Reaction mass of 1,3-diacetoxypropan-2-yl 12-acetoxyoctadecanoate and 2,3-diacetoxypropyl 12-acetoxyoctadecanoate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-08-12 to 2008-10-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study with no deviations and performed in accordance with GLP regulation

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies, 12 NohSan No 8147, 24 November 2000

Deviations:

no

Principles of method if other than guideline:

NA

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

451-530-8

EC Name:

-

Cas Number:

736150-63-3

Molecular formula:

Not applicable

IUPAC Name:

1,3-bis(acetyloxy)propan-2-yl 12-(carboxyoxy)octadecanoate; 2,3-bis(acetyloxy)propyl 12-(acetyloxy)octadecanoate

Details on test material:

- Name of test material (as cited in study report): TS-ED 532
- Lot/batch No.: 4010534806 (item 175540)
- Expiration date of the lot/batch: 2009-12-31

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Sprague-Dawley Crl:CD® (SD) IGS BR strain rats obtained from Charles River (UK) Limited, Margate, Kent
- Age at study initiation: ten to twelve weeks old
- Weight at study initiation: 200 to 300 g
- Fasting period before study: 5 days
- Housing: individually in solid-floor polypropylene cages with stainless steel lids furnished with softwood flakes (Datesand Ltd., Cheshire, UK)
- Diet (e.g. ad libitum): pelleted diet (Rodent PMI 5002 (Certified), BCM IPS Ltd, London, UK) ad libitum (certificate of analysis available in test report)
- Water (e.g. ad libitum): mains drinking water ad libitum (certificate of analysis available in test report)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2 ºC
- Humidity (%): 55 ± 15%
- Air changes (per hr): at least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): low intensity fluorescent lighting was controlled to give twelve hours continuous (07.00-19.00) light and twelve hours darkness (19.01-06.59).

Environmental conditions were continuously monitored by a computerised system, and print-outs of hourly mean temperatures and humidity are included in the study records.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

arachis oil

Remarks:

BP grade

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): Formulations (TS-ED 532 in Arachis oil BP) were prepared weekly.
- Mixing appropriate amounts with (Type of food): NA
- Storage temperature of food: Formulations were stored at approximately +4 ºC in the dark.

VEHICLE
- Justification for use and choice of vehicle (if other than water): TS-ED 532 was prepared at the appropriate concentrations (see below) as a solution in Arachis oil BP. This vehicle is usually used by Harlan Laboratories in this type of study.
- Concentration in vehicle: See below
- Amount of vehicle (if gavage): See below
- Lot/batch no. (if required): No information but usually used by Harlan Laboratories
- Purity: No information but usually used by Harlan Laboratories

Other:
TS-ED 532 was administered once daily, from Day 5 to 19 of gestation, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 ml/kg bw/day of Arachis oil BP. The volume of TS-ED 532 and Arachis oil BP administered to each animal was based on the most recent bodyweight. The following dose level, dose volume and dose concentrations were used:


Treatment Group Dose Level Dose Volume Concentration
Control 0 (mg/kg bw/day) 4 (ml/kg) 0 (mg/ml)
Low 100 (mg/kg bw/day) 4 (ml/kg) 25 (mg/ml)
Intermediate 300 (mg/kg bw/day) 4 (ml/kg) 75 (mg/ml)
High 1000 (mg/kg bw/day) 4 (ml/kg) 250 (mg/ml)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

TS-ED 532 was prepared at the appropriate concentrations as a solution in Arachis oil BP. The stability and homogeneity of TS-ED 532 formulations were determined by Harlan Analytical Laboratory. The nominal concentrations used for stability and homogeneity validation were 25 and 250 mg/ml. Results show ed the formulations to be stable for at least fourteen days. Formulations were therefore prepared weekly and stored at approxiately +4 ºC in the dark.

Samples were taken of each formulation and were analysed for concentration of TS-ED 532. The results indicated that the prepared formulations were within acceptable limits of the nominal concentration. Based on these results, it was concluded that the analytical method was satisfactorily validated in terms of linearity, specificity and accuracy for the purpose of the study.

Details on mating procedure:

- Impregnation procedure: purchased time mated female Sprague-Dawley Crl:CD® (SD) BR strain rats obtained from Charles River (UK) Limited, Margate, Kent. Animals were delivered in two batches containing females prior to Day 3 gestation. The day that positive evidence of mating was observed was designated Day 0 of gestation. On receipt the animals were examined for signs of ill-health or injury.

