Dibromomethane

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Although not performed according to known TG or GLP, the study was well documented and scientifically accepted

Data source

Reference

Reference 1

Reference Type:

review article or handbook

Title:

Unnamed

Year:

1982

Report date:

1982

Reference 2

Reference Type:

review article or handbook

Title:

Unnamed

Year:

1987

Report date:

1987

Reference 3

Reference Type:

secondary source

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Principles of method if other than guideline:

Groups of 115 male and 15 female Spargue- Dawley rats and groups of three male beagle dogs were exposed to 0, 25, 75 or 150 ppm (0, 178, 533, or 1067 mg/m3) methylene bromide for 6 hours/ day, 5 days/ week for a total of 62 (male rats), 63 (female rats) or 70 (dogs) exposures within a total of 90- day period.
After the 90- day exposure period, all dogs, 15 male rats and all the female rats were killed and given complete gross pathological examination. In addition,
comprehensive histological examination of 5 male and 5 female rats and all dogs from each of the control and 150 ppm groups were performed. Dogs in the 25 and 75 ppm groups were also examined microscopically for lesions of lung trachea thoracic lymph nodes. Of the remaining 100 male rats/group, 10 males/ group were observed for 1 year and 90 males/ group were observed for 2 years. All rats that died or were killed after 1 or 2 years were given complete necropsies. Comprehensive histological examinations were performed on 6, 2, 1 and 5 rats in the control, 25, 75 and 150 ppm groups, respectively, sacrificed after 1 year. Rats killed after 2 years were not examined histologically. In addition to pathological observations, the following parameters were monitored throughout the study: clinical signs, body weight changes, mortality, organ weights, hematology (90-day and 1-year). Dogs were also given ophthalmoscopic examinations., There were slight increases in liver weight in female rats at 75 or 150 ppm and dose-related increases in percent saturation of carboxyhemoglobin at >25 ppm in both sexes of rats and at 150 ppm in dogs in the 90-day study. The increased percent of carboxyhemoglobin saturation at 25 ppm was considered to be biologically significant. Decreased mean bodyweight gain beginning on day 121 occurred among rats exposed to 75 or 150 ppm for 90 days and observed for up to 2 years.

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Dibromomethane

EC Number:

200-824-2

EC Name:

Dibromomethane

Cas Number:

74-95-3

Molecular formula:

CH2Br2

IUPAC Name:

dibromomethane

Details on test material:

Purity: 99.6-99.7%

Test animals

Species:

other: rat and dog

Strain:

other: Rats: Sprague-Dawley. Dogs: Beagle

Sex:

male/female

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Details on inhalation exposure:

Inhalation exposures were conducted under dynamic airflow conditions in 4.1 cubic meter stainless steel chambers. Air temperature and humidity were controlled to 22oC and 50% humidity in the exposure chambers and holding rooms. Test animals were maintained on a 12 hour light/ dark cycle. Desired concentrations of DBM in each chamber were generated by metering the liquid at calculated rate into a warmed (~210oC) glass vaporization flaks. The resultant vapour was then swept into the chamber via the main airflow at a rate of ~825 litres/ minute. Nominal concentration of test article was calculated daily, as the ratio of the rate at which the liquid compound was dispensed into the vaporization flask to the ratio the total chamber airflow. The analytical concentration of DBM was determined by infrared spectrophotometry using 2 Miran I spectrophotometers in series. The first IR monitored concentrations of 25-75 ppm, and the second 150ppm DBM. Chamber concentrations were determined at least twice during each exposure day.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

6 hours per day

Frequency of treatment:

5 days per week for 62 (males) and 60 (females) exposures in 90 days

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 25, 75, 150 ppm
Basis:
no data

No. of animals per sex per dose:

Rats: 4 Groups of 115 male (15 for 90 days portion and 100 for 2 year recovery portion) and 15 female Sprague- Dawley albino rats were exposed to air containing 0, 25, 75 or 150 ppm DBM for 6 hours/day, 5 days/week for a total of 62 (male rats), 63 (female rats) exposures, over a period of three months.

