Radia 7838

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 October - 09 December 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test, July 1995.

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

- Name of test material (as cited in study report): RADIA 7838
- Physical state: Yellow to amber hazy viscous liquid
- Analytical purity: Not indicated by the sponsor; treated as 100% pure
- Lot/batch No.: OE 90922
- Expiration date of the lot/batch: 26 June 2014
- Stability under storage conditions: Stable
- Storage condition of test material: At room temperature in the dark
- Density: 1.103 g/ml
- pH: Not indicated
- Stability at higher temperatures: Yes
- Stability in vehicle; Propylene glycol:At least 5 hours (at room temperature over the concentration range 10 mg/mL to 200 mg/mL, NOTOX project 485789)
- Solubility in vehicle; Propylene glycol: Yes

Test animals

Species:

rat

Strain:

other: Crl:WI(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories France, L'Arbresle Cedex, France.
Nulliparous and nonpregnant females and untreated animals were used at initiation of the study.
- Age at study initiation: Approximately 12 weeks.
At the start of treatment, animals were approximately 12 weeks old instead of approximately 11 weeks. Mating started shortly after the animals had attained full sexual maturity according to the OECD 421 guideline and thus, a slight deviation in age does not affect the study’s integrity.
- Weight at study initiation: mean weight range at start of treatment was 333-340 gr (males) or 198-205 gr (females).
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages (MIV type, height 18 cm).
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages (MIII type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages (MIII type, height 18 cm).
Lactation: Pups were kept with the dam until termination in Macrolon cages (MIII type, height 18 cm).
General: Sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. Certificates of analysis were examined and then retained in the NOTOX archives. During activity monitoring, animals were housed individually in Macrolon cages (MIII type; height 15 cm) with sterilised sawdust as bedding material. No cage-enrichment was provided during activity monitoring.
- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). Results of analyses for nutrients and contaminants were examined and archived.
- Water (e.g. ad libitum): Free access to tap-water. Certificates of analysis (performed quarterly) were examined and archived.
- Acclimation period: At least 5 days prior to start of treatment.
- Randomization: Prior to commencement of treatment, by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.

A health inspection was performed prior to commencement of treatment to ensure that the animals were in a good state of health.

Analysis of bedding, paper, diet and water did not reveal any findings that were considered to have affected the study integrity.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.1 - 23.1°C
- Humidity (%): 31 - 96%
Temporary deviations from the minimum and maximum level of relative humidity occurred in the animal room. Laboratory historical data do not indicate an effect of the deviations.
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial light and 12 hours darkness per day.

IN-LIFE DATES: From: 24 October - 09 December 2010

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

Method of formulation: Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for the density of the test substance and the specific gravity of the vehicle.

Storage conditions of formulations: At ambient temperature.

Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at NOTOX.

Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Method of gavage: using a plastic feeding tube. Formulations were placed on a magnetic stirrer during dosing.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase, according to a validated method (NOTOX Project 485768). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).

The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.

The concentrations analyzed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). No test substance was detected in the Group 1 formulation and the formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Details on mating procedure:

- M/F ratio per cage: 1/1 (one female was cohabitated with one male of the same treatment group, avoiding sibling mating (Charles River supplied non-litter mates).
- Length of cohabitation: A maximum of 14 days was allowed for mating.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage, by staging of the estrous cycle and/or or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
- After successful mating each pregnant female was caged individually in Macrolon cages (MIII type, height 18 cm).

Duration of treatment / exposure:

Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 44-47 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). Some females (1,1,1 in Group 1,2,3, respectively) were not dosed during littering.

Pups were not treated directly, but were potentially exposed to the test substance in utero and through lactational transfer.

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.
Animals were dosed up to the day prior to scheduled necropsy.

Duration of test:

Males: 29 days
Females: 44-47 days

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 300, 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 selected based on the results of a 28-Day repeated dose toxicity study (NOTOX Project 485789, See 7.5.1 Repeated dose toxicity: oral) in which no toxicity was seen up to 1000 mg/kg.

