Bromoacetic acid

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From Juanuary to September 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD, EPA, etc)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

and EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Bromoacetic acid

EC Number:

201-175-8

EC Name:

Bromoacetic acid

Cas Number:

79-08-3

Molecular formula:

C2H3BrO2

IUPAC Name:

2-bromoacetic acid

Details on test material:

- Name of test material (as cited in study report): monobromoacetic acid - 80% monobromoacetic acid/20% water
- Substance type: yellow viscous liquid
- Physical state: suspended solution
- Purity test date: 80% monobromoacetic acid/20% water
- Lot/batch No.: 980028
- Expiration date of the lot/batch: 1 december 1998
- Stability under test conditions: stable at room temperature
- Storage condition of test material: room temperature

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Germany
- Age at study initiation: About 4 weeks old
- Weight at study initiation:
* Males: 134.2 g (120.2-151.1)
* Females: 109.9 g (98.3-120.3)
- Housing: Housing conditions were conventional.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: upon the arrival of the rats, the rats were places in a quarantine room and checked for overt signs of ill health and anomalies. period between rat arrival and initiation of the treatment: 12 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-24.5°C
- Humidity (%): 37-65%
- Air changes (per hr): 10 per hour
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark.

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Each concentration level was prepared by weighing the appropriate amount of the test substance (viz. 0.6, 1.5 and 3.75 g for the low-, mid- and high-dose level, respectively) and making up to 10 kg with tap water.
Fresh batches of test solutions were prepared on 26 January6 6 and 20 February, 3, 13 and 27 March, and 9 and 17 April 1998. The solutions were stored in plastic (polypropylene) 10- liter containers, in a refrigerator (2-10°C) until use.
The contents of the drinking bottles were refreshed once a week and filled as needed.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

no analytical verification

Duration of treatment / exposure:

13 consecutive weeks

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
10, 60 and 360 ppm
Basis:
other: drinking water ad libitum

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0, 48, 120 and 300
Basis:
other: based on bromoacetic acid

No. of animals per sex per dose:

Total 45 males, 45 females;
10 males and 10 females per group
The supernumerary rats (5/sex) were kept as sentinel animals but were not used in the study.

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: results from the 28-days oral toxicity study - limit test - refer to study V89.578
- Rationale for animal assignment (if not random): computer randomization program

Positive control:

No positive control

Examinations

Observations and examinations performed and frequency:

- Clinical signs:
Each animal was observed daily in the morning hours by cage-side observations and if necessary, handled to detect signs of toxicity. On working days all cages were checked again in the afternoon for dead or moribund animals.
- Mortality:
examination daily for 13 weeks
- Body weight:
At start of the study, once every week and at nominal days 91 (males) and 92 (females)
- Food consumption:
Food consumption was measured per cage by weighing the feeders and expressed in gram per rat per day. The consumption was measured over successive periods of 7 days. The efficiency of food utilization was calculated and expressed in gram weight gain per gram food consumed.
- Water consumption:
Water consumption was measured per cage by weighing the drinking bottles daily. The results are expressed in g per animal per day.
- Ophthalmoscopic examination:
- ophthalmoscopic observations were made prior to the start of treatment (on day 0) in all rats and in the last week of the treatment period (on day 86) in all rats of the control group and the high-dose group. Eye examination was carried out using an ophthalmoscope after induction of mydriasis by a solution of atropine sulphate.
- Haematology:
In all animals at the end of treatment (males day 91; females 92)
Parameters:
· haemoglobin
· packed cell volume
· red blood cell count
· rericulocytes
· trotal white blood cell count
· differential white blood cell count
· prothrornbin time
· thrombocyte count
· The following parameters were calculated:
* mean corpuscular volume (MCV)
* mean corpuscular haemoglobin (MCH)
* mean corpuscular haemoglobin concentration (MCHC).
- Clinical Chemisty:
In all animals - Clinical chemistry determinations were made on all rats. Fasting glucose was determined shortly before the end of the treatment period (on nominal day 88) in blood collected from the tip of the tail after deprivation of water for 24 hours and of food during the last 16 hours of
this period. The collection of fasting blood was combined with the urine concentration test.
At autopsy, blood was collected from the abdominal aorta of the rats whilst under ether anaesthesia. Blood was collected in heparinized plastic tubes and plasma was obtained by centrifugation. The following measurements were carried out the plasma:
· alkaline phosphatase activity (ALP)
· aspartate aminotransferase activity (ASAT)
· alanine aminotransferase activity (ALAT)
· gamma glutamvl transferase activity (GGT)
· total protein
· albumin
· ratio albumin/globulin
. urea
· creatinine
· bilirubin (total)
· cholesterol (total)
· triglycrides
· phospholipids
· calcium (Ca)
· sodium (Na)
· potassium (K)
· chloride (CI)
· inorganic phosphate
- Urinalysis:
All animals were handled shortly before autopsy (on day 87-88). The animals were deprived of water for 24hours and of food during the last 16 hours of this period. Urine was collected from individual rats whilst kept in stainless-steel metabolism cages (1 rat/cage) during the last 16 hours of the deprivation period. The following measurements were carried out:
· appearance,
· volume,
· osmolality,
· specific gravity,
· pH,
· protein,
· glucose,
· blood

