Octadecane-1,12-diol

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Nov 14 - Jan 15

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other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment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

2015

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Octadecane-1,12-diol

EC Number:

220-342-6

EC Name:

Octadecane-1,12-diol

Cas Number:

2726-73-0

Molecular formula:

C18H38O2

IUPAC Name:

octadecane-1,12-diol

Details on test material:

- Name of test material (as cited in study report): Sovermol 912
- Physical state: White solid
- Storage condition of test material: At room temperature

Test animals

Species:

rat

Strain:

other: Rat: Crl:WI(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: Approximately 11 (females) weeks
- Weight at study initiation: All animals were within ± 20% of the sex mean.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Tap-water, ad libitum
- Acclimation period: At least 5 days prior to start of treatment.

HOUSING
- Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).
- Mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm).
- Post-mating: Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
- Lactation: Pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm). During locomotor activity monitoring of the dams the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes.
- General: Sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and paper as cage-enrichment/nesting material (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24
- Humidity (%): 40 to 70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

2%

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenized to a visually acceptable level. No adjustment was made for specific gravity/density of the test substance, vehicle, and/or formulation. No correction was made for the purity/composition of the test substance. The test substance was ground prior to formulation.
- Storage temperature: At room temperature.

VEHICLE
- Justification for use and choice of vehicle: Based on trial formulations performed and in consultation with the Sponsor.
- Amount of vehicle: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of dose preparations were taken at the test facility on two occasions during the treatment period. At the first occasion (01 December 2014), freshly prepared formulations were sampled for determination of the accuracy of preparation, homogeneity and stability of the test substance in the formulations. As the accuracy of the formulations for Group 2 was found to be slightly below the target value, the accuracy of preparation for this group was also determined in the formulations prepared and sampled on 05 January 2015. The samples were dispatched on dry ice to ABL where they were analysed to assess accuracy of preparation, homogeneity and/or stability in vehicle over 5 hours at room temperature.

Details on mating procedure:

- Mating procedures: Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating occurred, the males and females were separated. A maximum of 14 days was allowed for mating, after which females who had not shown evidence of mating were separated from their males.
Detection of mating was not confirmed for animal nos. 46 and 69 which did deliver live offspring. The mating date of these animals was estimated at 21 days prior to the actual delivery date. This day was designated Day 0 post-coitum.
- Parturition: The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes and placentas cleaned up, nest build up and/or feeding of pups started). Females that were littering were left undisturbed.
Number of pups 442 pups.
- Identification of pups: On Day 1 of lactation, all pups were individually identified by means of subcutaneous injection of Indian ink.

Duration of treatment / exposure:

- Females were exposed for 41-55 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy).

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.

Duration of test:

Females: 55 days

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 300, and 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on a 14-day dose range finding study in which dose levels of 300 and 1000 mg/kg bw/day were tested and no serious adverse effects were seen.

Examinations

Maternal examinations:

MORTALITY: At least twice daily.

DETAILED CLINICAL OBSERVATIONS: At least once daily from treatment onwards up to the day prior to necropsy, detailed clinical observations were made in all animals. Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena. These clinical observations were made after dosing (at no specific time point as there was no peak occurrence of clinical signs after dosing in the dose range finding study. The time of onset, grade and duration of any observed sign was recorded.

BODY WEIGHT: Females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.

FOOD CONSUMPTION: Weekly, except for females which were housed together with males for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

WATER CONSUMPTION: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

CLINICAL LABORATORY INVESTIGATIONS:
Blood samples were collected at the end of the treatment period on the day of scheduled necropsy from the selected 5 animals/sex/group under anaesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) between 7.00 and 10.30 a.m. The animals were deprived of food overnight (start of deprivation maximally 24 hours before blood sampling), but water was available. Blood samples were drawn from the retro-orbital sinus and collected into tubes (Greiner Bio-One GmbH, Kremsmünster, Austria) prepared with K3-EDTA for haematological parameters (0.5 mL), with citrate for clotting tests (0.45 mL) and tubes treated with Li-heparin for clinical biochemistry parameters (0.5 mL). An additional blood sample (0.25 mL) was collected into serum tubes for determination of bile acids. Furthermore, from the selected 5 animals/group an additional blood sample (1.2 mL) was collected from the retro-orbital sinus into serum tubes for possible future measurement of thyroid-stimulating hormone (TSH) and the thyroid hormones triiodothyronine (T3) and thyroxine (T4). After clotting and centrifugation, serum samples were stored at ≤-75°C. Under these storage conditions the samples are stable for 2 months. The samples were discarded after 2 months because no treatment-related changes were observed at microscopic examination of the thyroid and, therefore, analysis of thyroid hormones in serum was not conducted.

