Potassium trifluorozincate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Remarks:

TNO Triskelion, Utrechtseweg 48, 3704 HE Zeist, the Netherlands

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Potassium trifluorozincate

EC Number:

237-537-7

EC Name:

Potassium trifluorozincate

Cas Number:

13827-02-6

Molecular formula:

F3Zn.K

IUPAC Name:

potassium trifluorozincuide

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Nocoloc Zn Flux
- Chemical name : Potassium trifluorozincate, KZnF3
- Appearance: white powder
- Batch number: BWF91112
- Purity: 99%
- Storage conditions: ambient temperature
- Expiry date: 31 December 2012

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan, Horst, the Netherlands.
- Age at study initiation: approx. 12 weeks
- Weight at study initiation: Male: about 360 g, Females: about 215 g
- Housing: Animals were housed in macrolon cages with a bedding of wood shavings (Lignocel) and strips of paper (Enviro-dri) and a wooden block as environmental enrichment. During the premating period, the animals were housed in groups of 4 of the same sex. For mating, one male and one female were housed together. Mated females were housed individually in macrolon cages.
- Diet: cereal-based rodent diet (Rat and Mouse No.3 breeding diet; RM3) from (SDS Special Diets Services, Witham, England), ad libitum
- Water: domestic mains tap-water suitable for human consumption, ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

Dilutions of the test substance in the vehicle were prepared weekly and stored in a refrigerator (2-10°C). Dilution and dividing of test dilutions was done under constant stirring on a magnetic stirrer. Eight aliquots (for 7 days plus 1 extra) were taken per dose level according to the daily volume required for each dosing, and stored in a refrigerator (2-10°C). The vehicle for dosing the controls (tap water) was similarly stored.
The dosing volume was 10 mL/kg body weight. Dose volume was adjusted to the latest recorded body weight for each individual animal to maintain a constant dose level in terms of the animal’s body weight. During the gestation period, dose volumes were not adjusted after GD 14.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Method: Analyses to determine the stability, homogeneity and content of the test substance in the test dilutions were conducted by Inductively coupled plasma atomic emission spectrometry (ICPAES) analysis. The test substance was quantified using Zn as a marker component. The following analyses were conducted in the batch of dosing formulations prepared in the study on 3 December 2012: Homogeneity (in 3 samples per dose level) and content at each dose level; Stability of the test substance under (simulated) experimental conditions (after storage for eight days in the refrigerator (2-10°C).
Results: Nocolok Zn Flux was homogeneously distributed in the gavage liquids and the content was close to intended at each dose level. Nocolok Zn Flux was stable in the gavage liquids under the experimental conditions (storage in the refrigerator (2-10°C) for more than 7 days).

Duration of treatment / exposure:

Male animals were dosed during a 2-week premating period, during mating and up to day 30 (the day before sacrifice on day 31). The female animals were dosed with the test substance during a 2-week premating period, and during mating, gestation and lactation up to the day before sacrifice (approx. day 4 of lactation).

Frequency of treatment:

once daily

Doses / concentrations

Remarks:

Doses / Concentrations:
7.5, 25, 75 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

Mating: At the end of the premating period, each female was caged with one male from the same group. Animals were caged together until mating occurred or 1 week had elapsed. If after one week no mating had occurred, the female was determined to be not mated. Because female 081 (high-dose group) died before the start of the mating period, the corresponding male animal (082) was not used for mating. Because male 060 (mid-dose group) died before the start of the mating period it was replaced by male 062 from the same dose group that already had successfully mated (hence male 062 mated with females 059 and 061). Every consecutive morning during the mating period, vaginal smears were made for determination of the presence of sperm. The day on which sperm was detected in the vaginal smear was considered as gestation day 0. Upon evidence of copulation the females were caged individually for the birth and rearing of their pups. Sperm positive females that turned out to be non-pregnant were killed not earlier than 24 days after copulation. Females that did not show evidence of copulation after the end of the one-week mating period were also housed individually until sacrifice (25 days after the last day of the mating period). Dams were allowed to raise their litter until sacrifice on day 4 of lactation or shortly thereafter.