Sacrifice and pathology:

- Organ Weights:
The following organs were examined:
· liver,
· kidneys,
· adrenals,
· testes,
· thymus,
· spleen,
· brain,
· heart
· thyroid (with parathyroid)
- Gross and histopathology:
In all dose groups, the following organs were examined:
· brain,
· spinal cord,
· pituitary,
· thyroid,
· parathyroid,
· thymus,
· oesophagus,
· salivary glands,
· stomach,
· small and large intestines,
· liver,
· pancreas,
· kidneys,
· adrenals,
· spleen,
· heart,
· trachea,
· lungs,
· aorta,
· uterus,
· female mammary gland,
· prostate,
· urinary bladder,
· peripheral nerve,
· skin,
· eyes
cecum
· epidymides
· exorbital lachrymal glands
· mesenteric lymph nodes
· ovaries
· rectum
· seminal vesicles with coagulating glands
· skeletal muscle (thigh)
· sternum with bone marrow
· testes
· vagina
· axilliary lymph nodes
· femur with joint
· mandibular lymph nodes
· gross lesions
the underlined organs were not examined histopathologically unless it was considered necessary from the gross observations. Histopathology was perfomed on all other organs in the high dose and control animals and on kidney, liver and lung of the mid dose animals. Gross changes were examined histopathologically in all dose groups.

Statistics:

The statistical procedures used to evaluate the results were as follows:
- Body weight: one-way analysis of covariance (covariate: body weight on day 0); when group means were significantly different (p<0.05), individual pair wise comparisons were made using Dunnett's multiple comparison method.
- Food consumption/efficiency, water consumption, red blood cell and coagulation variables, total white blood cell counts, absolute differential white blood cell counts, urinary volume and density, clinical chemistry values and organ weights: one way analysis of variance (ANOVA): when group means were significantly different (p<0.05), individual pair wise comparisons were made using least significant difference tests (LSD.; for food and water consumption and food efficiency) or Dunnett's multiple comparison method.
- Relative differential while blood cell counts. reticulocytes and urinalysis results (appearance. stix. sediment): Kruskal-Wallis non-parametric Anova followed by Mann-Whitney If-tests.
- Histopathological changes: Fisher's exact probability test.
All tests were two-sided. Group mean differences with an associated probability of less than 0.05 were considered to be statistically significant. Because numerous variables were subjected to statistical analysis, the overall false positive rate (Type I errors) is greater than suggested by a probability level of 0.05. Therefore, the final interpretation of results was based not only on statistical analysis but also on other considerations such as dose-response relationships and
whether the results were significant in the light of other biological and pathological findings.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

The daily observations did not reveal any treatment-related clinical signs. No mortality at any dose level.

Mortality:

no mortality observed

Description (incidence):

The daily observations did not reveal any treatment-related clinical signs. No mortality at any dose level.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights were statistically significantly decreased, by about 8% in the high-dose group in both sexes throughout the study.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

See below for further details

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

effects observed, treatment-related

Description (incidence and severity):

Water consumption was dose-dependently decreased in all groups (both sexes) given the test substance.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

The ophthalmoscopic examination of high-dose rats and controls in the last week of the treatment period did not reveal any ocular changes.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

increased mean corpuscular volume in high-dose males and decreased thrombocyte count in high-dose males.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

see below for details

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

See below for details

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At the highest-dose for female and male: decrease in the absolute weight of the liver and increase in the relative weight of the kidneys and brain.

Gross pathological findings:

no effects observed

Details on results:

MORTALITY: No mortalities at any dose level

Effect levels

1

Dose descriptor:

LOEL

Effect level:

20.6 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Reduced drinking water intake

2

Dose descriptor:

LOEL

Effect level:

24.2 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: reduced drinking water intake.

3

Dose descriptor:

NOAEL

Effect level:

10.2 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: average intake of bromoacetic acid through drinking water

4

Dose descriptor:

NOAEL

Effect level:

14.1 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: average intake of bromoacetic acid through drinking water

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Complement information on results:

* Food consumption and compound intake

-Food:

Throughout the study, male and female rats of the high-dose group consumed less food than did the corresponding controls. The differences were statistically significant at several stages in high-dose females and on day 7 in high-dose males. A statistically significant decrease in food consumption was also observed in females of the low- and mid-dose group on day 7 but in the weeks thereafter food consumption in these groups approximated that of controls.

Food conversion efficiency figures showed no consistent differences between treated rats and controls.

- Water (containing test substance):

Water consumption was dose-dependently decreased in all groups (both sexes) given the test substance. The reduction compared to the controls averaged about 10%, 20% and 40% in the low-, mid- and high-dose

groups, respectively. The reduced water consumption is likely caused by the palatability of the test substance.