HAEMATOLOGY:
In blood: White blood cells, Red blood cells, Reticulocytes, Red blood cell distribution width, Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelets.
In plasma: Prothrombin Time, Activated Partial Thromboplastin Time

CLINICAL CHEMISTRY:
In plasma: Alanine aminotransferase, Aspartate aminotransferase, Alkaline Phosphatase, Total protein, Albumin, Total Bilirubin, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate
In serum:Bile acids

NEUROBEHAVIOURAL EXAMINATION: The following tests were performed on 5 selected animals/sex/group:
- hearing ability, pupillary reflex and static righting reflex
- fore- and hind-limb grip strength were recorded as the mean of three measurements (Series M4-10, Mark-10 Corporation, J.J. Bos, Gouda, The Netherlands).
- locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway, USA). Total movements and ambulations were reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or more fine movements like grooming, weaving or movements of the head.
The selected females were tested towards the end of the scheduled lactation period (from lactation Day 4 onwards). These tests were performed after observation for clinical signs (incl. arena observation, if applicable) at no specific time point, but within a similar time period after dosing for the respective animals.

OTHER: GENERAL REPRODUCTION
Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Palpation was used to aid in confirmation of pregnancy. Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of pregnant females was examined to detect signs of abortion or premature birth. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.

GROSS PATHOLOGY: All animals were deprived of food overnight (with a maximum of 24 hours) prior to planned necropsy, but water was provided.
All animals were deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and subsequently exsanguinated and subjected to a full post mortem examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.

Necropsy was conducted on the following days:
- Females which delivered, Lactation Days 5-7.
- Females which failed to deliver, 21 days after the last day of the mating period (female without evidence of mating).

Samples of the following tissues and organs were collected from all animals and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands) : Ovaries, Adrenal glands, (Pancreas), (Aorta), Peyer's patches [jejunum, ileum] if detectable, Brain - cerebellum, mid-brain, cortex, Pituitary gland, Caecum, Preputial gland, Cervix, Clitoral gland, Rectum, Colon, (Salivary glands - mandibular, sublingual), Coagulation gland, Sciatic nerve, Duodenum, Skeletal muscle, Eyes (with optic nerve (if detectable) and Harderian gland), (Skin), Spinal cord -cervical, midthoracic, lumbar, Female (and male) mammary gland area, Spleen, Femur including joint, Sternum with bone marrow, Heart, Stomach, Ileum, Jejunum, Thymus, Kidneys, Thyroid including parathyroid if detectable, (Lacrimal gland, exorbital), (Tongue), (Larynx), Trachea, Liver, Urinary bladder, Lung, infused with formalin, Uterus, Lymph nodes - mandibular, mesenteric, Vagina, (Nasopharynx), All gross lesions and (Esophagus). Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.

HISTOPATHOLOGY: All organ and tissue samples, as defined under Histopathology (following section), were processed, embedded and cut at a thickness of 2-4 micrometers. These slides were stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands).
The following slides were examined by a pathologist:
- The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4.
- The mesenteric lymph node of the selected 5 females of Groups 2 and 3.
- All gross lesions of all animals (all dose groups).
- The reproductive organs (cervix, clitoral gland, coagulation gland, ovaries, preputial gland, uterus, and vagina) of all animals of Groups 1 and 4, including female no. 71 (Group 4) that failed to deliver healthy pups.

Ovaries and uterine content:

The numbers of former implantation sites and corpora lutea were recorded for all paired females.

Fetal examinations:

CAGE SIDE OBSERVATIONS: Mortality / Viability The numbers of live and dead pups were determined on Day 1 of lactation and daily thereafter. If possible, defects or cause of death were evaluated.

DETAILED CLINICAL OBSERVATIONS: Clinical signs At least once daily, detailed clinical observations were made for all animals.

BODY WEIGHT: Body weights Live pups were weighed on Days 1 and 4 of lactation.

OTHER: Sex was determined for all pups on Days 1 and 4 of lactation.

