched posture and piloerection, which were seen in 6 of 10 animals. No symptoms were seen in the 0.038 mg/L group. Body weights showed no treatment-related effects.

Excepted for local dermatitis and ulceration of skin in two rats of the 0.588 mg/L group, which were attributed to direct dermal contact with the test substance rather than to its inhalation, no treatment-related effects were reported. All the mean mass median aerodynamic diameters indicated that the test atmosphere was respirable. In fact, the reported mean values were 1.31, 1.99 and 6.66 µm, respectively for the 0.038, the 0.089 and the 0.588 mg/L groups.Conclusively, the LC50 was >0.588 mg/L and the NOEC was 0.038 mg/L. In this acute inhalation toxicity study no guideline was mentioned; it was not specified whether the study followed GLP or not. Nevertheless, it is classified as acceptable (key study) because it is well-documented and of scientific acceptability.

In an acute inhalation toxicity study (Dow, 2003) according to OECD TG 401 and GLP, five male and five female Wistar rats were exposed to an aerosol of 0, 0.12 and 1.14 mg/l for 4 hours. At 1140 mg/m3 4/5 males and 3/5 females died during exposure and the remaining animals died until end of day 3. At 120 mg/m3 one male rat died during exposure. Clinical signs during exposure included bloody nose and mouth breathing (at 1140 mg/m3 only) as well as labored breathing, and soiling of the hair coat (both concentrations). At the high concentration the observed gross nasal lesions and impaired respiration may be related to the deposition of the relatively large particles of Bronopol in the upper respiratory tract. Clinical signs after exposure included combinations of slow, noisy, deep and/or labored respiration; perinasal, perioral, perineal, abdominal and/or extensive body soiling; and swelling of the muzzle and chin. A cloudy eye observed for one male at 120 mg/m³ on day 9 (only) may be unrelated to treatment due to its isolated appearance more than a week after exposure. A slight loss of body weight was noted on day 2 but rats gained body weight afterwards. Necropsy findings in the animals that died during the study: congestion of the brain, kidneys, liver, pharynx, and lung; edema of the lung; froth in the tracheas well as gas in the gastrointestinal tract and stomach. No visible lesions were noted in animals that survived until the scheduled sacrifice. The acute inhalation LC50 in rats was >120 mg/m3 but <1140 mg/m3. As only two dose levels are available pinpointing the exact LC50 for classification is difficult.

 

Acute toxicity: dermal

In an acute dermal toxicity study (BASF AG, 2000), five male and five female Wistar rats were exposed to Bronopol. The test substance was applied as a suspension using 0.5% Tylose CB 30.000 in bidistilled aqua as vehicle and was used at a concentration of 80 g/100 mL vehicle. The administered volume was 2.5 mL/kg bw, corresponding to a dosage of 2000 mg/kg bw.

Animals were checked for mortality and were observed for clinical symptoms of toxicity. Body weights were recorded prior test start, weekly thereafter, and at the end of the observation period. The skin was examined 30 to 60 minutes following removal of the dressing, weekly thereafter, and at the end of the observation period. The assessment of skin findings was based on Draize JH (1959).

At the end of the observation period (about 14 days), the animals were sacrificed for the purpose of necropsy and were subjected to gross pathological examination.

No mortality occurred. Animals showed clinical symptoms of toxicity, which included poor general state, dyspnoea and apathy. These symptoms affected both, males and females, and lasted until day 1 of observation. Local effects at the application sites were seen in both, males and females and included white discoloration of the skin, erythema, oedema, eczematoid skin change, scaling and crust formation. These effects lasted until the end of the observation period. Body weights were inconspicuous. The skin of the application site showed incrustation and full thickness necrosis in 4/5 males and 5/5 females. No further abnormalities were seen.

No lethal effect was seen at the tested dose of 2000 mg/kg bw. Conclusively, the LD50 (males+females) was determined as >2000 mg/kg bw.

This GLP-conform study is classified as acceptable (key study), since it satisfies the guideline requirement for an acute dermal study according to OECD 402.

Reference: Draize JH (1959). Appraisal of the safety of chemicals in food, drugs and cosmetics. The association of food and drug officials of the United States Austin, Texas

The dermal LD50 of >2000 mg/kg in rat was confirmed in another GLP conform acute dermal toxicity study according to OECD 402 (Lanxess, 2000).

The test substance was applied occlusive as a suspension in water. The animals received a dose of 2000 mg/kg bw. Two females died on day 2-3. In one female decreased motility and reactivity, laboured breathing and piloerection was observed on days 2 and 3. No clinical signs were observed in the remaining females and in males. Body weight of males was not affected by treatment. Slightly retarded body weight development was noted for some females but was attributed to the occlusive dressing and not directly to treatment. No gross lesions were apparent in necropsy examination of the surviving animals. Dark-red discoloration of the adrenal glands was observed in the two females that died during the study. Signs of skin irritation were seen in most of the animals from day to up to the end of the observation period: reddening, scale formation, encrustations (detached in females), yellow, grey or brown discolorations and swelling (females). Acute dermal LD50 in rats is >2000 mg/kg bw in males, females and both sexes combined.

Two further supporting studies with methodological deficiencies are available for the test substance and are named for the sake of completeness.


Justification for selection of acute toxicity – oral endpoint
Key study: Reliable GLP-conform study according to GL OECD 401 (Dow, 2001).
Supporting studies provide results within an acceptable range (Safepharm Laboratories Limited, 1987) Boots Pharmaceuticals, 1992; Boots Pharmaceuticals UK, 1992).


Justification for selection of acute toxicity – inhalation endpoint
WoE study (Hazleton Laboratories Europe Ltd, 1986): No guideline was mentioned. It was not specified whether the study did follow GLP. The study was however well-documented and of scientific acceptability.

WoE study (Dow, 2003): according to OECD TG 401 and GLP.

No exact LC50 could be determined. Therefore, the classification is based on a weight of evidence approach considering the LC50 (4h) > 580 mg/m3 determined in the study by Hazleton Laboratories Europe Ltd (1986) and the LC50 (4h) > 120 - < 1140 mg/m3 determined in the study by Dow (2003). An aerosol was tested in both studies.

Other studies show methodological deficiences (Huntingdon Research Centre, 1971) or tested product possessed only a small portion of the test substance (PSL, 1999).


Justification for selection of acute toxicity – dermal endpoint
Key study: Guideline study conducted in accordance with GLP (BASF AG, 2000).

The dermal LD50 of the key study was confirmed in a supporting study also conducted in accordance with GLP and guideline (Lanxess, 2000).

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 considered to be classified for acute oral toxicity Cat. 3 and acute inhalation toxicity Cat. 3 under Regulation (EC) No. 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.

Furthermore, Bronopol is included in Annex VI of Regulation (EC) No. 1272/2008 with the following legal classification:

  • Acute toxicity - oral Cat 4 (H302: Harmful if swallowed).
  • Acute toxicity - dermal Cat. 4 (H312: Harmful in contact with skin).
  • STOT SE Cat. 3 (H335: May cause respiratory irritation).