Bromoacetic acid

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study under GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Monobromo acetic acid

IUPAC Name:

Monobromo acetic acid

Constituent 2

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

Test material form:

solid: crystalline

Details on test material:

Purity: 98%, batch 210892

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga, Sulzfeld, Germany
- Age at study initiation: About 6 weeks old,
- Weight at study initiation: 164.3-212.3 g for males and 127.0-159.0 g for
females
- Fasting period before study: no
- Housing: during acclimatisation housing sexes separated, 5 animals per cage, stainless steel wire mesh cages, during premating in groups of 4 animals. Mating: one male and one female in suspended stianless steel nesting cages, after mating males were returned to group cages, females remained in the nesting cages. A few days before littering females were moved to macrolon cages with saw dust and bedding material added. After weaning females were returned to the group cages.
- Use of restrainers for preventing ingestion (if dermal): no
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.5 to 24.5
- Humidity (%): 50 to 70%
- Photoperiod 12 hrs dark /12 hrs light):

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test solution with the highest concentration (360 mg/kg) was prepared first. Then the 120 and 40 mg/kg solutions were prepared by diluting the 360 mg/kg solution with the appropriate amounts of tap-water. All solutions and the tap-water for the controls were stored in plastic (polyethylene) 10 liter vessels. Fresh solutions were prepared when necessary, generally at c. I-week intervals, and stored in a refrigerator (c. 4 °C) until used.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: until detection of vaginal plug, maximum 21 days
- Proof of pregnancy: vaginal plug or sperm cells in vagina referred to as day 0 of pregnancy
- After 21.. days of unsuccessful pairing replacement of first male by another male with proven fertility.
- After successful mating each pregnant female was caged individually in the nesting cage

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of bromoacetic acid in tap-water upon storage at ambient temperature (animal room> for 7 days or at c. 5 °C (refrigerator) for 7 or 21 days was determined in samples taken from the first batch of test solutions prepared. The homogeneity was also determined in the first
batch of solutions prepared by analysing three samples per concentration ttaken from the top, the middle and the bottom of the vessel. In addition
the content of the test substance in four other batches of solutions was checked by analysis (viz. once in the premating and gestation periods of both generations).
The test substance concnetration was determined by HPLC analysis

Duration of treatment / exposure:

10 weeks premating, during mating, and thereafter continously through 2 generations

Frequency of treatment:

Continously, 7d/week

Details on study schedule:

- F1 parental animals weeks sacrificed after 55 d males, 61 days females (after weaning of the F1 on day 21 post partum.
- Selection of parents from F1 generation on day 21 post partum. (24 males and females per dose group at random from as many litters as possible).
- Age at mating of the mated animals in the study: F0 16, F1: 13 weeks

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 10, 40, 120, 360 mg/kg bw
Basis:
nominal in water

No. of animals per sex per dose:

24

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale:
The oral route was selected because this is an anticipated route of human exposure. Drinking-water was used as the carrier, since bromoacetic acid is not stable when mixed with feed. The dose selection was based on a 28-day range finding study with bromo acetic acid in rats.

- Actual dose levels based on drinking water consumption: The intake of Bromoacetic acid per kg body weight per day during the
premating period showed the expected decrease with increasing study duration for each of the three test groups. The mean intake levels in this
period were 4, 10 and 25 mg/kg body weight/day in males, and 5, 14 and 27 mg/kg body weight/day in females at nominal concentrations of
40, 120 and 360 mg/kg. The intake remained similar for males and females until lactation. During lactation the test substance intake was calculated to be 11, 32 and 83 mg/kg bw per day for the low, mid and high dose group females respectively.

Positive control:

no

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
The general condition and behaviour of all animals were checked daily by cage-side observations. All abnormalities, signs of ill health or reaction to treatment were recorded. On working days, all cages were checked again in the afternoon for dead or moribund animals. At weekends and public and company holidays only one check per day was carried out.

BODY WEIGHT: Yes
- Time schedule for examinations:
The individual body weights of all parent males and of females which did not mate were recorded weekly throughout the study. Parent females were weighed weekly during the premating and mating periods, on days 0, 7, 14 and 21 of pregnancy, and during lactation on days 1/ 7, 14 and 21 post partum (females that did not deliver a litter were not weighed after day 21 of gestation).
FOOD AND WATER CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food and water consumption were measured on a cage basis over I-week periods (except for the first interval post partum that lasted 6 days),
and subsequently the consumption per rat per day was calculated. Measurements were made throughout the study, except during the mating
period, by weighing the feeders or bottles.
The mean intake of the test substance per kg body weight per day in successive weeks was calculated on the basis of the nominal concentration of the test substance in the drinking-water, the amount of water consumed and the mean body weight in the week for which the intake was calculated.