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, placentas cleaned up, nest built up and/or feeding of pups started). Females that were littering were left undisturbed.

Identification of pups:
On Day 1 of lactation, all pups were randomized per litter and individually identified by means of subcutaneous injection of Indian ink.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS
- Time schedule: At least once daily, detailed clinical observations were made for all animals. The time of onset, grade and duration was recorded. All symptoms were recorded and graded according to fixed scales.

NEUROBEHAVIOURAL EXAMINATION
No

BODY WEIGHT
- Time schedule for examinations: Males and 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.
For one female of Group 4 no body weight and food consumption was determined during the post-coitum period as mating of this female was overlooked. Sufficient data is available to make a thorough assessment.

FOOD CONSUMPTION
Weekly, for males and females. Food consumption was not recorded during the mating period. 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.

FOOD EFFICIENCY
(Average food consumption [per animal per day]/average body weight per cage)x1000

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

GENERAL REPRODUCTION DATA
- Male number paired with, mating date, confirmation of pregnancy, and delivery day was recorded.
- Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of these 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.

HAEMATOLOGY
No

CLINICAL CHEMISTRY
No

URINALYSIS
No

GROSS PATHOLOGY
Animals surviving to scheduled necropsy were deeply anaesthetized using an isoflurane (Abbott B.V., Hoofddorp, The Netherlands) in nitrous oxide/oxygen (Air Products, Amsterdam, The Netherlands) combination and subsequently exsanguinated. Necropsy was conducted on the following days: males: following completion of the mating period (29 days of dose administration), females which delivered: Lactation Days 6-7, females which failed to deliver <1> (1, Group 4): Post-coitum Day 27. The males were deprived of food overnight, but water was provided. This has no effect on the study’s integrity because fasting did not impact the parameters investigated in the study. The females were not deprived of food overnight.

All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded. The number of former implantation sites and corpora lutea were recorded for all paired females.

Samples of the following tissues and organs were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands):
Identification marks (not processed) , Cervix, Clitoral gland, Coagulation gland, Epididymides <2>, Ovaries, Preputial gland, Prostate gland, Seminal vesicles, Testes <2>, Uterus, Vagina, all gross lesions.

<1 > In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites (Salewski staining prepared at NOTOX using Ammoniumsulfide-solution 20% (Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)).
<2> Fixed in modified Davidson's solution (prepared at NOTOX using Formaldehyde 37-40%, Ethanol, Acetic acid (glacial) and Milli-Ro water and transferred to formalin after fixation for at least 24 hours.

ORGAN WEIGHTS
The following organ weights and terminal body weight were recorded from all males on the scheduled day of necropsy:
Epididymides and Testes

HISTOTECHNOLOGY
All organ and tissue samples, as defined under Histopathology (following), were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands). Of the selected 5 males of the control and high dose group, additional slides of the testes were prepared to examine staging of spermatogenesis. The testes were processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin (Klinipath, Duiven, The Netherlands).

HISTOPATHOLOGY
The following slides were examined by a pathologist:
- The ovaries, testes and epididymides of the animals of Groups 1 and 4.
- The additional slides of the testes of the males of Groups 1 and 4 to examine staging of spermatogenesis.
- All gross lesions of all animals (all dose groups).
- The reproductive organs* of one male and one female of Group 4 that failed to sire or deliver healthy pups.
* Reproductive organs included the cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testis, uterus, and vagina.

All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.

Inadvertently, the tail of one male of Group 2 was not available for histopathology because it was erroneously not collected at necropsy. Sufficient data was available for evaluation.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No

Fetal examinations:

The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross abnomalies, weight gain, physical or behavioural abnormalities.
- Mortality: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were made in all animals.
- Body weights: Live pups were weighed on Days 1 and 4 of lactation.
- Sex: Sex was determined for all pups on Days 1 and 4 of lactation (by assessment of the ano-genital distance).