Dogs: 4 groups of 3 male beagle dogs were exposed to air containing 0, 25, 75 or 150 ppm (0, 178, 533, or 1067 mg/m3) DBM for 6 hours/day, 5 days/week for a total of 70 exposures, over a period of three months.

Control animals:

yes

Details on study design:

Rats: 4 Groups of 115 male (15 for 90 days portion and 100 for 2 year recovery portion) and 15 female Sprague- Dawley albino rats were exposed to air containing 0, 25, 75 or 150 ppm (0, 178, 533, or 1067 mg/m3) DBM for 6 hours/day, 5 days/week for a total of 62 (male rats), 63 (female rats) exposures, over a period of three months.
Dogs: 4 groups of 3 male beagle dogs were exposed to air containing 0, 25, 75 or 150 ppm (0, 178, 533, or 1067 mg/m3) DBM for 6 hours/day, 5 days/week for a total of 70 exposures, over a period of three months.

Examinations

Observations and examinations performed and frequency:

Rats: Animals were observed for clinical signs during each exposure day. Rats in the recovery group were examined monthly for tumours. Rats were weighed twice weekly during the first two months of the study, weekly during the rest two weeks. During the two year recovery, male rats were weighed twice during the first month of recovery and monthly thereafter. Haematological parameters were evaluated on 7 rats per exposure level, both before initiation of exposures and at study termination. Blood samples were taken from the tail veins of the rats. Urinalysis were also performed on each animal (pH, glucose, protein, ketones, bilirubin, urobilinogen, and occult blood, as well as specific gravity and urine sediment exam). Clinical chemistry determinations included blood urea nitrogen, serum glutamic pyruvic transaminase, serum glutamic oxaloacetic transaminase, and alkaline phosphatase. Clinical chemistries were also conducted on 10 male rats/ exposure level at the time of 1 year interim sacrifice. Specific ion electrode analysis of bromide levels (inorganic bromide) in plasma of 3 rats/ sex/ level were performed prior to exposure and at 41 days of exposure. Total bromine levels in plasma were determined by neutron activation analysis prior to initiation and at days 30, 51 and 61 of exposure for rats, and at days 31, 40,50, and 60 days of exposure for dogs. Carboxyhemoglobin levels were made on tail vein samples of 3 rats/ sex/ exposure after 90 experimental days. All rats which died spontaneously during the study were subjected to full gross pathological examination. After 61 (males) and 62 (females) exposures, complete gross necropsies were performed on 10 rats per sex per exposure level. Complete gross necropsy was also conducted on 10 male rats per level at the one year interim sacrifice, and on all surviving male rats at the 2 year sacrifice.


Dogs: All dogs were given eye exams prior to initiation of exposures and at termination. Animals were observed for clinical signs during each exposure day. Dogs were weighed weekly during the test. Haematological parameters (RBCs, haemoglobin concentration, packed cell volume, WBC) were evaluated on all dogs both before initiation of exposures, at 11/2 months into the study and at study termination. Blood samples were taken from the jugular vein of the dogs. Urinalysis was also performed on each animal (pH, glucose, protein, ketones, bilirubin, urobilinogen, and occult blood, as well as specific gravity and urine sediment exam). Clinical chemistry determinations were made on blood of dogs twice before study initiation and at termination and included blood urea nitrogen, serum glutamic pyruvic transaminase, serum glutamic oxaloacetic transaminase, and alkaline phosphatase. Specific ion electrode analysis of bromide levels (inorganic bromide) in plasma of all dogs was made twice prior exposure, at 31 and 50 exposures. Total bromine levels in plasma were determined by neutron activation analysis prior to initiation and at days 31, 40, 50, and 60 days of exposure. Carboxyhemoglobin levels were made on jugular vein samples of all dogs on test after 90 experimental days. At necropsy, weights of brain, heart, liver, kidneys, and testes were recorded. Representative portions of the organs and tissues were collected from each animal and preserved in formalin.

