Bromoacetic acid

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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:

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

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

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

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: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

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

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'

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'

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

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

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

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

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'

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'

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

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

Reproductive effects observed:

not specified

see attached table reproduced from the biocide dossier.

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Study duration:

subchronic

Species:

rat

Quality of whole database:

Based on a guideline study and additional literature data indicating no effects on fertility. Supported by exisiting repeated dose studies with no effects on the reproductive organs.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A two-generation reproduction study in Wistar rats is available (OECD 416; performed under GLP) where Bromoacetic acid was examined for its possible effects on male and female reproductive performance and on the growth and development of the offspring. The Bromoacetic acid was administered ad libitum 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 Bromoacetic acid was administered at concentrations of 0 (control), 40, 120 and 360 mg/kg in the drinking-water (tap-water) for 7 days per week for a period of 10 weeks pre-mating period. The mean intake levels (both generations) 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 Bromoacetic acid/kg in the drinking-water. During lactation the intake was 11, 32, and 83 mg/kg bw respectively. There was no treatment-related mortality. Treatment-related clinical signs were seen in the topdose 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 top-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 pre-mating period in males and during gestation and the first two weeks of lactation in females, in both generations. Throughout the study, top-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. According to the authors this effect likely has resulted from reduced palatability of the test solutions. The reduction amounted to about 40-60 % throughout the study in both sexes at the top-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. The number of implantation sites was slightly, though statistically significantly decreased in the top-dose group of the Fl-generation. This finding was accompanied by a small decrease in litter size, though the difference from controls was not statistically significant.

The effect at F1 high dose group in the decrease in implantation sites was slight, the litter size was not statically significant, no dose-relationship was observed and it was occurring in the presence of some systemic effects, therefore it is not considered to costitute a specific effect on fertility.

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 was very low, resulting in a live birth index of 100, or nearly 100, in all groups. Postnatal death between days l and 4 post-partum was also low, and the viability index day 4-21 high (viz. 98-100 %). The sex ratio varied between 48 and 56 % in the F0- generation and between 44 and 56 % in the Fl-generation, but the differences did not show a clear dose-response relationship.

Mean pup weight was statistically significantly decreased in the top-dose group from day 4 post partum in the F0-generation, and from day 14 post partum 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 top-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 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 seen. The study did not show any specific effects of the test substance on reproductive performance and development.[EDJ58] It was concluded that a concentration of 120 mg Bromoacetic acid/kg drinking-water was a noadverse- effect level for reproductive performance and development of the offspring. This concentration was equivalent to an average compound intake of approximately 10 and 14 mg/kg body weight/day in males and females, respectively (viz. the mean intake levels during the premating periods). Furthermore, it was concluded that 40 mg/kg, which provided about 4 (males) or 5 (females) mg of Bromoacetic acid/kg body weight/day, was the NOAEL for parental toxicity, based on decreased food and water consumption and lower body weight.

Short description of key information:
No test substance related effects on male or female fertility were observed in a guideline conform 2-generation study in rats receiving Mono bromo acetic acid in drinking water up to the maximum concentration that could be administered. The LOEL and NOAEL values established from this study are related to the direct effects of the test substance (food and drinking water reduction, body weight reduction, slight signs of irritation) at the highest dose level. The NOAEL was 10 mg/kg bw for males and 14 mg/kg bw for females.

Justification for selection of Effect on fertility via oral route:
Valid guideline conform 2-generation study

Effects on developmental toxicity

Description of key information

Bromoacetic acid, when administered in the drinking water from day 6 up to and including day 15 of gestation at levels up to 360 mg/kg water (mean actual daily intake: 21.9 mg/kg bw  maximum applicable dose) is not embryo/foetotoxic and does not induce any teratogenic effect.
Consequently, the no-adverse effect level for prenatal development is 21.9 mg/kg bw day or higher. In view of the effects seen in the pregnant rats, the no-adverse effect level of Bromoacetic acid for maternal toxicity is 40 mg Bromoacetic acid per kg water, which is equivalent to 4.7 mg/ kg
bw day.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From August to October 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD, EPA, etc)

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga, Sulzfeld, Germany
- Age at study initiation: About 14 weeks old
- Weight at study initiation: 190.5-249.8 g
- Housing: Upon arrival in the study room, the males were housed individually in suspended stainless steel cages fitted with wire mesh fronts and floors of 18x32x18 cm and the females were housed in groups of four per sex in similar cages of 45x32x18 cm. During mating, 2 females were caged with a male. During gestation, the females were housed individually in cages of l8x32x18 cm.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.5-24°C
- Humidity (%): 50-70%
- Air changes (per hr): 10 changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: the test substance was administred dissolved in tap water.