GROSS EXAMINATION OF DEAD PUPS: Yes
If possible, defects or cause of death were evaluated.

Statistics:

- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Dunnett, 1955) (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. (Ref. 1)
- The Steel-test (Miller, 1981) (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution. (Ref. 3)
- The Fisher Exact-test (Fisher, 1950) was applied to frequency data. (Ref. 2)

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values. No statistical analysis was performed on histopathology findings.

References:
Ref. 1 Dunnett C.W., A Multiple Comparison Procedure for Comparing Several Treatments with a Control, J. Amer. Stat. Assoc. 50, 1096-1121 (1955).
Ref. 2 Fisher R.A., Statistical Methods for Research Workers, Oliver and Boyd, Edinburgh (1950).
Ref. 3 Miller R.G., Simultaneous Statistical Inference, Springer Verlag, New York (1981).

Indices:

Reproductive 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

Offspring indices:
- Percentage live males at First Litter Check: Number of live male pups at First Litter Check/Number of live pups at First Litter Check x 100
- 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 Days 0-4 of lactation: Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check x 100
- Viability index (%): Number of live pups on Day 4 of lactation / Number of pups born alive x 100

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
MORTALITY
No mortality occurred during the study period.

CLINICAL SIGNS
No clinical signs of toxicity were noted during the observation period.
Salivation seen after dosing for all animals at 1000 mg/kg during several occasions of the treatment period was considered to be a physiological response rather than a sign of systemic toxicity, considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign may be related to irritancy or taste of the test substance and was not reflective of treatment related toxicity.
Incidental findings seen for control and/or treated animals included rales, alopecia, scabs and/or scales on various body areas, chromodacryorrhoea of the snout or eye, gasping, a nodule noted on the right inguinal region, and a bent tail apex. 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 not considered to be toxicologically relevant.

BODY WEIGHTS
No toxicologically relevant changes in body weights and body weight gain were noted.
Body weight gain was slightly reduced for males at 300 mg/kg on Day 1 of the mating period. Because the difference between controls was slight, and in the absence of a dose response relationship, this was not considered to be toxicologically relevant.

FOOD CONSUMPTION
No toxicologically relevant changes in food consumption (before or after allowance for body weight) were noted.
At 1000 mg/kg, females had an increase in absolute food consumption over Days 11-14 and an increase in relative food consumption Days 11-20 of the post coitum period, respectively. At 100 and 300 mg/kg, relative food consumption was increased from Days 0-4 and Days 4-14 (not always statistically significant at 300 mg/kg) of the post coitum period, respectively. The relative food consumption seen for control females was slightly lower than the historical control data available for this parameter, and in the absence of any effects seen for body weights for treated animals, the higher food consumption was not considered to be toxicologically relevant.

MACROSCOPIC EXAMINATION
Macroscopic observations at necropsy did not reveal any alterations that were considered to be attributable to treatment with RADIA 7838.
Agenesis of the left testis and left epididymis was noted for one animal at 300 mg/kg. As this animal sired viable pups, it was not considered to be toxicologically relevant.
Incidental findings among control and treated animals included alopecia of various body areas, red-brown focus on the left clitoral gland, yellowish soft nodule on the left epididymis, yellowish hard nodule on the epididymal adipose tissue found in the abdominal body cavity, and a bent tail. The incidence of these findings was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. As such, these necropsy findings were therefore not considered to be toxicologically relevant.

ORGAN WEIGHTS
Organ weights and organ to body weight ratios of treated animals were similar to those of control animals.

MICROSCOPIC EXAMINATION
There were no treatment related microscopic findings.
At 1000 mg/kg, there was one male and one female of Group 4 that failed to conceive or sire; there were no macroscopic or histopathological findings attributable to the test item that could account for their infertility.
The spermatogenic staging profiles were normal for all Group 1 and Group 4 males and all microscopic findings recorded were within the normal range of background pathology encountered in Wistar Han rats of this age and strain.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth, and no deficiencies in maternal care were observed

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There was no evidence of teratogenic effects based on the absence of relevant clinical signs and external macroscopic findings.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

REPRODUCTIVE DATA

The mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.