Results and discussion

Results of examinations

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

rats were slightly affected. Dogs were not affected.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

elevated carboxyhemoglobin both rats and dogs

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

liver weight increased in rats

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

Rats:
Exposure related effects were not observed at any exposure level during the study. In the 90- day study, body weights on male rats were generally comparable to those of controls during the study except for the 5th day of exposure when the mean body weight of the 75 and 150ppm groups were lower than the control. There was also a significantly decreased mean body weight in male rats of the 75 ppm group on the 47th experiment day. This was not considered exposure related. Body weights of female rats were comparable to those of controls rats for the entire exposure period except for an isolated decrease in mean body weight in female150 ppm group.
In the 2-year recovery portion, male rats in the 150 ppm exposure group showed decreased mean body weight during days 121 to 607 of the study. In these intervals, there were 9 occasions on which the body weights were statistically decreased from the control values. Rats of the 75 ppm group showed trend to decreased mean body weights from days 121 to 361 of the study, but there were no statistically significant differences from the control values. Rats in the 75 ppm group had 2 instances of statistically significant differences from the control values. There was no pattern or trend to these differences.
At necropsy, there was a significant decreased mean relative liver weight in male rats of the 25 ppm male rats compared to the controls rats. This may have been due to the greater body weight of these animals compared to controls. This was not observed among male rats exposed to 75 or 150 ppm DBM.
Organ weight data for all other groups were comparable to controls. In female rats at necropsy, there was a trend towards increased mean absolute and relative liver weights in the 150 ppm group. The mean liver weight of females exposed to 75 ppm also showed a trend towards an increased absolute weight and also statistically significant increase in relative liver weight. This trend towards slightly increased liver weight in female rats exposed to 75 and 150 ppm was considered related to exposure.
At the 1-year interim sacrifice, fasted body weights of the male rats of 75 and 150 ppm groups were slightly decreased. However, there were no significant differences in the weights of the brain, heart, liver, kidneys or testes.
Haematological values at the 90- day termination in treated males were comparable to controls. The total WBC counts were greater at the pre-exposure bleeding than at termination. This was not thought to be exposure related. The same effect was seen in female rats. There were no significant differences in values of treated animals in the 1-year interim sacrifice. Urinalysis values for both sexes did not show exposure related alterations in the 90-day and in the 1-year measurements. Clinical chemistry values for exposed rats were comparable to control in the 90-day and in the 1-year measurements. DBM consistently increased blood % carboxyhemoglobin saturations in rats exposed to 75 ppm.

Dogs:
Exposure related effects were not observed at any exposure level during the study. Body weights on all dogs were comparable to those of controls during the study. At necropsy, terminal body weights, organ weights, and organ to body weights ratio were comparable to controls. Haematological values in treated dogs were comparable to those of controls. Clinical chemistry values of exposed dogs were comparable to control dogs. All gross and histopathologic observations on control and treated dogs were considered spontaneous in nature, and not as a result of treatment. Among spontaneous lesions was a frequent occurrence of chronic inflammatory lesions of the lungs that varied in severity. In some animals, the lesions were clearly related to aspiration of food material, and in others typical of a parasitic infestation common in dogs. There was some discrepancy in values for bromide ions and for total bromides.

Effect levels

1

Dose descriptor:

NOAEL

Remarks:

rats

Effect level:

25 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: based on carboxyhemoglobin

2

Dose descriptor:

LOAEL

Remarks:

rats

Effect level:

75 ppm

Sex:

male/female

Basis for effect level:

other: based on elevated carboxyhemoglobin, increased liver weight, and decreased body weight

3

Dose descriptor:

NOAEL

Remarks:

dogs

Effect level:

75 ppm

Sex:

male/female

Basis for effect level:

other: based on elevated carboxyhemoglobin

4

Dose descriptor:

LOAEL

Effect level:

150 ppm

Sex:

male/female

Basis for effect level:

other: based on elevated carboxyhemoglobin

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The NOAEL for dogs was determined to be 75 ppm (based on elevatedcarboxyhemoglobin), and the LOAEL was set to 150 ppm.
The NOAEL for rats was determined to be 25 ppm (mostly based on carboxyhemoglobin) and the LOAEL was set to 75 ppm (based on elevatedcarboxyhemoglobin, increased liver weight, and decreased body weight).

Executive summary:

A repeated dose inhalation study was performed by DOW (Keyes et al 1982). This was a 90-day repeated inhalation study in rats and dogs with a subsequent 2-year holding period for the rats. The study was described in US EPA, 1987 and in US EPA 2006. The study did not follow the accepted EPA or OECD testing guidelines or GLP, but was well documented and QA audited, therefore, is considered reliability Klimisch 2.