VEHICLE: Tap water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Drinking water containing test substance was prepared twice and samples for analyses were taken once.

Details on mating procedure:

Females were placed with males, i.e. two females with one male. vaginal smears were taken the next morning to determine whether mating had occurred. If no sperm cells were detected, the female in question was placed again with the male until observation of a vaginal smear or vaginal plug (several days) (considered as the day 0 of gestation)

Duration of treatment / exposure:

The test substance solutions were offered from day 6 up to and including day 15 of gestation.

Remarks:

Doses / Concentrations:
0, 4.7, 11.6, 21.9 mg/kg bw
Basis:
other:

Remarks:

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

No. of animals per sex per dose:

96 females - 24 number of animals per group

Control animals:

yes

Details on study design:

On day 21 of gestation the females were killed by cervical dislocation preceded by ether anaesthesia, opened through a midline incision in the abdominal and thoracic wall and examined for gross abnormalities. Subsequently, the uterus and ovaries were removed for further examination.
The fetuses were removed from the uterus, dried of amniotic fluids and examined grossly, upon which they were further processed.

Maternal examinations:

- Body weight: On day 0, 6, 11, 16 and 21 of gestation.
- Food consumption: Periods day 0-6, 6-11, 11-16 and 16-21
_ Clinical signs/ checked daily, twice daily during weekdays, once daily on weekends.

Ovaries and uterine content:

- Gravid uterine weight, net uterine weight
- Placental weight
- Number of corpora lutea
- Number of implantations

Fetal examinations:

- Litter Size,
- Nr. of dead Foetuses,
- Foetal Weight, length
- Sex Ratio
- Grossly vivible malformations

Statistics:

- Clinical findings of the female rats were evaluated by Fisher's exact probability test.
- Body weights and food and water consumption were subjected to one-way analysis of variance (ANOVA) followed by Dunnett's Multiple Comparison test.
- The mating data were calculated for the females from each group and compared by Fisher's exact test.
* Day 21 sacrifice data:
- Mean gravid and empty uterus weight, mean ovary and carcass weight were analysed for each group by ANOVA + Dunnett-test.
- Numbers of corpora lutea, implantation sites, resorptions, and live and dead fetuses were analysed for each group by the Kruskal-Wallis test followed by the Mann Whitney U-test.
- Mean fetal body weights and lengths, and placenta weights were calculated for each litter and subjected to ANOVA + Dunnett-test.
- Numbers of male and female fetuses, and visceral and skeletal findings were analysed by the Fisher exact test.

Indices:

- Pre implantation loss (%) = (a-b)/a x 100
- Post implantation loss (%) = (b-c)/b x 100
where:
a = number of corpora lutea
b = total number of implantation sites
c = number of live fetuses