DEVELOPMENTAL DATA

No toxicologically relevant effects on gestation index and duration were observed.

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, and no deficiencies in maternal care were observed.

EARLY POSTNATAL PUP DEVELOPMENT

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.

MORTALITY - PUPS

Three pups of the control group, two pups at 100 mg/kg, two pups at 300 mg/kg and one pup at 1000 mg/kg were found dead or were missing during the first days of lactation. Missing pups were most likely cannibalized. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

CLINICAL SIGNS - PUPS

Incidental clinical symptoms of pups consisted of small size, pale appearance, no milk in the stomach, and cold. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore not considered to be toxicologically relevant.

BODY WEIGHT - PUPS

Body weights of pups were considered to have been unaffected by treatment up to 1000 mg/kg.

MACROSCOPY - PUPS

The absence of milk in the stomach, an incidental finding, was the only macroscopic finding noted for pups that were found dead. The only macroscopic finding among surviving pups was small size; this was incidental in nature. Missing was noted for a single pup in the control group on her scheduled day of necropsy. The pup was present at the last litter check, but went missing by the time the macroscopic examination was conducted for that litter. The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore not considered to be toxicologically relevant.

Applicant's summary and conclusion

Conclusions:

Based on these results, a developmental No Observed Adverse Effect Level (NOAEL) of 1000 mg/kg/day was derived.

Executive summary:

Title

Reproduction/developmental toxicity screening test of RADIA 7838 in rats by oral gavage.

 

Guidelines

The study was based on the following guidelines:

1) Organisation of Economic Co-operation and Development Guidelines (OECD) for testing of Chemicals Guideline 421, Reproduction/Developmental Toxicity Screening Test, July 1995.

2) The United States Environmental Protection Agency (EPA) Health Effects Test Guidelines OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000.

Rationale for dose levels

Based on the results of a 28-Day repeated dose toxicity study (NOTOX Project 485789), the dose levels for this reproduction/developmental toxicity screening test were selected to be 100, 300 and 1000 mg/kg.

 

Study outline

After acclimatization, four groups of ten male and ten female Wistar Han rats were exposed by oral gavage to the test substance at 0, 100, 300 and 1000 mg/kg/day. Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 44-47 days, i.e. during 2 weeks prior to mating, during mating, duringpost-coitum, and during at least 4 days of lactation.

 

Evaluated parameters

The following parameters were evaluated: mortality / viability, clinical signs, body weights, food consumption, reproduction/developmental parameters, observations pups, macroscopy, organ weights, and histopathology. Chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity.

Results/discussion

Accuracy and homogeneity of formulations were demonstrated by analyses.

 

Parental results:

No parental toxicity was observedat any dose level.

 

No toxicologically significant changes were noted in any of the parental parameters investigated in this study (i.e. clinical appearance, body weight, food consumption, macroscopic examination, organ weights, and microscopic examination).

 

Reproductive results:

No reproduction toxicity was observedat any dose level.

 

No treatment related changes were noted in any reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, precoital time, and numbers of corpora lutea and implantation sites).

 

Developmental results:

No developmental toxicity was observedat any dose level.

 

No toxicologically significant changes were noted in any of the developmental parameters investigated in this study (i.e.gestation index and duration, parturition, maternal care and earlypostnatalpup development consisting of mortality, clinical signs, body weight and macroscopy).

 

Conclusion

Treatment with RADIA 7838 by oral gavage in male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg body weight/day revealed no parental toxicity up to 1000 mg/kg body weight/day. No reproduction and developmental toxicity was observed for treatment up to 1000 mg/kg body weight/day.

 

Based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg/day was determined.