GROSS PATHOLOGY: Pups surviving to planned termination were killed by decapitation on Days 5-7 of lactation. All pups were sexed and examined for external abnormalities. The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated. Terminal body weight was recorded from all animals. The following organ weights were recorded from the following animals on the scheduled day of necropsy (Selected 5 animals/sex/group): Adrenal glands, Spleen, Brain, Testes, Epididymides, Thymus, Heart, Uterus (including cervix), Kidneys, Prostate, Liver, Seminal vesicles including coagulating glands, Ovaries, Thyroid including parathyroid.

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.

Indices:

For each group, the following calculations were performed:
- Mating index (%): Number of females mated/Number of females paired x 100
- Fertility index (%): Number of pregnant females/Number of females paired x 100
- Conception index (%): Number of pregnant females/Number of females mated x 100
- Gestation index (%): Number of females bearing live pups/Number of pregnant females x 100
- Duration of gestation: Number of days between confirmation of mating and the beginning of parturition.
- Percentage live males at First Litter Check: Number of live male pups at First Litter Check/ Number of live pups at First Litter Check x100
- Percentage live females at First Litter Check: Number of live female pups at First Litter Check / Number of live pups at First Litter Check x 100
- Percentage of postnatal loss: Number of dead pups before planned necropsy / Number of live pups at First Litter Check x 100
- Viability index: Number of live pups before planned necropsy / Number of pups born alive x 100

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY
No mortality occurred during the study period. No clinical signs of toxicity were noted up to 1000 mg/kg bw/day. Incidental findings that were noted included alopecia, piloerection and rales. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological relevance.

BODY WEIGHT AND WEIGHT GAIN
No toxicologically relevant changes in body weights and body weight gain were noted. In females at 100 mg/kg bw/day statistically significantly higher body weight gain was noted at postcoitum Days 4, 11 and 14. The weight gain values showed no dose-related response and mean body weights of treated females were similar to those of controls. Therefore, thedifferences in body weight gain were not attributed to treatment.

FOOD CONSUMPTION
Food consumption before or after allowance for body weight showed no toxicologically or statistically significant differences between treated and control animals.

HAEMATOLOGY
Haematological parameters of treated rats were not affected by treatment. It was noted that one female at 1000 mg/kg bw/day (no. 75) had several abnormal values (lower values for WBC, red blood cells, haemoglobin and haematocrit; higher values for reticulocytes, RDW and MCV). The other females at 1000 mg/kg bw/day had normal haematology values. Therefore, it was concluded that no toxicologically relevant changes occurred in haematology parameters of treated rats.

CLINICAL CHEMISTRY
No toxicologically relevant changes occurred in clinical biochemistry parameters of treated rats. Statistically significant differences between treated rats and controls were limited to a higher fasting glucose level in females at 1000 mg/kg bw/day. The glucose values at 1000 mg/kg bw/day remained in the normal range for rats of this age and strain (values in concurrent controls were at the lower (glucose) end of the normal range). Moreover, there were neither changes in other clinical biochemistry parameters nor corroborative histopathological changes. Therefore, these difference in glucose was considered not to be adverse. Further it was noted that the mean plasma levels of ALP, urea and bile acids in females at 1000 mg/kg bw/day were somewhat higher than in controls. These findings were not attributed to treatment because the differences from control were not statistically significant and remained in the normal range for females of this age and strain.

NEUROBEHAVIOUR
Hearing ability, pupillary reflex, static righting reflex and grip strength were not affected by treatment. Lower mean forelimb and hind limb grip strength values were noted in females at 1000 mg/kg bw/day. The differences from control were not statistically significant and remained in the normal range for female rats of this age and strain. Moreover, the lower hind limb grip strength was particularly due to a low value in one animal (no. 75). Therefore, these findings were not attributed to treatment.The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with high activity in the first interval that decreased over the duration of the test period. In females the mean activity reached a minimum after about 50 minutes (interval 10-11) and increased slightly thereafter (activity during the last interval (no. 12) remained well below the activity level at the start of the test period).

ORGAN WEIGHTS
Females at 1000 mg/kg bw/day had lower uterus weights. The organ to body weight ratio, but not the absolute uterus weight, differed statistically significantly from controls. In a reproduction/ developmental toxicity screening test, uterus weights of individual animals may vary widely due to differences in the time between delivery and weighing of the uterus and the normal involution of the uterus after delivery. Uteri of the five selected control females were weighed at Day 5 of lactation while uteri of the selected females at 1000 mg/kg bw/day were weighed at Day 7 (4/5 females) or Day 5 (1/5 females) of lactation. Hence, uterus weights of 4/5 females at 1000 mg/kg bw/day were determined later during the involution process than those of control females. Therefore, the lower uterus weights 1000 mg/kg bw/day were considered to be unrelated to treatment. At 300 mg/kg bw/day females had a statistically significantly higher heart weight (absolute and relative to body weight). In the absence of a dose-related response, these slight differences were considered not to be related to treatment. The other organ weights and organ to body weight ratios of treated animals were similar to those of control animals.