- Any other deviations from standard protocol: No deviations

Duration of treatment / exposure:

TS-ED 532 was administered daily, from Day 5 to 19 of gestation, by gavage. Control animals were treated in an identical manner with the vehicle (Arachis oil) only.

Frequency of treatment:

TS-ED 532 was administered once daily by gavage.

Duration of test:

From Day 0 to 20 of gestation.

Doses / concentrations

Remarks:

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

No. of animals per sex per dose:

A total of ninety-six time mated female Sprague-Dawley rast were used:
Control (vehicle): 24
250 mg/kg bw/day: 24
500 mg/kg bw/day: 24
1000 mg/kg bw/day: 24

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels (100 , 300 and 1000 mg/kg bw/day) were chosen based on the data from a preliminary study (See section 7.8.2). The oral route was selected as the most appropriate route of exposure, based on the physical properties of TS-ED 532.

- Rationale for animal assignment (if not random): The animals were randomly allocated to treatment groups using a randomisation procedure based on stratified bodyweight to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomised. The animals were uniquely identified within the study by an ear punching system routinely used in Harlan Laboratories.

Examinations

Maternal examinations:

DETAILED CLINICAL OBSERVATIONS: All animals were examined for overt signs of toxicity, ill-health or behavioural change once daily on non-treatment days, immediately before dosing and one and five hours after dosing during the working week of the treatment period. Animals were observed
immediately before dosing and one hour after dosing at weekends and public holidays. All observations were recorded.

BODY WEIGHT: Individual bodyweights were recorded on Day 3 (before the start of treatment) and on Days 5, 6, 7, 8, 11, 14, and 17. Bodyweights were also recorded at terminal kill (Day 20).

FOOD CONSUMPTION: Food consumption was recorded for each surviving individual animal at Day 3, 5, 8, 11, 14, 17 and 20 of gestation.

POST-MORTEM EXAMINATIONS: All females were killed by carbon dioxide asphyxiation followed by cervical dislocation on Day 20 of gestation. All animals were subjected to a full external and internal examination and any macroscopic abnormalities were recorded.

Ovaries and uterine content:

The ovaries and uteri of pregnant females were removed, examined and the following data recorded:

i) Gravid uterus weight
ii) Number of corpora lutea
iii) Number, position and type of intra-uterine implantation
iv) Foetal sex
v) External foetal appearance
vi) Foetal weight
vii) Placental weight

The uteri of any apparently non-pregnant females were immersed in 10% ammonium sulphide to reveal evidence of implantation.

Implantation types were divided into Early Death (No visible distinction between placental/decidual tissue and embryonic tissue), Late Death ( Separate embryonic/foetal and placental tissue visible) and Dead Foetus (A foetus that had died shortly before necropsy, these were included as late deaths for reporting purposes)

All implantations and viable foetuses were numbered according to their intra-uterine position.

Fetal examinations:

- External examinations: All animals were subjected to a full external and internal examination and any macroscopic abnormalities were recorded.
- Soft tissue examinations: Foetuses from each litter were divided into two groups and examined for skeletal alterations and soft tissue alterations.
- Skeletal examinations: Foetuses from each litter were divided into two groups and examined for skeletal alterations and soft tissue alterations.
- Head examinations: Foetuses from each litter were divided into two groups and examined for skeletal alterations and soft tissue alterations.

The foetuses were killed by subcutaneous injection of sodium pentobarbitone. Foetuses from each litter were divided into two groups and examined for skeletal alterations and soft tissue alterations. Alternate foetuses were identified using an indelible marker and placed in Bouin’s fixative. Foetuses were transferred to 90% industrial methylated spirits (IMS) in distilled water and examined for visceral anomalies under a low power binocular microscope. The remaining foetuses were identified using colour coded wires and placed in 70% IMS in distilled water. The foetuses were eviscerated, processed and the skeletons stained with alizqarin red. The foetuses were examined for skeletal development and anomalies. Following examination, foetuses that were examined for skeletal development were placed in 100% glycerol.

Statistics:

Where appropriate group mean values was calculated as follows:
Mean 1: Includes data from all surviving pregnant females including those with total resorption.
Mean 2: Includes data from surviving pregnant females with live foetuses on Day 20 of gestation.

Where appropriate the data was statistically analysed. In-life data was analysed by parametric analysis of variance followed by pairwise comparison where appropriate. Caesarian data were analysed using comparable non-parametric methods.

The following parameters were analysed statistically, where appropriate, using the test methods outlined below.