Examinations

Observations and examinations performed and frequency:

- General clinical observations: Each animal was observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity. All cages were checked again in the afternoon for dead or moribund animals to minimize loss of animals from the study. On Saturdays, Sundays and public holidays only one check per day was carried out. All abnormalities, signs of ill health or reactions to treatment were recorded.
- Neurobehavioural testing: Detailed clinical examinations outside the home cage: In addition to the above daily general clinical observations, detailed clinical examinations outside the home cage were performed on all rats of all groups prior to the first exposure and then once weekly throughout the study (in the last week of the study, detailed clinical examination was part of the Functional Observational Battery tests in the animals concerned. Signs noted included but were not limited to changes in skin and fur, piloerection, changes in the eyes, gait (including posture), and presence of clonic or tonic movements, stereotypies and bizarre behaviour. FOB and motor activity assessment: On day 29 of the study (males) or on post-natal day 4 (females), FOB and motor activity testing was performed in 5 animals/sex/group, namely: 5 males/ group selected from each of 3 cages/sex/group (2, 2 and 1 surviving animals with the lowest animal number of each cage/group, respectively); 5 females/group with a litter, taking into account the day of delivery. During neuro-behavioural testing, the observer was unaware of the treatment of the animals.
- Body weight: Body weights of male and female animals were recorded one day before the start of the treatment and at the start of the study (day 0). Males were weighed weekly until sacrifice. Females were weighed once per week during the premating period. Mated females were weighed on days 0, 7, 14 and 21 during presumed gestation and on day 0 and 4 of lactation. Non-mated females were weighed once per week after the mating period. All surviving animals were weighed on their scheduled necropsy date in order to calculate the correct organ to body weight ratios.
- Feed consumption: Feed consumption was measured per cage over weekly intervals during the study, except for the mating period, during which no food consumption was registered. The results were expressed in g per animal per day.
- Haematology: Haematology was conducted in samples collected prior to the end of the premating period (on day 13) in 5 females/group, and on day 30 (one day before necropsy) in 5 males/group. The samples were collected in 2, 2 and 1 rats/sex with the lowest animal number in each cage. The rats were fasted overnight (water was freely available) and blood was taken, whilst CO2/O2 anaesthesia by orbita punction. EDTA was used as anticoagulant. In each sample the following determinations were carried out: haemoglobin, packed cell volume, red blood cell count, reticulocytes, total white blood cell count, differential white blood cell counts (neutrophils, lymphocytes, eosinophils, basophils, monocytes), prothrombin time, thrombocyte count, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration.
- Clinical chemistry: Clinical chemistry was conducted in samples collected prior to the end of the premating period (on day 13) in 5 females/group, and on day 30 (one day before necropsy) in 5 males/group. The samples were collected in 2, 2 and 1 rats/sex with the lowest animal number in each cage. The rats were fasted overnight (water was freely available) and blood was taken, whilst CO2/O2 anaesthesia by orbita punction. Blood was collected in heparinized plastic tubes and plasma was prepared by centrifugation. The following measurements were made in the plasma collected at necropsy: alkaline phosphatase activity, aspartate aminotransferase activity, alanine aminotransferase activity, gamma glutamyl transferase activity, total protein, albumin, ratio albumin to globulin, urea, creatinine, glucose (fasting), bilirubin (total), cholesterol (total), triglycerides, phospholipids, calcium, sodium, potassium, chloride, inorganic phosphate.

Sacrifice and pathology:

- Gross necropsy and histology of parental animals: All surviving male and female parent animals were sacrificed by exsanguination from the abdominal aorta whilst under CO2/O2 anaesthesia at necropsy and then examined grossly for pathological changes. Male animals were sacrificed after the mating period (on day 31). Female animals were sacrificed at or shortly after day 4 of lactation. A necropsy was also performed on animals that died intercurrently (if not precluded by autolysis) or that had to be killed because they were moribund. Samples of the following tissues and organs of all F0 animals were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde; except for the testes which were preserved in Bouin's fixative: ovaries (after counting the corpora lutea), uterus (after counting of the implantation sites), testes*, epididymides*, seminal vesicles, prostate, all gross lesions. (* These organs of all surviving animals were weighed (paired organs together) as soon as possible after dissection to avoid drying.). In addition the following organs of five animals/sex/group (2, 2 and 1 males with the lowest animal number in each cage, and females with a litter) were preserved: adrenals**, bone marrow (femur), brain (including sections of cerebrum, cerebellum, medulla/pons)**, heart**, small and large intestines (including Peyer’s patches), kidneys**, liver**, lungs, mesenterial and axillary lymph nodes, peripheral nerve (sciatic or tibial), spinal cord (cervical, mid-thoracic, and lumbar), spleen**, stomach, thymus**, thyroid, trachea, urinary bladder. (** These organs of five animals/sex/group were weighed (paired organs together) as soon as possible after dissection to avoid drying).
- Microscopic examination: Tissues for microscopic examination were embedded in paraffin wax, sectioned at 5µm, and stained with haematoxylin and eosin, except for sections of the testes which were stained with PAS haematoxylin. Microscopic examination was performed on the collected organs of all animals of the control (group 1) and high- concentration group (group 4). Because treatment-related changes were observed in stomach in the high-dose group, the evaluation of this organ was extended to the selected rats of the intermediate-dose groups (2 and 3). In addition, organs showing gross lesions were microscopically examined in animals of all groups. Reproductive organs (ovaries, uterus, testes, epididymides, seminal vesicles and prostate) of males that failed to sire (did not mate or mated females were not pregnant) and females that were non-mated or non-pregnant, of the low- and mid-dose groups, were also examined microscopically.