Test system: Inhalation exposures were conducted under dynamic airflow conditions in 4.1 cubic meter stainless steel chambers. Air temperature and humidity were controlled to 22^oC and 50% humidity in the exposure chambers and holding rooms. Test animals were maintained on a 12 hour light/ dark cycle. Desired concentrations of DBM in each chamber were generated by metering the liquid at calculated rate into a warmed (~210^oC) glass vaporization flaks. The resultant vapour was then swept into the chamber via the main airflow at a rate of ~825 litres/ minute. Nominal concentration of test article was calculated daily, as the ratio of the rate at which the liquid compound was dispensed into the vaporization flask to the ratio the total chamber airflow. The analytical concentration of DBM was determined by infrared spectrophotometry using 2 Miran I spectrophotometers in series. The first IR monitored concentrations of 25-75 ppm, and the second 150ppm DBM. Chamber concentrations were determined at least twice during each exposure day.

Rats

4 Groups of 115 male (15 for 90 days portion and 100 for 2 year recovery portion) and 15 female Sprague- Dawley albino rats were exposed to air containing 0, 25, 75 or 150 ppm (0, 178, 533, or 1067 mg/m³) DBM for 6 hours/day, 5 days/week for a total of 62 (male rats), 63 (female rats) exposures, over a period of three months. Control animals were housed in the holding room during exposure periods. Animals were acclimated for at least one week prior to exposures. Rats were housed in groups of 4 per cage and were not fed during exposure periods.

Animals were observed for clinical signs during each exposure day. Rats in the recovery group were examined monthly for tumours. Rats were weighed twice weekly during the first two months of the study, weekly during the rest two weeks. During the two year recovery, male rats were weighed twice during the first month of recovery and monthly thereafter. Haematological parameters were evaluated on 7 rats per exposure level, both before initiation of exposures and at study termination. Blood samples were taken from the tail veins of the rats. Urinalysis were also performed on each animal (pH, glucose, protein, ketones, bilirubin, urobilinogen, and occult blood, as well as specific gravity and urine sediment exam). Clinical chemistry determinations included blood urea nitrogen, serum glutamic pyruvic transaminase, serum glutamic oxaloacetic transaminase, and alkaline phosphatase. Clinical chemistries were also conducted on 10 male rats/ exposure level at the time of 1 year interim sacrifice. Specific ion electrode analysis of bromide levels (inorganic bromide) in plasma of 3 rats/ sex/ level were performed prior to exposure and at 41 days of exposure. Total bromine levels in plasma were determined by neutron activation analysis prior to initiation and at days 30, 51 and 61 of exposure for rats, and at days 31, 40,50, and 60 days of exposure for dogs. Carboxyhemoglobin levels were made on tail vein samples of 3 rats/ sex/ exposure after 90 experimental days.All rats which died spontaneously during the study were subjected to full gross pathological examination. After 61 (males) and 62 (females) exposures, complete gross necropsies were performed on 10 rats per sex per exposure level. Complete gross necropsy was also conducted on 10 male rats per level at the one year interim sacrifice, and on all surviving male rats at the 2 year sacrifice.

Results: Exposure related effects were not observed at any exposure level during the study. In the 90- day study, body weights on male rats were generally comparable to those of controls during the study except for the 5^thday of exposure when the mean body weight of the 75 and 150ppm groups were lower than the control. There was also a significantly decreased mean body weight in male rats of the 75 ppm group on the 47^thexperiment day. This was not considered exposure related. Body weights of female rats were comparable to those of controls rats for the entire exposure period except for an isolated decrease in mean body weight in female150 ppm group.

In the 2-year recovery portion, male rats in the 150 ppm exposure group showed decreased mean body weight during days 121 to 607 of the study. In these intervals, there were 9 occasions on which the body weights were statistically decreased from the control values. Rats of the 75 ppm group showed trend to decreased mean body weights from days 121 to 361 of the study, but there were no statistically significant differences from the control values. Rats in the 75 ppm group had 2 instances of statistically significant differences from the control values. There was no pattern or trend to these differences.

