4-Penten-2-ol, 3,3-dimethyl-5-(2,2,3-trimethyl-3-cyclopenten-1-yl)-, (4E)-

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From June 20 to November 02, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline 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 421 (Reproduction / Developmental Toxicity Screening Test)

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Programme (inspected on June 18-20, 2012/ signed on September 19, 2012)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

(+)-(2R,4E)-3,3-Dimethyl-5-[(1S)-2,2,3-trimethyl-3-cyclopenten-1-yl]-4-penten-2-ol

Cas Number:

163579-65-5

Molecular formula:

C15H260

IUPAC Name:

(+)-(2R,4E)-3,3-Dimethyl-5-[(1S)-2,2,3-trimethyl-3-cyclopenten-1-yl]-4-penten-2-ol

Constituent 2

Reference substance name:

(+)-(2S,4E)-3,3-Dimethyl-5-[(1S)-2,2,3-trimethyl-3-cyclopenten-1-yl]-4-penten-2-ol

Cas Number:

163748-45-6

Molecular formula:

C15H260

IUPAC Name:

(+)-(2S,4E)-3,3-Dimethyl-5-[(1S)-2,2,3-trimethyl-3-cyclopenten-1-yl]-4-penten-2-ol

Test material form:

liquid

Details on test material:

- Physical state: colourless liquid
- Storage condition of test material: At ambient temperature in the dark

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

The rat (sexually mature, virgin) was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Crl:CD(SD) strain was used because of the historical control data available at this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: Approximately 71 days
- Weight at study initiation: 350-395 g for males; 234-265 g for females
- Housing: Pre-pairing - 5/sex/cage; Pairing – 1 male:1 female per cage; Males after mating – 5 males/cage; Gestation – 1 female /cage; Lactation – 1 female/cage (+ litter) in polycarbonate cages
- Diet: Standard pelleted rodent diet (SDS VRF1 Certified diet), ad libitum
- Water: Potable water taken from the public supply, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-23 °C
- Humidity: 40-70 %
- Air changes: At least fifteen air changes per hour
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: From: June 20, 2012 To: November 02, 2012

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Test material was prepared for administration as a series of graded concentrations in the vehicle. Starting with the lowest concentration (20 mg/mL), the required amount of test material was weighed into a suitable container. Formulations were prepared by adding approximately 50% of the final volume of vehicle to the test material. The suspension was then magnetically stirred until thoroughly mixed. The suspension was made up to the required volume with vehicle. The formulation was placed into a container and stirred using a magnetic stirrer until homogenous; any samples that were required were taken at this point. During magnetic stirring, the suspension was transferred in daily aliquots to the final containers. Remaining concentrations were prepared in ascending group order using the same method.
- Stability assessment confirmed that formulations were stable for eight days following ambient storage and for 15 days following refrigerated storage (2-8 °C). All formulations were prepared in weekly batches, up to five days in advance of first use and stored refrigerated prior to use.

VEHICLE
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw/day

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: Following two weeks of treatment, males and females were paired on a one-to-one basis from within the same treatment group for a period of up to two weeks.
- Proof of pregnancy: Day on which evidence of mating was found (at least one copulation plug or a sperm positive vaginal smear) was designated Day 0 of gestation.
- After successful mating each pregnant female was caged (how): 1/cage
- The pre-coital interval was calculated for each female as the time elapsing between initial pairing and detection of mating.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The homogeneity and stability was confirmed for test material in corn oil formulations at nominal concentrations of 1 and 200 mg/mL during distribution between the bottles, during magnetic stirring for 2 hours, ambient temperature storage for 8 days and refrigerated storage for up to 15 days. The storage times represented the expected maximum time from preparation to completion of administration. In addition, the stability of discrete 1 mL samples was confirmed following refrigerated storage for 15 days. The mean concentrations of test material in test formulations analysed for the study was within +10%/-15% of nominal concentrations, confirming accurate formulation.