Other examinations:

- Parturition and litter evaluation: At the end of the gestation period (GD 21), females were examined twice daily for signs of parturition. Any difficulties occurring during parturition were recorded. To keep nest disturbance to a minimum the litters were examined only once daily for dead pups.
- Litter size, sexes and weight: The total litter size and numbers of each sex as well as the number of stillbirths, live- and dead pups and grossly malformed pups was evaluated on days 0 and 4 of lactation. The pups were individually weighed on days 0 and 4 of lactation. Mean pup weight was calculated per sex and for both sexes combined.
- Signs and pathology of pups: Any abnormal behaviour of pups was recorded on day 0 and 4 of lactation. Grossly malformed pups were sacrificed and examined. A necropsy was performed on stillborn pups and pups that died during the study; macroscopic abnormalities were recorded. At necropsy of the dams, pups were killed by appropriate techniques and examined externally for gross abnormalities. Pups were stored in a freezer for possible skeletal analyses until finalisation of the report, thereafter they were discarded.
- Fertility and reproductive performance: For each mating the following data was recorded for each group: number of females mated (= placed with males), number of males mated (= placed with females), number of females inseminated (= showing evidence of mating), number of males with pregnant females, number of pregnant females as demonstrated by the presence of implantation sites observed at necropsy, number of females surviving delivery, number of females with liveborn and (all) stillborn pups, number of pups delivered (live- and stillborn), number of live pups at day 0 and 4, number of pups lost, number of litters lost entirely, number of male pups at day 0 and 4, number of corpora lutea, number of implantation sites, number of lost implantations, litter size. The following parameters were calculated: pre-coital time = time between the start of mating and successful copulation, duration of gestation = time between gestation day 0 and day of delivery, female mating index = (number of females inseminated/number of females placed with males) x 100, male fertility index = (number of males with pregnant females/number of males placed with females) x 100, female fertility index = (number of pregnant females/number of inseminated females) x 100, gestation index = (number of females with live pups / number of females pregnant) x 100, live birth index = (number of pups born alive/number of pups born) x 100, viability index day 0 - 4 = (number of pup surviving 4 days/number of liveborn on day 0) x100, pup mortality day 0 or 4 = (number of dead pups on day 0 or 4/ total number of pups on day 0 or 4) x 100, sex ratio day 0 or 4 = [(number of live male pups on day 0 or 4 / number of live pups on day 0 or 4)] x 100, pre-implantation loss = [(number of corpora lutea – number of implantation sites) / number of corpora lutea] x 100, number of lost implantations = number of implantation sites – number of pups born alive, post-implantation loss = [(number of implantation sites - number of pups born alive) / number of implantation sites] x 100, prenatal loss = (Total number of Implantations - Total number of pups delivered) x 100 / Total number of Implantation Sites, perinatal loss = (Total number of pups delivered - Total number of alive pups delivered) x 100 / Total number of pups delivered.