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: For details, see below

Details on maternal toxic effects:
- Clinical signs:
Alopecic areas were observed in 1 animal of the control group, the midand high-dose group respectively. No other clinical signs were observed.
- Marternal necropsy:
No remarkable findings were observed in most of the animals (23 of the control, 20 of the low and high dose group and 18 of the mid dose group). Four females with a pronounced lobular pattern in the liver were observed, 2 in the mid-dose and 2 in the high-dose group. One female in the mid-dose group was observed to have a stomach with a haemorrhagic content. Unilateral kidney cysts were observed in 3 females, one in the control group, mid-dose and high dose group respectively. Ovary cysts were observed of one animal in each the lowor mid-dose group. A swollen uterus was observed in 2 females of the low-dose and 1 female of the high-dose group and uteri of one female each of the low- or mid-dose group were observed to be filled with blood. None of the effects was considered to be related to the treatment. No other relevant observations were made at autopsy.
- Body weight:
Maternal body weight was statistically significantly decreased in the high-dose animals on gd 16. Body weight gain was affected in both the mid- and high-dose groups during the administration period. Mean body weight change was reduced compared to controls from day 6 to 11 in the mid dose group and from day 6 to 16 in the high dose group, whereas in the latter an increase in body weight change compared to controls was observed between gestation day 16 and 21.
- Food consumption:
At start of the study (day 0-6) food consumption was comparable between all groups.. A statistically significant reduction in food consumption was obseved in the mid dose group between day 6 and 11 of gestation and in the high dose group from day 6 to 16 of gestation, whereas it was significantly increased in the latter group from gd 16 to 21.
- Water consumption:
At start of the study (day 0-6) water consumption was comparable between all groups. Mean water consumption was statistically significantly reduced in the
mid-dose group between gestation days 6 and 11, and in the high-dose group between days 6 and 16. Mean water consumption was slightly increased in the high-dose group between day 16 and 21.
- Maternal performance and organ weights:
None of the females had an abortion or died during the study. In each group one female delivered before scheduled sacrifice, and 3 to 4 females per group were not pregnant. All pregnant females had litters with viable foetuses. No statistically significant differences were observed in mean gravid and mean empty
uterus weight and in mean ovary weight.
- Reproduction findings and litter data
No significant differences were observed between the dose groups and the control group concerning the numbers of corpora lutea, implantation sites, and live and dead foetuses, and in the pre- and postimplantation loss. The number of dead foetuses was very low (1 foetus only, in the low-dose group). The count of early and late resorptions alone or taken together did not show any differences amongst the groups. The sex ratio in all groups was, within the normal limits, close to 1:1.
- Autopsy findings:
Placental haemorrhages were observed on 3 occasions, in a single case of each treatment group. Fused placentas were observed on 1 occasion, in the high-dose group.

Dose descriptor:

NOEL

Effect level:

21.9 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: other:

Dose descriptor:

NOEL

Effect level:

4.7 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

LOEL

Effect level:

21.9 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

LOEL

Effect level:

21.9 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: other:

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects. Remark: For details, see below

Details on embryotoxic / teratogenic effects:
- Autopsy findings:
Foetal crown-rump lengths, placental weight and fetal body weights were similar in all groups. At Caesarian section 842 foetuses were found in 78 litters. Only 1 foetus was dead (low-dose group). Umbilical hernia was observed in 1 foetus in the mid-dose group. Hypogenesis of the tail was observed in 1 foetus of the high-dose group. A swelling on the head was observed in 1 foetus of the high-dose group.
Flexed limbs were observed in 1 foetus of the control group, in 2 foetuses of the low-dose group and in 1 foetus of the mid-dose group. A dysmature appearance (i.e. a foetus smaller/lighter than 25/of the mean) was observed in 1 foetus of the low-dose group. A purple appearance was observed in 5 foetuses of the high-dose group and a pale appearance in 2 foetuses from low-dose group. One large foetus was observed (i.e. a foetus bigger/heavier than 25\ of the mean), in mid-dose group.
Subcutaneous haemorrhages were observed in many foetuses, distributed about equally over all groups. Due to the location, it could be decided that these haemorrhages were probably caused by the method with which the foetuses were identified individually. None of the observations described above, being few and distributed evenly over the groups, was considered to be related to the treatment.
- Visceral malformations:
The only malformation found was one case of encephalocele in the high dose group. This finding is not considered treatment related.
- Visceral anomalies:
The number of viceral anomalies was small and none of them was considered the result of treatment. One incidence each of defects of the retina or lens were observed in the control and high dose group respectively. One incident of haemorrhagein the brain was observed in the high dose group and haemorrhage in the liver was observed in one case in both the control and the high dose group. One incidence of hydronephrosis was reported in the high dose group and hydorurether was found in one fetus of the control and high dose group respectively. One incidence of thoracic cavity filled with blood was observed in the high dose group.
- Visceral variations:
The visceral variations observed included esophagectasia (2 incidences in the control, 1 in the high dose group), kidneys with increased pelvis cavitation (12 in the control, 9 in the high dose group), crooked tails (3 in both the control and high dose group), subcutaneous haemorrhages (1 in the control 7 in the high dose group), and a haemorrhage in the salivary gland in the control group. The number of visceral variations was small and none of the observed variations could be ascribed to the treatment.
- Skeletal malformations and variations:
Neither skeletal malformations nor variations were observed in the cotnrol and high dose group. Variations in ossification were present in approximately equal quantities in both groups.