Duration of treatment / exposure:

Males were treated 15 days before pairing up to necropsy after a minimum of four consecutive weeks.
Females were treated for 15 days before pairing, throughout mating and gestation, until Day 6 of lactation.
The F1 generation received no direct administration of the test substance; any exposure was in utero or via the milk.

Frequency of treatment:

Once each day at approximately the same time each day, seven days per week.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected based on the previously conducted 28-day toxicity study. In that study, a high dose level of 300 mg/kg bw/day was employed and did not induce any adverse effects. In this current study, the high dose level of 1000 mg/kg bw/day was selected as this is the limit dose in most circumstances for OECD421 studies. The doses of 100 and 300 mg/kg bw/day were selected as the low and intermediate dose levels to establish a suitable dose relationship for any treatment-related changes and to give an approximate three fold interval between doses; the 300 mg/kg bw/day dose level was also selected as an anticipated No Observed Adverse Effect Level.
- Rationale for animal assignment: Allocation to study was revised before treatment commenced to reduce inter/intra-group bodyweight variation. Animals were replaced with spares from the same delivery to achieve this. On Day 1 (before dosing) variations in individual bodyweights were confirmed to be within ± 20% of the mean for each sex.

Positive control:

None

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages and cage-trays were inspected daily for evidence of ill-health amongst the occupant(s).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A detailed physical examination was performed weekly on each F0 animal and additionally for the F0 females on Days 0, 7, 14 and 20 after mating and on Days 1 and 7 of lactation to monitor general health.
- Detailed observations were conducted daily during the first week of treatment and weekly thereafter for all adult animals. For the F0 females, these observations were also performed on Days 0, 7, 14 and 20 after mating and Days 1 and 6 of lactation. These detailed observations were recorded at the following times in relation to dose administration:
Immediately before dosing; at the end of dosing each group; on completion of dosing of all groups; between one and two hours after completion of dosing of all groups; as late as possible in the working day

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight of each adult animal was recorded during acclimatisation, before dosing on the day that treatment commenced (Week 0), weekly throughout the treatment period and before necropsy. The weight of each F0 female was also recorded on Days 0, 3, 7, 10, 14, 17 and 20 after mating and on Days 1, 4 and 7 of lactation.

FOOD CONSUMPTION:
- The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded on a weekly basis for males and females (from the first day of treatment until pairing). From these records the mean weekly consumption per animal (g/rat/week) was calculated for each cage.
- For each F0 female after mating, the weight of food supplied, that remained and an estimate of any spilled was also recorded for the periods, Days 0-2, 3-6, 7-9, 10-13, 14-16 and 17-19 after mating and Days 1-3 and 4-6 of lactation. From these records the mean daily consumption (g/rat/day) was calculated for each animal.

WATER CONSUMPTION: Yes

OTHER:
PARTURITION OBSERVATIONS AND GESTATION LENGTH:
From Day 20 after mating, females were inspected three times daily for evidence of parturition. The progress and completion of parturition was monitored, numbers of live and dead offspring were recorded and any difficulties observed were noted. The duration of gestation was calculated as the time elapsing between the detection of mating and commencement of parturition.

Oestrous cyclicity (parental animals):

For 15 days before pairing, daily vaginal smears (dry) were taken from all females, using cotton swabs moistened with saline. The smears were subsequently examined to establish the duration and regularity of the oestrous cycle. After pairing with the males, smearing was continued using pipette lavage, until evidence of mating was observed.

Sperm parameters (parental animals):

For the assessment of the testes, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects, such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the lumen. Any cell- or stage-specificity of testicular findings was reported.

Litter observations:

PARAMETERS EXAMINED
All litters were examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter.
The following parameters were examined in F1 offspring:
- Clinical signs: Daily records were maintained for evidence of ill health or reaction to maternal treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate.
- Litter size: Daily records were maintained of mortality and consequent changes in litter size from Days 1-7 of age.
- Sex ratio: The sex ratio of each litter was recorded on Days 1, 4 and 7 of age.
- Bodyweight: Individual offspring bodyweights were recorded on Days 1, 4 and 7 of age.