Statistics:

Tests were generally performed as two-sided tests with results taken as significant where the probability of the results is p<0.05 (*) or p<0.01 (**). Continuous data were subjected to a decision tree or parametric statistical test. Dichotomous data were evaluated using the statistical test Chi-square-Fisher. Non-mated and non-pregnant females were excluded from mean data tables presenting data from the gestation and lactation periods

Results and discussion

Results of examinations

Details on results:

- Mortalities and clinical signs: One male (number 60) in the mid-dose group was found dead on day 12 of the premating period. Prior to death, the animal had shown no clinical signs indicative for a deteriorating condition. One female (number 81) in the high-dose group was killed moribund on day 12 of the premating period. The animal showed hunched posture, piloerection, red ears, encrustation round the nose and general weakened condition leading to a preterm sacrifice. One female (number 75) in the high-dose group was found dead on day 2 of lactation. This rat showed no clinical signs indicating a deteriorating condition. White discoloration of the lower incissors (fluorosis) was noted in all males and in all females of the mid- and high-dose group. Furthermore, all surviving high-dose females showed discoloration of the upper incisors. These findings were confirmed at macroscopic examination although not in all rats. The overall distribution and incidence of clinical observations did not indicate any other treatment- related effects.
- Neurobehavioural testing: Detailed clinical observations, Functional Observation Battery (FOB) and motor activity assessment did not indicate any neurotoxic potential of Nocolok Zn Flux in rats.
- Body weight and body weight change: Body weight were statistically significantly lower in the males of the high-dose group at the end of the premating period (day 14 of dosing) and during the post-mating period. No effects on body weight were observed in the low-and mid-dose groups as compared to the control group. In females no effects on body weight were observed at any dose level during premating and gestation. During lactation, Nocolok Zn Flux treated animals of all groups showed higher mean body weights as compared to the control animals on both day 0 and day 4 of lactation, but there was no dose-response relationship. These higher body weights compared to controls are considered not to represent an adverse effect.
- Feed consumption: The feed consumption was slightly, but statistically significantly decreased in males of the high-dose group during the premating period. An incidental increase in feed consumption was noted in males of the mid-dose group in the first week of the premating period. In females no effects on feed consumption were observed during premating and gestation. During lactation, feed consumption was elevated in all Nocolok Zn Flux treatment groups, although not statistically significantly. There was no dose-response relationship. This finding, which reflected the higher body weights in the treatment groups during lactation, is not considered to represent an adverse effect.
- Haematology: Thrombocytes tended to be increased in the high-dose group. This finding was statistically significant in the high-dose females only. There were no changes in red blood cell variables. There were no relevant changes in total and differential white blood cell counts. A statistically significant increase in the percentage eosinophils in females of the high-dose group was not reflected in significant changes in absolute eosinophil counts, and therefore not ascribed to the treatment.
- Clinical chemistry: Mean total protein and albumin concentrations were statistically significantly decreased in males and females of the high-dose group and in males of the mid-dose group. The albumin globulin ratio was increased in the high-dose group; this finding was statistically significant in males only. The effects occurred in both sexes and were generally below the range of historical control data obtained in RccHanTM:WIST rats at similar stages. Therefore they were considered to be treatment-related. A statistically significantly lower glucose plasma was observed in females of the high-dose group.
- Fertility and reproductive performance: Fertility and reproductive performance were not affected by the treatment. A number of 12, 12, 12 and 11 females were placed with males for mating, resulting in 7, 12, 11 and 8 pregnant females in the control group, low-dose, mid-dose and high-dose group, respectively. Two females (5 and 21) in the control group and two females (77 and 89) in the high-dose group showed no signs of insemination. Three females in the control group (3, 7 and 13), one female in the mid-dose group (71) and one female in the high dose group (85) did not deliver and were found not pregnant at necropsy. This resulted in 7, 12, 11 and 8 litters in the control, low-dose, mid-dose and high-dose group, respectively. All litters included viable pups and there were no litters comprising only stillborn pups. There were no relevant differences in pre-coital time, mating index, male or female fertility index, gestation index and the duration of gestation between the groups. No effects were found on the mean number of corpora lutea and implantation sites between the groups. Pre-implantation loss tended to be elevated in all treatment groups. Because there was no dose-response relationship and the values were within the historical control range (mean 12.1%, range 5.3 - 23.8) this finding is not ascribed to the treatment. Postimplantation loss (prenatal and perinatal loss together) was comparable in all groups.