*Clinical chemistry - Clinical chemistry values showed the following statistically significant differences between treated rats and controls:

- increased plasma alkaline phosphatase activity in males of the mid- and high-dose group and in females of all treatment groups; the differences showed a dose-response relationship in males but not clearly in females,

- decreased plasma levels of total protein, cholesterol and phospholipids in males of the high-dose group,

- increased plasma level of bilirubin in females of the high-dose group,

- increased plasma level of chloride in both sexes of the highdose group.

* Urinalysis - The results of the renal concentration test showed a decreased urinary volume and an increased urinary density in males and females of the high-dose group. Except for the lower volume in females, the differences with the controls were statistically significant.

The semi-quantitative urinary observations showed no treatment-related intergroup differences. Microscopic examination of the urinary

sediment showed a decrease in the number of crystals in males of the high-dose group.

Applicant's summary and conclusion

Conclusions:

The study, using the guidelines OECD 408 or EC B.26, was performed to examine the possible sub-chronic oral toxicity of Bromoacetic acid by administering this substance in the drinking water to groups of 10 male and 10 female Wister rats for 13 consecutive weeks, at constant concentrations of 0 (controls), 48, 120 or 300 mg Bromoacetic acid/kg drinking solution.
LO(A)EL:
20.6 mg/kg bw/day males
24.2 mg/kg bw/day females
NO(A)EL:
10.2 mg/kg bw/day males
14.1 mg/kg bw/day females.
At the highest dose (360 ppm about 20 mg Bromoacetic acid/kg bw/day) a reduction of food intake and body weight in both males and females was observed. Haematological effects were also limited to the high-dose group, being an increased mean corpuscular volume and an decreased in thrombocyte count in high-dose males only. Clinical chemistry showed statistically significant differences for an increased plasma alkaline phosphatase activity in males of the mid- and
high-dose group and in females of all treatment groups; the differences showed a dose-response relationship in males but not clearly in females.
Also decreased plasma levels of total protein, cholesterol and phospholipids in males were seen in the high-dose group, an increased plasma level of bilirubin in females of the high-dose group and increased plasma level of chloride in both sexes of the high-dose group. In addition an decrease in the absolute weight of the liver, and an increase in the relative weight of the kidneys and brain was found.. Most of these observations might be explained by the reduced drinking
water intake, and all effects observed are slight changes that did not lead to any histopathological organ changes and are not considered adverse.
The value of 20.6 or 24.2 mg/kg bw respectively is therfore considered a LOEL rahter than a LOAEL. It reflects the highest dose that could be administered due to palatability reasons.
No toxicologically significant changes were observed in the low- and mid-dose groups for both males and females. The mid-dose at 120 mg Bromoacetic acid/kg drinking water resulting in an average intake of about 10 mg Bromoacetic acid/kg body weight/day is therefore considered as no-observed-adverse-effect level (NOAEL) of Bromoacetic acid under the conditions of this study.

Executive summary:

A 90 -day drinking water study was performed according to guidelines OECD 408 or EC B.26 and under GLP. Bromoacetic acid was administered in the drinking water to groups of 10 male and 10 female Wister rats for 13 consecutive weeks, at constant concentrations of 0 (controls), 48, 120 or 300 mg Bromoacetic acid/kg drinking solution. The choice for application in drinking water is based on the corrosive properties of Bromoacetic acid.

From the start of the study, there was a dose-related decrease in water consumption in both sexes, which averaged about 10, 20 and 40% in the low-, mid- and high-dose group, respectively. This effect was considered to be due to reduced palatability of drinking water containing the test substance. In addition to the markedly decreased water consumption, animals of the high-dose group showed several changes, which were judged to be secondary to the low water consumption:

slight decreases in food consumption and body weight, an increased relative kidney weight, and a decreased volume and increased density of the urine. These urinary changes indicate a good ability of the kidneys to concentrate the urine in response to the lower fluid intake. The increased relative brain weight, decreased absolute liver weight and possibly the decreased plasma levels of total protein, cholesterol and phospholipids in the high-dose group were ascribed to the decreased body weight and food consumption in this group. Apart from these secondary changes males and/or females of the high-dose group showed an increased mean corpuscular volume, decreased thrombocyte count, decreased number of crystals in the urinary sediment, and increased plasma levels of alkaline phosphatase, chloride and bilirubin.

Treatment-related changes observed in the low- and mid-dose group were limited to decreased water consumption and an increased plasma alkaline phosphatase activity in both sexes. The significance of the changes in alkaline phosphatase was not clear. They were not clearly dose-related, nor consistently correlated with the changes in water consumption. In the rat, ALP activity in plasma is mainly of intestinal origin and altering the feeding pattern may influence plasma ALP and has been reported before. Such a phenomenon might have occurred in the present study due to the lower water consumption. In the absence of any treatment-related histopathological alterations in animals treated with Bromoacetic acid, no toxicological significance was attached to the

increases in plasma ALP. The folloing LO(A)ELs and NO(A)ELs were established: LO(A)EL 20.6 mg/kg bw/day males

24.2 mg/kg bw/day females. NO(A)EL: 10.2 mg/kg bw/day males; 14.1 mg/kg bw/day females.