GROSS EXAMINATION OF DEAD PUPS: Yes

Postmortem examinations (parental animals):

SACRIFICE
All adult animals were killed by carbon dioxide asphyxiation.
Male animals: All surviving F0 males were killed during Week 5 of treatment.
Maternal animals:
- F0 females surviving until the end of the scheduled study period were killed on Day 7 of lactation.
- F0 females that failed to produce a viable litter were killed on Day 25 after mating.
- F0 females whose litter died before Day 7 of lactation were killed on the day the last offspring died.

GROSS NECROPSY
All adult animals were subject to a detailed necropsy, which involved the following:
- After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. After ventral mid-line incision, the neck and associated tissues and the thoracic, abdominal and pelvic cavities and their viscera were exposed and examined in situ. Any abnormal position, morphology or interaction was recorded.
- For F0 females, the number of uterine implantation sites was recorded. For females failing to produce a viable litter, the number of uterine implantation sites was re-checked after staining with ammonium sulphide (modification of the Salewski staining technique (Salewski, E, 1964)).

ORGAN WEIGHTS
The following organs, taken from each adult animal, were dissected free of adjacent fat and other contiguous tissue and the weights recorded:
- Epididymides (L&R), ovaries (L&R), prostate, seminal vesicles, testes (L&R), L&R Bilateral organs weighed individually.
Organ weights were also adjusted for terminal bodyweight, using the weight recorded before necropsy.

HISTOPATHOLOGY
Fixation:
Testes were fixed initially in modified Davidson’s fluid. Samples (or the whole) of the other tissues listed below from all F0 animals were preserved in 10% neutral buffered formalin.
- Epididymides, mammary - caudal (only females whose litter dies), ovaries, pituitary, prostate, seminal vesicles, stomach (including forestomach), testes, uterus with cervix and oviducts and vagina
Samples of any abnormal tissues were also retained and processed for examination. In those cases where a lesion was not clearly delineated, contiguous tissue was fixed with the grossly affected region and sectioned as appropriate.
- Tissue samples were dehydrated, embedded in paraffin wax, sectioned at approximately four to five micron thickness and stained with haematoxylin and eosin, except the testes which were stained using a standard periodic acid/Schiff (PAS) method. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required for microscopic pathology.
Light microscopy:
Microscopic examination was performed as follows:
The testes, epididymides, ovaries and stomach were examined for all animals of Groups 1 (Control) and 4 (1000 mg/kg bw/day) sacrificed on completion of the scheduled treatment period. Tissues reported at macroscopic examination as being grossly abnormal were examined for all F0 animals in line with current practice.

Postmortem examinations (offspring):

SACRIFICE
All offspring were killed by an intraperitoneal injection of sodium pentobarbitone.

GROSS NECROPSY
For offspring surviving to scheduled termination, a careful external examination was performed for gross abnormalities and externally normal offspring were discarded without internal examination. Externally abnormal offspring were internally examined and any abnormal tissues were retained in an appropriate fixative.
Additionally the following procedures were applicable:
Premature deaths: Missing offspring and those grossly autolysed or grossly cannibalised could not be examined. All other offspring dying before Day 7 of age were examined as detailed above. The necropsy also included an assessment for the presence of milk in the stomach, where this was possible.

Statistics:

Statistical analyses were performed on for bodyweight, food consumption, organ weights and litter data were performed. For some parameters, including oestrous cycles, pre-coital interval, mating performance and fertility and gestation length the similarity of the data was such that analyses were not considered to be necessary. All statistical analyses were carried out separately for males and females.