Female bodyweight change, food consumption and gravid uterus weight: Bartlett’s test for homogeneity of variance and one way analysis of variance, followed by Dunnet’s multiple comparison test or, if unequal variances were observed, on alternative multiple comparison test.

All caesarian necropsy parameters and foetal parameters: Kruskal-Wallis non-parametric analysis of variance; and a subsequent pairwise analysis of control values against treated values using the Mann-Whitney ‘U’ test, where significance was seen.

Foetal evaluation parameters, including skeletal or visceral findings: Kruskal-Wallis nonparametric analysis of variance and Mann-Whitney ‘U’ test.

Probability values (p) are presented as follows:
P<0.001 ***
P<0.01 **
P<0.05 *
p≥0.05 (not significant)

Indices:

Percentage pre-implantation loss was calculated as:
Number of corpora lutea - Number of implantations/ Number of corpora lutea x 100

Percentage post-implantation loss was calculated as:
Number of implantations - Number of live foetuses/Number of implantations x 100

Sex Ratio was calculated for each litter:
Number of males/Number of offspring x 100

Historical control data:

Yes, background control data for Litter Data in the Sprague-Dawley Crl:CD® (SD) IGS BR Strain Rat are included in the test report. The data are collected between July 2006 and June 2008 from 25-45 animals.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
One female treated with 1000 mg/kg bw/day was killed in extremis on Day 17. This death was considered not to be related to treatment but considered to be due to an accident during the dosing procedure.One further female from this treatment group was also terminated early on Day 19 due to the early onset of littering. There were no further unscheduled deaths.

No clinically observable signs of toxicity were detected in terminal kill animals throughout the study period. The female that was killed in extremis on Day 17 showed hunched posture, pilo-erection, fur staining, respiratory pattern changes and a mass under the fore-leg.

No adverse effect on bodyweight development was detected. Statistical analysis did not reveal any significant differences.

No adverse effect on dietary intake was detected. Statistical analysis did not reveal any significant differences.

No treatment-related macroscopic abnormalities were detected in treated females at terminal kill. One control female had red/brown staining around the ano-genital region.

The female treated with 1000 mg/kg bw/day that was killed in extremis on Day 17 had a hole in the oesophagus and a mass present in the upper right thorax. The female that was terminated early on Day 19 showed an enlarged spleen and a distended stomach with red contents.

Effect levels (maternal animals)

1

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOEL

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:
Litter Data and Litter Placental and Foetal Weights:
There was no adverse effect on in-utero offspring survival, as assessed by the mean numbers of early or late resorptions, live litter size and post-implantationlosses. Although the percent of males present in each litter from all treatment groups shows lower mean values compared to controls this is not considered to represent an adverse effect of treatment. The differences in mean values are considered to be within expected biological variation when performing a study with the number of animals present in this study. A chemical effect upon sexual development can be discounted as there is no indication of disruption of the reproductive tract for offspring of either sex. Females from all treatment groups showed an increase in foetus weight when compared with control animals. An increase in bodyweight is not considered of toxicological significance (See Tabel 2).

Foetal Examination:
For all dose groups, there were no significant treatment-related trends in the proportion of foetuses (or litters) with evidence of visceral or skeletal anomalies. The type of visceral and skeletal anomalies were those commonly observed for this type of study. Females treated with 300 and 100 mg/kg bw/day showed a statistically significant reduction in the percent of foetuses showing ovoid eye lens. In the absence of a true dose-related response the intergroup differences were considered attributable to the increased number of control foetuses showing the effect with the exception of the following observations, all intergroup differences in foetal variations were within the range observed amongst background control values. Due to a significant interval between conduct of similar studies within this laboratory the actual number of control foetuses for comparison is lower than normal but reflects a relevant reference point for background data.

Females from all treatment groups showed a statistically significant increase in the number of foetuses showing greater than four ossified sternebrae and greater than six ossified metacarpals. Subsequently these females also showed a statistically significant reduction in the number of foetuses showing four ossified sternebrae and six ossified metacarpals (See Table 3).

The standard number of ossified sternebrae in Day 20 foestuses is greater than four and the standard number of ossified metacarpals in Day 20 foetuses is greater than six. Therefore the intergroup differences indicate a higher number of foetuses showing a normal number of ossified sternebrae and ossified metacarpals compared to control and as such, are considered of no toxicological importance.