Oestrous cyclicity (parental animals):

not determined

Sperm parameters (parental animals):

not reported as such. Part of pathology assessment

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 offspring:
- number and sex of pups
- stillbirths
- live births
- presence of gross anomalies
- weight gain
- physical or behavioural abnormalities
For each litter:
- litter size (dead and live pups) on days It 4 (before and after culling), days 7, 14 and 21 post partum
- number of male and female pups on days I and 4 (before and after culling), days 7, 14 and 21 post partum
- pups with external abnormalities on days 1 and 4 (before culling), 7, 14 and 21 post partum. To judge the size of the PUPS pup body weight was
used as criterium (see Tables 47 en 48).
The litters were weighed on days 1 and 4 (after culling), 7 and 14 post partum. At weaning, i.e. day 21 post partum, all pups were weighed
individually.
GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals see above for scheduled deaths
- Maternal animals: All surviving animals after weaning of the last litter

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
Samples of the following tissues and organs of all parental animals were preserved in a neutral, aqueous, phosphate-bUffered, 4 % solution of formaldehyde (F) or Bouin's fixative (B):
- ovaries, F
uterus including cervix (after counting implantation sitesJ, F
- vagina, F
- testes, B
- epididymides, B
- seminal vesicles, F
- prostate, F
- coagulating glands, F
- pituitary, F
organs or tissues showing macroscopic abnormalities, F
The tissues for examination by light microscopy were embedded in paraffin wax, sectioned at 5 micro-m and stained with haematoxylin and eosin. Detailed
microscopic examination was conducted on the collected organs of all animals of the control group and the top-dose group.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 21 days of age.
- These animals were not subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

Statistics:

All analyses were two-sided.
FO- and Fl-generation adult data
Clinical findings were evaluated by Fisher's exact probability test.
Body weight and food and water consumption data were subjected to one-way
analysis of variance (ANOVA) followed by Dunnett's multiple comparison
tests.
Mating and litter data
Fisher's exact probability test was used to evaluate the numbers of: mated and pregnant females, females with liveborn pups, females surviving
delivery, females with stillborn pups or lost litters, liveborn and stillborn pups, pups lost at various stages, pups surviving 21 days, and
male pups on days 1 and 21.
Pre-coital time, duration of gestation and litter size were evaluated by Kruskal-wallis nonparametric analysis of variance followed by the MannWhitney u-test, and pup body weights by analysis of variance followed by Dunnett's multiple comparison tests.
Histopathology:
The number of implantation sites per litter and post-implantation loss nwere analysed by Kruskal-wallis nonparametric analysis of variance
followed by the Mann-Whitney u-test.
For pathological changes Fisher's exact probability test was used.

Reproductive indices:

Mating index:
number of females mated / numer of females placed with males x 100
females mated: number of females with successful copulation as evidenced by a positive vaginal smear or vaginal plug.

Fertility index: number of females pregnant / number of females placed with males x 100

Fecundity index: number of females pregnant / numbe rof females mated x 100

Offspring viability indices:

Gestation index: Number of females with live pups / Number of females pregnant x 100

Live birth index: Number of pups born alive / total Number of pups x 100

Weaning index: Number of live pups on day 21 / Number of live pups on day 4 post partum (after culling) x 100

Sex ratio day n: Number of live male pups on day n / Total number of live pups on day n x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

nasal discharge reduced grooming

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

both decreased in top dose

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

both decreased in top dose

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Test substance intake: see below