LITTER DATA
- Litter data and sex: Litter data and sex were not affected by the treatment. The mean number of pups per litter (11.7, 10.8, 10.2 and 11.1 for the control, low-dose, mid-dose and high-dose groups, respectively) was comparable in all groups. The number of male and female litter mates was comparable in all groups. No effects were observed on the live born index or on the number of stillborn pups. The viability index was not affected by the treatment. One female in the control group (23) and one female in the high-dose group (79) completely lost their litter during lactation. Another female in the high-dose group (75) was found dead during lactation and the surviving pups were sacrificed for humane reasons.
- Pup observations: Clinical observations in pups during lactation did not reveal any relevant effects. A number of pups in the control group were cold or had no milk in the stomach. These pups were litter mates of litters 23 (control group) and 75 and 79 (high-dose group) that were lost during lactation.
- Pup weight: Mean pup weight was statistically significantly higher in the low-dose males and in the mid-dose males and females on day 0 of lactation. On day 4 of lactation, mean pup weight was statistically significantly higher in the low-dose and mid-dose female pups. Because no statistically significant effects were noted in the high-dose group and in absence of a dose response relationship, this was considered not to be treatment-related.
- Macroscopic observations of stillborn pups or pups that died during lactation: Macroscopic examination of stillborn pups or pups that died during lactation did not reveal any treatment-related effects.
PARENTAL NECROPSY OBSERVATIONS
- Organ weights of F0 animals: The relative weight of the kidneys was statistically significantly increased in males of the high-dose group. In females of this group, the absolute weight of the kidneys was statistically significantly increased and the relative weight tended to be increased although not statistically significantly. The relative weight of the adrenals was statistically significantly increased in males of the high-dose group. The following statistically significant changes in absolute or relative organ weights were considered not to be treatment-related: Elevated absolute heart weight in females of the mid- and high-dose group. This finding was ascribed to the higher terminal body weights in these groups. The relative weights of this organ were not affected; Increased relative testes weight in high-dose males, and decreased relative brain weights in mid- and high-dose females. These findings were related to the lower and higher terminal body weights, respectively, and the well-known inverse correlation between the weight of these organs and the body weight; Increased absolute and relative thymus weights in males of the low-dose group. These findings were not confirmed at the higher dose levels.
- Animals found dead or killed in moribund condition: One male (number 60) in the mid-dose group and one female (number 75) in the high-dose group were found dead. The cause of death could not be established. Another female (number 81) in the high-dose group was killed in moribund condition. Its poor health condition was most probably caused by moderate myocardial mineralization, accompanied by multifocal mixed inflammatory cell infiltrates (observed at microscopy). Furthermore, a haemorrhage (unknown cause) in the cranial cavity was found macroscopically. Because none of the other high-dose animals showed myocardial mineralization, the moribund condition of animal number 81 was considered a chance finding and not attributed to treatment.
- Macroscopic examination of F0 animals: At necropsy white discoloration of the lower incisors was observed in 5/12 mid-dose and 8/12 high-dose males and in 12/12 mid-dose and 11/12 high-dose females. Furthermore, 1/12 mid-dose and 11/12 high-dose females also showed discoloration of the upper incisors. The incisors were not processed for microscopical examination. The stomach of several mid- and high-dose males and females showed red or pale discoloration and/or mucosal swelling, which was attributed to treatment. Incidental other gross findings were observed in the stomach of single control and low-dose animals, but these are considered chance findings. The gross findings in other organs and tissues were unremarkable.
- Microscopic examination of F0 animals: Microscopic examination revealed treatment related histopathological changes in the stomach. They were characterized by minimal to moderate mixed inflammatory cell infiltrate, mild hyperaemia, mild haemorrhage(s), mild epithelial necrosis and increased mitotic activity in the glandular stomach and by minimal to mild epithelial vacuolization in the non-glandular stomach, mainly in the limiting ridge. The epithelial necrosis in the glandular stomach was characterized by several scattered necrotic cells in the top layer of the mucosa, indicated by the presence of pyknotic nuclei. The histopathological changes occurred as single finding or in combination with others. Generally, the highest incidences were found in the mid- and high-dose males. Inflammation, hyperaemia and epithelial vacuolization were also present in several low-dose animals. The other histopathological changes observed were about equally distributed amongst the different treatment groups or occurred in only one or a few animals. They are common findings in rats of this strain and age. Therefore, they were considered not to be related to treatment.