Females from all treatment groups showed a statistically significant increase in the percent of foetuses showing bilateral/unilateral wavy 13th rib(s). The finding was seen in isolation and whilst this type of anomaly may well be indicative of toxicity to the foetus, it is apparent that there were no other indicators of such an effect. There is no evidence of a reduction in foetal weight, no reduction in the degree of ossification of skull bones and no decline in the amount of ossification of metatarsals/metacarpals. Direct toxic effects upon the foetus that reflect a true effect of treatment are normally observed as a pattern
or syndrome of effects and therefore the differences in variation values for one parameter are considered not to be reflective of a true effect of treatment. This is also supported by the fact that the proportion of affected foetuses do not follow a true response across all groups (See Table 4).
The biological significance of this finding has been discussed (Nishimura. M. et al (1982). Arzneim-Forsch./Drug Res. 32 (II), Nr 12, 1518-1522
and Hayasaka. I. et al (1985). Cong. Anom 25, 121-127) and the suggestion is that this is a transient effect during gestation that is resolved during early post natal growth and development.

Females treated with 300 mg/kg bw/day also showed a statistically significant reduction in the percent of foetuses showing incomplete ossification of one cranial bone. In the absence of a true dose-related response the intergroup differences were considered of no toxicological importance (data not shown).
Females treated with 1000 mg/kg bw/day also showed a statistically significant increase in the percent of foetuses showing one thoracic vertebral centre bipartite (data not shown). The observation of one variant at a higher incidence compared with controls is not significant when evaluated in isolation.
The number of parameters evaluated makes it highly likely that one finding will be seen at a higher level in dosed groups compared with controls. A true developmental effect is only seen when a number of variants or a syndrome of variance is observed. Therefore this is of no biological significance.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Table 1. Animals were allocated to treatment groups as follows:

Treatment Group	Dose level (mg/kg bw/day)	Dose volume (ml/kg bw)	Concentration (mg/ml)	Animal numbers
Control	0	4	0	24 (1 -24)
Low	100	4	25	25 (25 -48)
Intermediate	300	4	75	24 (49 -72)
High	1000	4	250	24 (72 -96)

Table 2. Litter data - Group mean values.

Dose Level (mg/kg bw/day)	Number of Litter		Number of Corpora Lutea	Number of Implants	Number of Embryonic/Foetal Deaths			Implantation Loss %		Number of Live Implants			% MaleFoetuses	Mean Male Foetal Weight (g)	Mean Female Foetal Weight (g)	Mean Foetal Weight (g)	MeanPlacentalWeight (g)	Litter Weight (g)	TotalPlacentalWeight (g)
					Early	Late	Total	Pre	Post	Male	Female	Total
0 (control)	23/22•	mean	13.8	11.7	0.3	0.1	0.4	18.1	3.8	6.3	5.0	11.3	59.2	3.96	3.76	3.91	0.57	43.48	6.11
		sd	3.0	3.8	0.6	0.3	0.7	19.0	6.0	2.6	2.9	3.7	18.2	0.34	0.25	0.30	0.13	13.15	1.71
100	22/19■	mean	14.2	12.5	0.3	0.0	0.3	13.8	2.2	6.6	5.6	12.2	52.4	*4.24	*4.01	*4.14	0.53	*50.55	6.35
		sd	2.4	2.8	0.6	0.0	0.6	14.1	4.81	2.7	2.2	2.7	18.3	0.31	0.28	0.29	0.05	11.13	1.31
300	22/21♦	mean	12.5	10.9	0.2	0.1	0.3	14.4	2.7	5.2	5.4	10.7	49.0	**4.32	***4.18	***4.25	0.56	44.47	5.78
		sd	3.0	3.6	0.5	0.3	0.6	18.9	4.8	2.4	2.4	3.5	15.2	0.25	0.30	0.24	0.09	14.85	1.97
1000	20	mean	14.5	13.3	0.3	0.0	0.3	8.8	2.1	5.9	7.1	13.0	46.5	*4.23	*3.86	4.15	0.55	**53.72	7.12
		sd	1.9	2.3	0.6	0.0	0.6	10.1	3.9	1.5	2.5	2.2	14.6	0.48	0.41	0.43	0.05	9.12	1.33