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

Parent males
Mean body weights were decreased in the high-dose group in both generations, the differences from controls being statistically significant from the first week of the study, weight gain was decreased most markedly during the first two weeks of the premating period in males in both generations.
Throughout the study, high-dose males of both generations consumed less feed than the corresponding controls. There was a dose-dependent decrease in water consumption in both generations. This effect might have resulted from reduced palatability of the test solutions. The reduction amounted to about 40-60 % throughout the study at the high-dose level, and to about 10-25 % throughout the study at the mid-dose level. At the low-dose level, mean water
consumption was slightly decreased during most weeks, but only statistically significantly F1-generation males. LOEL = appr. 25 mg/kg bw/day
Parent females
Mean body weights were decreased in the high-dose group in both mgenerations, the differences from controls being statistically significant from the fifth week of the study, weight gain was decreased most markedly during the during gestation and the first two weeks of lactation, in both generations. It should be noted that during lactation the drinking water intake increases and the amount of test substance taken up increases consequently in parallel.
Throughout the study, high-dose females of both generations consumed less feed than the corresponding controls.
There was a dose-dependent decrease in water consumption in both generations. This effect might have resulted from reduced palatability of the test solutions. The reduction amounted to about 40-60 % throughout the study at the high-dose level, and to about 10-25 % up to gestation at the mid-dose level. At the low-dose level, mean water consumption was slightly decreased during most weeks, but only statistically significantly in F0-generation females.

LOEL = appr. 27-34 mg/kg bw/day appr. 57 mg/kg day during lactation

Effect levels (P0)

1

Dose descriptor:

LOEL

Effect level:

ca. 25 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: see 'Remark'

2

Dose descriptor:

LOEL

Effect level:

27 - ca. 34 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks:

57 mg/kg bw during lactation

Sex:

female

Basis for effect level:

other: see 'Remark'

3

Dose descriptor:

NOAEL

Effect level:

ca. 4 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: see LOEL

4

Dose descriptor:

NOAEL

Effect level:

ca. 5 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: see LOEL

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

see below

Sexual maturation:

no effects observed

Description (incidence and severity):

see below

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

see below

Gross pathological findings:

no effects observed

Description (incidence and severity):

see below

Histopathological findings:

no effects observed

Description (incidence and severity):

see below

Details on results (F1)

F1 males:
Mean pup weight was statistically significantly decreased in the high dose group from day 4 of lactation in the F0-generation, and from day
14 of lactation in the F1-generation. Treatment-related clinical findings in pups were limited to an increase in the number of small pups from day 14 post partum in the high-dose group in both generations. As these effects occurred at a time where the pups start drinking and feeding independently the effects are likely to be related to a direct effect of the substance rather than a developmental effect.
LOEL = appr. 22 mg/kg bw/day
F1 females Mean pup weight was statistically significantly decreased in the high dose group from day 4 of lactation in the F0-generation, and from
day 14 of lactation in the F1-generation. Treatment-related clinical findings in pups were limited to an increase in the number of small pups from
day 14 post partum in the high-dose group in both generations. As these effects occurred at a time where the pups start drinking and feeding independently the effects are likely to be related to a direct effect of the substance rather than a developmental
effect. LOEL = appr. 38 mg/kg bw/day

Effect levels (F1)

1

Dose descriptor:

LOEL

Generation:

F1

Effect level:

ca. 22 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: see 'Remark'

2

Dose descriptor:

LOEL

Generation:

F1

Effect level:

ca. 38 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: see 'Remark'

3

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

ca. 10 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: see LOEL

4

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

ca. 14 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: see LOEL

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

see attached table reproduced from the biocide dossier.

Applicant's summary and conclusion

Conclusions:

Treatment of rats in this two-generation reproduction study with Bromoacetic acid caused no specific reprotoxic or teratogenic effects in the offspring. At the highest dose all animals had a decreased body weight and a statistically lower food intake. Drinking water consumption was also reduced. The observed effects at higher dose-levels are typical for Bromoacetic acid and are mainly the result of the reduced water and food consumption. The effects on body weight in
the pups from lactation day 4 can be attributed to a direct ingestion of the test substance as the pups start feeding and drinking directly in this period as well, rather than to any postnatal developmental effect. The LO(A)EL and NO(A)EL values derived are in this case rather LOEL and NOEL values, that indicating any
true adverse effects. The effects observed are most likely secondary to palatability problems of the test substance and the resulting reduction in water and
food consumption.
Parent males
Mean body weights were decreased in the high-dose group in both generations, the differences from controls being statistically significant from the first week of the study, weight gain was decreased most markedly during the first two weeks of the premating period in males in both generations.
Throughout the study, high-dose males of both generations consumed less feed than the corresponding controls. There was a dose-dependent decrease in water consumption in both generations. The reduction amounted to about 40-60 % throughout the study at the high-dose level, and to about 10-25 % throughout the study at the mid-dose level. At the low-dose level, mean water consumption was slightly decreased during most weeks, but only statistically significantly F1-generation males.
Parent females
Mean body weights were decreased in the high-dose group in both generations, the differences from controls being statistically significant from the fifth week of the study, weight gain was decreased most markedly during the during gestation and the first two weeks of lactation, in both generations. It should
be noted that during lactation the drinking water intake increases and the amount of test substance taken up increases consequently in parallel.
Throughout the study, high-dose females of both generations consumed less feed than the corresponding controls.There was a dose-dependent decrease in water consumption in both generations.
The reduction amounted to about 40-60 % throughout the study at the high-dose level, and to about 10-25 % up to gestation at the mid-dose level. At the lowdose level, mean water consumption was slightly decreased during most weeks, but only statistically significantly in F0-generation females.
The number of implantation sites was slightly, though statistically significantly decreased in the high-dose group of the F1-generation. This finding was
accompanied by a small decrease in litter size, though the difference from controls was not statistically significant. Other reproductive indices showed no
treatment-related changes. The finding is mos tlikely not of biological relevance.