At necropsy, there was a significant decreased mean relative liver weight in male rats of the 25 ppm male rats compared to the controls rats. This may have been due to the greater body weight of these animals compared to controls. This was not observed among male rats exposed to 75 or 150 ppm DBM.

Organ weight data for all other groups were comparable to controls. In female rats at necropsy, there was a trend towards increased mean absolute and relative liver weights in the 150 ppm group. The mean liver weight of females exposed to 75 ppm also showed a trend towards an increased absolute weight and also statistically significant increase in relative liver weight. This trend towards slightly increased liver weight in female rats exposed to 75 and 150 ppm was considered related to exposure.

At the 1-year interim sacrifice, fasted body weights of the male rats of 75 and 150 ppm groups were slightly decreased. However, there were no significant differences in the weights of the brain, heart, liver, kidneys or testes. 

Haematological values at the 90- day termination in treated males were comparable to controls. The total WBC counts were greater at the pre-exposure bleeding than at termination. This was not thought to be exposure related. The same effect was seen in female rats. There were no significant differences in values of treated animals in the 1-year interim sacrifice. Urinalysis values for both sexes did not show exposure related alterations in the 90-day and in the 1-year measurements. Clinical chemistry values for exposed rats were comparable to controlin the 90-day and in the 1-year measurements. DBM consistently increased blood % carboxyhemoglobin saturations in rats exposed to 75 ppm.

The NOAEL was determined to be 25 ppm (mostly based on carboxyhemoglobin) and the LOAEL was set to 75 ppm (based on elevatedcarboxyhemoglobin, increased liver weight, and decreased body weight).

Dogs

4 groups of 3 male beagle dogs were exposed to air containing 0, 25, 75 or 150 ppm (0, 178, 533, or 1067 mg/m³) DBM for 6 hours/day, 5 days/week for a total of 70 exposures, over a period of three months. Control animals were housed in the holding room during exposure periods. Animals were acclimated for at least one week prior to exposures. Dogs were housed in groups of 3 during non-exposure periods, and individually during exposures.

All dogs were given eye exams prior to initiation of exposures and at termination. Animals were observed for clinical signs during each exposure day. Dogs were weighed weekly during the test. Haematological parameters (RBCs, haemoglobin concentration, packed cell volume, WBC) were evaluated on all dogs both before initiation of exposures, at 11/2months into the study and at study termination. Blood samples were taken from the jugular vein of the dogs. Urinalysis was also performed on each animal (pH, glucose, protein, ketones, bilirubin, urobilinogen, and occult blood, as well as specific gravity and urine sediment exam). Clinical chemistry determinations were made on blood of dogs twice before study initiation and at termination and included blood urea nitrogen, serum glutamic pyruvic transaminase, serum glutamic oxaloacetic transaminase, and alkaline phosphatase. Specific ion electrode analysis of bromide levels (inorganic bromide) in plasma of all dogs was made twice prior exposure, at 31 and 50 exposures. Total bromine levels in plasma were determined by neutron activation analysis prior to initiation and at days 31, 40, 50, and 60 days of exposure. Carboxyhemoglobin levels were made on jugular vein samples of all dogs on test after 90 experimental days. At necropsy, weights of brain, heart, liver, kidneys, and testes were recorded. Representative portions of the organs and tissues were collected from each animal and preserved in formalin.

Results: Exposure related effects were not observed at any exposure level during the study. Body weights on all dogs were comparable to those of controls during the study. At necropsy, terminal body weights, organ weights, and organ to body weights ratio were comparable to controls. Haematological values in treated dogs were comparable to those of controls. Clinical chemistry values of exposed dogs were comparable to control dogs. All gross and histopathologic observations on control and treated dogs were considered spontaneous in nature, and not as a result of treatment. Among spontaneous lesions was a frequent occurrence of chronic inflammatory lesions of the lungs that varied in severity. In some animals, the lesions were clearly related to aspiration of food material, and in others typical of a parasitic infestation common in dogs. There was some discrepancy in values for bromide ions and for total bromides. The NOAEL was determined to be 75 ppm (based on elevatedcarboxyhemoglobin), and the LOAEL was set to 150 ppm.