•= n=22 for number of corpora lutea and pre-implantation loss

■= n=19 for number of implantations and pre and post implantation losses

♦= n=21 for number of live implants and % male foetuses

sd= standard deviation

*= significantly different from control group p<0.05

**= significantly different from control group p<0.01

***= significantly different from control group p<0.001

Table 3. Foetal skeletal development - Group incidence

		Dose Level (mg/kg bw/day)
		0			100			300			1000
Skeletal Development		Number of Foetuses (litters) Examined
		126 (23)			130 (22)			111 (22)			123 (20)
		NF	NL	%†	NF	NL	%†	NF	NL	%†	NF	NL	%†
Number of ribs
	0/0	0	0	0.0	0	0	0.0	1	1	1.0	0	0	0.0
	13/13	125	23	99.4	128	22	98.5	110	22	99.0	120	20	97.3
	13/14	0	0	0.0	1	1	0.8	0	0	0.0	1	1	1.0
Number of fully ossified sternebrae	<4	0	0	0.0	1	1	0.8	1	1	1.0	0	0	0.0
	4	33	14	30.2	9	5	**6.6	10	9	*11.9	7	6	**5.5
	>4	91	21	68.4	119	122	**91.9	100	21	*87.1	116	20	**94.5
Number of post lumbar vertebral centra	<7	3	2	2.1	3	2	1.9	3	3	2.5	2	2	1.7
	³7	123	23	97.9	0.7	22	98.1	108	22	97.5	121	20	98.3
Number of post lumbar vertebral arches	<5	9	4	6.2	6	3	4.5	4	4	3.5	3	2	1.8
	³5	115	22	92.5	124	22	95.5	107	22	96.5	120	20	98.2
Number of metacarpals	<6	1	1	0.7	0	0	0.0	0	0	0.0	0	0	0.0
	6	85	22	66.8	34	14	***29.9	29	16	**34.9	38	16	**34.8
	>6	40	16	32.5	96	20	***70.1	82	19	**65.1	85	17	**65.2

†= group mean per litter

NF= Number of foetuses

NL= Number of litters

NOTE: afoetus may appear in more than one category

*= significantly different from control group p<0.05

**= significantly different from control group p<0.01

***= significantly different from control group p<0.001

Table 4. Foetal skeletal findings - Group incidence

	Dose Level (mg/kg bw/day)
	0			100			300			1000
	Number of Foetuses (litters) Examined
	126 (23)			130 (22)			111 (22)			123 (20)
	NF	NL	%†	NF	NL	%†	NF	NL	%†	NF	NL	%†
RIBS
k - Bilateral/unilateral wavy 13^thrib(s)	1	1	1.4	12	9	**9.2	9	6	*7.9	13	12	***10.5
l - Bilateral/unilateral wavy rib(s)	4	3	3.0	2	1	1.5	1	1	0.6	1	1	1.0
m - Bilateral/unilateral rudimentary 14^thrib(s)	0	0	0.0	1	1	0.8	0	0	0.0	1	1	1.0
n - 6^th, 7^th, 8^th ribs nodulated (both sides)	1	1	1.1	0	0	0.0	0	0	0.0	0	0	0.0
o - Bilateral/unilateral 13^thrib(s) short	0	0	0.0	4	2	3.0	0	0	0.0	0	0	0.0
p - No ribs present	0	0	0.0	0	0	0.0	1	1	0.9	0	0	0.0

†= group mean per litter

NF= number of foetuses in category

NL= number of litters in category

NOTE: afoetus may appear in more than one category

*= significantly different from control group p<0.05

**= significantly different from control group p<0.01

***= significantly different from control group p<0.001

Applicant's summary and conclusion

Conclusions:

The oral administration (gavage) of TS-ED 532 to pregnant rats during organogenesis at dose levels of 100, 300 and 1000 mg/kg bw/day did not result in any toxicological significant maternal effects at any dose level. The NOEL was therefore, considered to be 1000 mg/kg bw/day. No toxicological significant changes were detected in the offspring parameters measured. The NOEL for developmental toxicity was therefore considered to be 1000 mg/kg bw/day.

Executive summary:

The study was performed according to OECD guideline 414 with no deviations and was designed to investigate the effects of TS-ED 532 on embryonic and foetal development following repeated oral administration by gavage to the pregnant female rats during the period of organogenesis. TS-ED 532 was administered to three groups each of twenty-four time mated Sprague-Dawley Crl:CD® (SD) IGS BR strain rats at dose levels of 100, 300 and 1000 mg/kg bw/day. A further group of twenty-four time mated females was exposed to the vehicle (Arachis oil) only to serve as a control.

No macroscopic abnormalities were detected in treated females at terminal kill and there were no treatment-related effects detected in the uterine parameters examined, in foetal viability or in growth and development. Furthermore, no treatment-related effects were detected on skeletal development or in the type and incidence of skeletal or visceral findings.

The NOEL for maternal and developmental toxicity was established as 1000 mg/kg bw/day in this study, based on no toxicologically significant changes in the maternal and offspring parameters measured at the highest dose tested (1000 mg/kg bw/day).