GROSS PATHOLOGY
There were no test item-related gross observations. Incidental findings among control and treated animals were within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related trend. These necropsy findings were therefore considered to be unrelated to treatment.

HISTOPATHOLOGY: There were no test item-related microscopic observations. There was one finding of note: The right kidney of female no. 59 (100 mg/kg bw/day) showed a nephroblastoma which correlated with a yellowish, hard nodule of 5x6 mm recorded at necropsy. Nephroblastomas can be seen as a spontaneous background finding in Wistar Han rats. Nephroblastoma is a renal tumor originating from nephrogenic blastema and results from abnormal differentiation of the kidney during embryogenesis. The macrophage foci recorded in the mesenteric lymph node of some females at a minimal or slight degree (3/5 at 100 mg/kg bw/day, 1/5 at 300 mg/kg bw/day and 1/5 at 1000 mg/kg bw/day) did not show a dose relationship and can be seen as a spontaneous background finding in animals of this age and strain. This finding was therefore regarded not to be related to the treatment with the test substance. The findings mentioned above and all remaining histologic changes were considered to be incidental findings. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

REPRODUCTION DATA
No toxicologically relevant effects on reproductive parameters were noted. The mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment. There were 10, 10, 10 and 9 pregnant females at 0, 100, 300 and 1000 mg/kg bw/day, respectively, and all pregnant females delivered litters with live pups. For a few females the number of pups born was slightly higher than the number of implantations and/or corpora lutea (female nos. 44, 56 and 68 of Groups 1, 2 and 3, respectively). This was considered to be due to normal resorption of these areas as these enumerations were performed on Days 5-7 of lactation.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Details on embryotoxic / teratogenic effects:
DEVELOPEMENTAL DATA
No toxicologically relevant effects on gestation index and duration, parturition, maternal care and early postnatal pup development (mortality, clinical signs, body weight and macroscopy) were observed.
- Gestation: The gestation index and duration of gestation were unaffected by treatment up to 1000 mg/kg bw/day.
- Parturition/maternal care: No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.
- Early postnatal pup developmen:The number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings.
It was noted that the number of pups (total and mean) at 100 and 1000 mg/kg bw/day was slightly lower than in the control group. These differences were not attributed to treatment because there was no dose-related response and values were within normal limits.
- Mortality: Four pups of the control group and three pups at 100 mg/kg bw/day went missing during the first days of lactation and one pup at 1000 mg/kg bw/day was found dead at the first litter check. No pups went missing or were found dead at 300 mg/kg bw/day. Pups missing were most likely cannibalised. These mortality incidences did not indicate a relation with treatment and remained within the range considered normal for pups of this age.
- Clinical signs: Pups that went missing showed no clinical signs. Incidental clinical signs seen in surviving pups consisted of scabs on the snout, pale appearance, missing tail apex, and blue (spot on) abdomen. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.
- Body weights: Body weights of pups were unaffected by treatment.
- Macroscopy: The single pup (of female no. 73 of Group 4) found dead at first litter check was decapitated. Findings among surviving pups were limited to a missing tail apex (one pup of female no. 61 of Group 3). The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Under the conditions of this assay the NOAEL for maternal and developmental toxicity was found to be greater than 1000 mg/kg bw/day.

Executive summary:

A combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test was performed according to OECD 422 and in compliance with GLP. The test substance was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg bw/day (10 rats/sex/dose level). Concurrent controls (10 rats/sex) received the vehicle, 2% aqueous carboxymethyl cellulose, alone. Accuracy, homogeneity and stability of formulations were demonstrated by analyses. The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 41-55 days). No maternal toxicity was observed up to the highest dose level tested (1000 mg/kg bw/day) as evidenced by the absence of clinical signs of toxicity or adverse changes in functional observational results, body weight (gain), food consumption, haematology and clinical chemistry parameters, organ weights, macroscopic findings, and microscopic findings. No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight and macroscopy). Under the conditions of this assay the NOAEL for maternal and developmental toxicity was found to be greater than 1000 mg/kg bw/day.