See further details in section “Any other information on materials and methods incl. tables”

Reproductive indices:

Percentage mating = (Number animals mating / Animals paired) x 100
Conception rate (%) = (Number animals achieving pregnancy / Animals mated) x 100
Fertility index (%) = (Number animals achieving pregnancy / Animals pairing) x 100
Gestation index (%) = (Number of live litters born / Number pregnant) x 100

Offspring viability indices:

Post-implantation survival index (%) = (Total number of offspring born / Total number of uterine implantation sites) x 100
Live birth index (%) = (Number of live offspring on Day 1 after littering / Total number of offspring born) x 100
Viability index (%) = (Number of live offspring on Day 7 after littering / Number of live offspring on Day 1 after littering) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

- There were no clinical signs observed at routine physical examination that were related to treatment.
- Signs observed in relation to dose administration were limited to salivation in males and females receiving 300 or 1000 mg/kg bw/day from Day 2-3 of treatment, with a very low sporadic incidence at 100 mg/kg bw/day, and an associated low incidence of chin rubbing at 1000 mg/kg bw/day. Post-dosing salivation and/or transient chin rubbing are commonly observed where animals are dosed by oral gavage and the reaction is generally regarded as reflecting distaste of the dosing formulations rather than a sign of toxicity.

Mortality:

no mortality observed

Description (incidence):

- Treatment with test material at doses up to and including 1000 mg/kg bw/day was well tolerated and there were no mortalities.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- There was no effect of treatment on mean bodyweight gain of males at any dose level investigated.
- Among males receiving 1000 mg/kg bw/day it was noted that overall mean bodyweight gain was 15% lower than control, with mean weight gain during three of the four recording periods being slightly lower than control. Review of the individual animal data did not reveal a clear difference in the pattern of weight gain, with the exception of the period between Week 3 and Week 4, where 5 of the ten males gained less weight than the lowest control. None of the differences in mean weight gain from control attained statistical significance however, and therefore the differences were considered to be of no toxicological or biological significance.
- There was no effect of treatment on the bodyweight gain of females during the first week of dosing. During the second week of treatment, mean bodyweight gain of females receiving 1000 mg/kg bw/day was statistically significantly higher than in controls and as a consequence, mean absolute bodyweights from Week 2 to termination were slightly but significantly higher than in controls. There was no effect of treatment at 1000 mg/kg bw/day on bodyweight gain during gestation or lactation. Treatment of females with test material at 300 or 100 mg/kg bw/day did not affect bodyweight gain.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- During the first week of treatment, mean food intake for males receiving 1000 mg/kg bw/day was marginally lower than control; during Week 2, food intake was slightly higher than control and was considered to be unaffected by treatment. Among males receiving 100 or 300 mg/kg bw/day there was no effect of the administration of test material on mean food intake.
- Food intake for all groups of treated females was considered unaffected by the administration of test material. It was noted that females receiving 1000 mg/kg bw/day showed slightly higher food intake than controls during Week 2 of treatment, during gestation and during the first three days of lactation, with the majority of the recording periods attaining statistical significance. The slightly higher food intake during Week 2 of treatment was considered to be responsible for the higher weight gain in these females during this period. The subsequent slightly higher food consumption of these females during gestation and lactation may reflect the slightly greater size of these animals compared with controls.

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

During Week 1 of dosing, a daily qualitative assessment of water consumption indicated a suggestion of an increase in fluid intake for animals in Groups 3 and 4 when compared to Controls. Increased water intake (along with post-dosing salivation) had been seen in previous studies conducted with this test material was considered most likely to be a response to the flavour of the test formulations. In an attempt to reduce any potential effects of the taste of the formulations, once the syringe had been loaded with formulation, the catheter was wiped with a dry tissue, dipped in fresh tap-water and wiped with a separate dry tissue (which
will be replaced as deemed necessary).