Effect levels

1

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on mortality and decreased plasma levels of total protein and albumin in the mid- and high-dose group.

2

Dose descriptor:

LOAEL

Remarks:

local

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on the pathological changes in the stomach in all test groups.

3

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related effects were seen in any of the reproductive endpoints.

4

Dose descriptor:

NOAEL

Remarks:

developmental

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related effects were seen in any of the developmental endpoints.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

A NOAEL of 7.5 mg/kg bw for systemic effects and a NOAEL of 75 mg/kg bw for fertility and developmental toxicity was established in a combined oral repeated dose toxicity study with reproduction/developmental toxicity screening test (OECD 422, GLP).

Executive summary:

In a GLP compliant combined oral repeated dose toxicity study with reproduction/developmental toxicity screening test performed according to OECD 422, Nocolok Zn Flux (KZnF3) was administered daily by oral gavage to Wistar rats. The animals (12 per sex per dose) were exposed to 0 (control), 7.5, 25 and 75 mg/kg bw/day KZnF3 dissolved in water. Male animals were dosed during a 2-week premating period, during mating and up to day 30 (the day before sacrifice on day 31). The female animals were dosed with the test substance during a 2-week premating period, and during mating, gestation and lactation up to the day before sacrifice (approx. day 4 of lactation). At the end of the premating period, each female was caged with one male from the same group. Animals were caged together until mating occurred or 1 week had elapsed. Upon evidence of copulation the females were caged individually for the birth and rearing of their pups. Sperm positive females that turned out to be non-pregnant were killed not earlier than 24 days after copulation. Females that did not show evidence of copulation after the end of the one-week mating period were also housed individually until sacrifice (25 days after the last day of the mating period). Dams were allowed to raise their litter until sacrifice on day 4 of lactation or shortly thereafter. The following endpoints were evaluated to assess general toxicity: daily clinical observations, neurobehavioural examination, body weight, food consumption, routine haematology and clinical chemistry, organ weights, macroscopic examination and histopathological examination of a wide range of tissues and organs (except for the stomach which was examined in all groups, only high-dose animals and controls were subjected to histopathology). Endpoints to assess reproductive / developmental toxicity included parental fertility and reproductive performance (weight and morphology of sex organs, mating index, pre-coital time, male and female fertility indices, gestation index and length, pre- and post-implantation loss) and litter data (numbers of stillborn, live and dead pups, external abnormalities, pup weight on lactation days 0 and 4, necropsy of stillborn pups and pups that died during the study).Three rats died during the study, two females in the high-dose group and one male in the mid-dose group. The death of one of these high-dose females was caused by myocardial mineralization, a chance finding which was not ascribed to treatment. The cause of death of the other high-dose female and the mid-dose male could not be established. Because there were no signs of a dosing error, these deaths are ascribed to the treatment. White discoloration of the incissors (fluorosis) was noted in all males and in all females of the mid and high-dose group. These findings were confirmed at macroscopic examination. These mild changes are considered not to be adverse when they do not affect the functional structure of the teeth. The decreased body weights and feed consumption, observed in males of the high-dose group are ascribed to the treatment. Males and females of the high-dose group and males of the mid-dose group showed decreased levels of total protein and albumin in plasma. The differences from controls increased with dose. Because these effects occurred in both sexes and were not within the range of historical control values, they were ascribed to the treatment. Data from the present study did not indicate a plausible mechanism for these decreases in plasma proteins; the present study revealed no clear evidence of hepatotoxicity, as assessed by liver weight, pathology and relevant clinical chemistry values. Microscopic findings in the kidneys could not explain the decrease in plasma protein either. The relative weight of the kidneys was elevated in the high-dose group. Although not corroborated by microscopic findings in the kidneys, the increase in kidney weight was ascribed to treatment. The significance of the increased relative weight of the adrenals in males of the high-dose group is not clear. Treatment of the animals with the test substance caused a range of pathological changes in the stomach in all test groups. Based on the presence of inflammation, hyperaemia and epithelial vacuolization in several low-dose animals, a no-observed-adverse-effect-level could not be established. These changes in the stomach are considered local effects resulting from direct contact of the test substance with the stomach wall. Microscopic findings in the other organs examined revealed no treatment-related changes. There were no effects of the test substance on fertility and reproductive performance, litter data or pup observations in any group. In conclusion, no treatment-related effects were seen in any of the reproductive or developmental endpoints. Hence, the no-observed-adverse-effect-level (NOAEL) for reproductive or developmental effects was placed at the highest level tested, namely 75 mg/kg bw/day. Based on mortality and decreased plasma levels of total protein and albumin in the mid- and high-dose group, the NOAEL for systemic effects was placed at 7.5 mg/kg bw/day. Because of pathological changes in the stomach in all test groups, the NOAEL for local effects was below 7.5 mg/kg bw/day.