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

The oral administration of bromoacetic acid, dissolved in the drinking water at levels up to 360 mg/kg water (mean actual daily intake: 21.9 mg/kg body weight) from day 6 through 15 of pregnancy induced no clinical signs and adverse findings at autopsy of the pregnant females.
In addition and on the basis of the results abtained it can be concluded that:
- bromoacetic acid is not embryo/fetotoxic and does not induce any teratogenic effect. Consequently, the no-adverse effect level for prenatal development is
21.9 mg/kg bw per day or higher.
- In view of the effects seen in the pregnant rats the no-adverse effect level of bromo acetic acid for maternal toxicity is 40 mg/kg water, which is equivalent to 4.7 mg/kg body weight per day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

21.9 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The data base is consistent with regard to developmental toxicity. A guideline conform developmental toxicity study in rats and a 2-generation study in rats, both after oral administration in drinking water revealed consistent results.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

A guideline conform developmental toxicity study (OECD 414) was performed under GLP where Bromoacetic acid was examined for its embryotoxic/fetotoxic and teratogenic potential in Wistar rats. Bromoacetic macid was administered to mated female rats (24 animals per dose group) dissolved in the drinking water at levels of 0, 40, 120 and 360 mg/kg water from day 6 up to and including day 15 of gestation.

Female rats exposed to Bromoacetic acid during gestation (days 6-15) had reduced food and mwater consumption in both the mid- and high-dose. Mean substance intake, calculated for gestation day 6 to 16 on basis of the nominal level of the test substance in the drinking water, were on average 4.7 mg/kg bw/day in the low-dose group (40 mg/kg water), 11.6 mg/kg bw/day in the mid-dose group (120 mg/kg water) and 21.9 mg/kg bw/day for the high-dose group (360 mg/kg water).

Maternal body weight was statistically significant decreased in the high-dose group. Body weight gain was affected in both the mid- and high-dose groups during the administration period. Food intake and water intake showed a statistically significant decrease in the high and medium dose groups during the administration period. None of the females had an abortion or died during the study. In each group one female delivered before scheduled sacrifice, and 3 to 4 females per group were not pregnant. All pregnant females had litters with viable foetuses. No statistically significant differences were observed in mean gravid and mean empty uterus weight and in mean ovary weight. No treatment related clinical signs were observed during the study and no macroscopic organ changes were observed at necropsy. No significant differences were observed between the dose groups and the control group concerning the numbers of corpora lutea implantation sites, and live and dead foetuses, and in the pre- and post implantation loss. The number of dead foetuses was very low (1 foetus only, in the low-dose group). The count of early and late resorptions alone or taken together did not show any differences amongst the groups. The sex ratio in all groups was, within the normal limits, close to 1:1.

· Upon macroscopic foetal examination no compound-related defects were observed.

· Upon microscopic foetal examination no visceral and skeletal malformations, anomalies or variants were observed that could be related to the treatment with Bromoacetic acid.

On the basis of the results obtained it can be concluded that: Bromoacetic acid, when administered in the drinking water from day 6 up to and including day 15 of gestation at levels up to 360 mg/kg water (mean actual daily intake: 21.9 mg/kg bw maximum applicable dose) is not embryo/foetotoxic and does not induce any teratogenic effect.Consequently, the no-adverse effect level for prenatal development is 21.9 mg/kg bw day or higher.

In view of the effects seen in the pregnant rats, the no-adverse effect level of Bromoacetic acid for maternal toxicity is 40 mg Bromoacetic acid per kg water, which is equivalent to 4.7 mg/ kg.

Justification for selection of Effect on developmental toxicity: via oral route:
A guideline conform developmental toxicity after oral (drinking water) adminstration to rats is available and used as the key stidy. The effects are corroborated by the 2-generation study in rats.

Justification for classification or non-classification

No test substance related effects on fertility and developmental toxicity were observed when the substance was administered to rats via the oral route (drinking water) up to dose levels that induced parental toxicity. Therfore a classification for reproductive endpoints is not warranted.