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

- There were no histopathological findings which were considered to be related to treatment with test material.
- Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage-specific abnormalities were noted.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

- Oestrous cycle activity was unaffected by treatment with test material, with all females showing regular 4-day or 4/5 day cycles.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

- Mating performance was unaffected by treatment with test material, with all pairs mating within four days (ie. at the first oestrus opportunity). There was no evidence of dystocia, and with the exception of one non-pregnant female receiving 300 mg/kg bw/day, all females successfully gave birth.
- One female receiving 300 mg/kg bw/day gave birth prematurely on Day 20 of gestation (normal gestation length 22-23 days). Two externally normal dead pups were found in the cage on the afternoon of Day 20 of gestation, which weighed 3.7 g and 3.8 g and evidence of two cannibalised pups was found on the morning of Day 21 of gestation. The female was despatched to necropsy as a Total Litter Loss, where macroscopic examination revealed 18 implantation sites in the uterus, pale ovaries, enlarged spleen (most likely due to physiological state), pale liver, fetal material in the stomach and pale/inactive mammary tissue. Although it is not possible to ascertain the aetiology of this event, in the absence of similar premature births in the high dose group (at more than 3 times the intermediate dose level) it was considered that this premature birth was unrelated to treatment with test material.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Remarks:

(Screening for fertility)

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

- There were no clinical signs observed among the F1 offspring which were attributable to parental treatment with test material.

Mortality / viability:

no mortality observed

Description (incidence and severity):

- There was no effect of parental treatment with test material at any dose level investigated on mean litter size, sex ratio or offspring survival to Day 7 of age.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

- Offspring absolute bodyweight on Day 1 of age was similar in all groups, and mean weight gain between Day 1 and Day 4 of age was unaffected by parental treatment. For offspring in the 1000 mg/kg bw/day group, mean weight gain between Days 4 and 7 of age was slightly lower than control, although statistical significance was not attained.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

- There were no macroscopic findings observed in the low number of offspring that died prior to scheduled termination or among those offspring killed on Day 7 of age, that were attributable to parental treatment with test material.

Histopathological findings:

not examined

Other effects:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks:

(Screening for development (foetal and pup growth survival until day 6))

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Result tables are included in "Attached background material"

Applicant's summary and conclusion

Conclusions:

Under the test conditions, the NOAEL for fertility and development (foetal and pup growth until day 6) is 1000 mg/kg bw/day in the rat.

Executive summary:

In a Reproduction / Developmental Toxicity Screening Test conducted according to OECD Guideline 421 and in compliance with GLP, test item was administered to groups of Crl:CD(SD) rats (10/sex/dose) at 0, 100, 300 and 1000 mg/kg bw/day by oral (gavage). Males were treated 15 days before pairing up to necropsy after a minimum of four consecutive weeks. Females were treated for 15 days before pairing, throughout mating and gestation, until Day 6 of lactation. The F1 generation received no direct administration of the test substance; any exposure was in utero or via the milk. A control group of ten males and ten females was dosed with vehicle alone (corn oil). Clinical signs, body weight change, food and water consumption were monitored during the study. All animals were subjected to a gross necropsy examination, selected organs were weighed and histopathological evaluation of selected tissues was performed. The clinical condition of offspring, litter size and survival, sex ratio and offspring bodyweight were assessed and macroscopic pathology investigations were undertaken. 

Oral administration of the test material at dose levels up to and including 1000 mg/kg bw/day was well tolerated. There were no deaths, no treatment-related clinical signs or dosing signs observed and no treatment-related effects on bodyweight performance or food intake of males. Females receiving 1000 mg/kg bw/day consumed slightly more food and gained more weight during the second week of treatment compared with Controls, and as a consequence, absolute bodyweights of these females during gestation and lactation were slightly greater than in Controls; these findings were not adverse.

Oestrous cycle length, mating performance, fertility, reproductive capacity and gestation length were unaffected by treatment with the test material at all dose levels investigated. The weights of the reproductive organs were unaffected, and there were no treatment-related macroscopic or microscopic abnormalities detected.

Bodyweight gain, survival and development of the offspring to Day 7 of age was considered unaffected by parental treatment. In the 1000 mg/kg bw/day group, the weight gain of offspring between Day 4 and Day 7 of age was slightly lower than Control; differences were small, and may reflect the slightly higher litter size in this group, however, and in the absence of any changes in clinical condition or offspring survival, these minor differences in weight gain were considered not to be adverse.

 

Under the test conditions, the NOAEL for fertility and development (foetal and pup growth until day 6) is 1000 mg/kg bw/day in the rat.