Executive summary:

To obtain information on the possible effects of Bromoacetic acid on male and female reproductive performance and on the growth and

development of the offspring, a two-generation reproduction study was conducted. The test substance was administered in the drinking-water at concentrations of 40, 120 and 360 mg/kg over two successive generations. In each generation one litter was raised.

The two-generation study was performed before, but similar the OECD/EU protocol. For that reason the oestrus cycle and sperm

parameter were not evaluated. However a publication by Linder et al., 1994 (see acute oral toxicity section) investigated the spermatogenic affect of Bromoacetic acid in male rats and did not find adverse effects for (mono)Bromoacetic acid.

Results and discussion

There was no treatment-related mortality. Treatment-related clinical signs were seen in the top-dose group only, viz. symptoms of decreased grooming in males and females of the F1-generation in the first week of the premating period, and nasal discharge (probably also resulting from decreased grooming) in females of the F0-generation towards the end of lactation.Mean body weights were decreased in the high-dose group in both sexes in both generations, the differences from controls being statistically significant from the first (males) or fifth (females) week of the study, weight gain was decreased most markedly during the first two weeks of the premating period in males and during gestation and the first two weeks of lactation in females, in both generations. Throughout the study, high-dose males and females of both generations consumed less feed than the corresponding controls.There was a dose-dependent decrease in water consumption in both generations. This effect might have resulted from reduced palatability of the test solutions. The reduction amounted to about 40-60 % throughout the study in both sexes at the high-dose level, and to about 10-25 % throughout the study in males or up to gestation in females at the mid dose level. At the low-dose level, mean water consumption was slightly decreased during most weeks, but only statistically significantly in F0 -generation females and F1-generation males.

Mating in most (F0-generation) or all (F1-generation) pairs of each group. No treatment related changes were observed in pre-coital time,number of pregnant females, female fertiltiy index and fecuditiy index.

Apart from one low dose female (one stillborn pup) and one mid dosefemale (delivered no pups) of the F1-generation, all pregnant females delivered live litters and thus the gestation index was 100 or close to 100 in all groups.

The number of implantation sites was slightly, though statistically significantly decreased in the high-dose group of the F1-generation.

This finding was accompanied by a small decrease in litter size, though the difference from controls was not statistically significant. Other reproductive indices showed no treatment-related changes.The mean number of pups born per litter did not show any statistically significant intergroup differences. The number of stillborn pups and postnatal deaths between days 1 and 4 post partum was also low in all groups. The sex ratio varied between 48 and 56% in the F0-generation and between 44 and 59% in the F1-generation, but the difference did not show a dose response and was therefore not considered treatment related.

Mean pup weight was statistically significantly decreased in the highdose group from day 4 post partum in the F0-generation, and from day 14 of lactation in the F1-generation. This effect was accompanied by a decrease in weight gain throughout the lactation period in the F0 -generation and from day 7 of lactation in the F1 generation. Treatment-related clinical findings in pups were limited to an increase in the number of small pups from day 14 post partum in the high-dose group in both generations.

Gross necropsy findings in stillborn pups or pups that died or were examined on account of clinical findings did not indicate the presence of any treatment-related effects.

Gross examination of parent rats of the F0 and F1-generation did not reveal any treatment-related abnormalities.

Upon microscopic examination of the pituitary and various organs of the reproductive tract, no treatment-related abnormalities were observed.