Reaction mass of sodium 3-{[3-(diethylamino)propyl]carbamoyl}-2-(dodecylamino)propanoate and sodium 3-{[3-(diethylamino)propyl]carbamoyl}-3-(dodecylamino)propanoate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 May 2008 - 10 July 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP study conducted according to OECD Guideline 421 without some deviations: 2 QC samples were non-GLP.

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

targeted dose-level of group 4 was not reported exactly; concentration analysis was performed in Week 5 instead of Week 4

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of sodium 3-{[3-(diethylamino)propyl]carbamoyl}-2-(dodecylamino)propanoate and sodium 3-{[3-(diethylamino)propyl]carbamoyl}-3-(dodecylamino)propanoate

EC Number:

939-579-8

Molecular formula:

Empirical Formula : C11H21N3O3Na C2nH4n+1 (n=4 – 9), C11H21N3O3NaC18H35, C11H21N3O3NaC18H33 Molecular formula of the two main constituents : C23H46N3O3Na

IUPAC Name:

Reaction mass of sodium 3-{[3-(diethylamino)propyl]carbamoyl}-2-(dodecylamino)propanoate and sodium 3-{[3-(diethylamino)propyl]carbamoyl}-3-(dodecylamino)propanoate

Details on test material:

- Name of test material (as cited in study report): CHIMEXANE HB
- Physical state: Yellowish thick gel
- Analytical purity: 53.8 %
- Correction factor: 1.86
- Lot/batch No.: 0137998
- Date of receipt: 21 April 2008
- Expiration date of the lot/batch: April 2009
- Storage condition of test material: Room temperature

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France.
- Age at study initiation: 11 weeks
- Weight at study initiation: Males: 372-450 g (mean 416 g); Females: 246-294 g (mean 264 g)
- Housing: Animals were housed individually in wire-mesh cages (43.0 x 21.5 x 18.0 cm); Towards the end of gestation and during lactation, the females were individually housed, and with their litter after birth, in polycarbonate cages (43.0 x 21.5 x 20.0 cm) containing autoclaved wood shavings (SICSA, Alfortville, France) as nesting material.
- Diet: A04 C powdered maintenance diet (SAFE, Augy, France) distributed weekly, ad libitum
- Water: Tap water (filtered with a 0.22 μm filter), ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 50 ± 20 %
- Air changes: Approximately 12 cycles/h of filtered, non-recycled air
- Photoperiod: 12 h dark / 12 h light

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: dietary admixture, in A04 C powdered maintenance diet

Details on exposure:

DIET PREPARATION
- A premix at 20000 ppm of test item in the diet was prepared and then each concentration was prepared by diluting the premix with the diet. On each day of check of concentration, the premix prepared at 20000 ppm was kept for possible analysis until knowledge of the results.
- Storage temperature of food: Dietary admixtures were prepared on a weekly basis and were stored in closed bags at room temperature and protected from light prior to use.

STABILITY
- Satisfactory homogeneity and stability of dosage forms prepared at 500 or 20000 ppm was demonstrated during the study (Study No. 34831 AHS) over an 8-day storage period in open feeders at room temperature and over a 15-day period in closed bags at room temperature.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 1-14 days
- Proof of pregnancy: Presence of vaginal plug / sperm in vaginal lavage referred to as Day 0 post-coitum (p.c.).
- Pre-coital time was calculated for each female.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Concentration of the test item in samples of each control and test item dietary admixture prepared for use in Weeks 1, 2, 5, 6 and 8 was determined.
- As data of the check of concentrations given during Week 5 was outside the acceptance criteria (one quality control sample was outside the acceptance criteria of ± 10 % compared to the nominal value), it was decided to check the concentration of dietary admixtures prepared in Week 6.
- At the end of the treatment period, a check of concentration was performed on the premix at 20000 ppm.
- Acceptance criterion: Measured concentration = nominal concentration ± 20 %.

Results:
- Test concentrations in the administered dietary admixtures analyzed in Weeks 1, 2 and 8 remained within the acceptable range of -19.0 to -0.1 % of variation compared to the nominal values.
- At the end of the treatment period, the deviation from nominal concentration for the premix prepared at 20000 ppm was -22.4 %.

Duration of treatment / exposure:

Males:
- 2 weeks before mating, during the mating (maximum of 2 weeks) and post-mating periods (at least 2 weeks) until sacrifice (at least 6 weeks in total).

Females:
- 2 weeks before mating, during the mating period (maximum of 2 weeks), during pregnancy and lactation, until Day 4 post-partum inclusive, (or until sacrifice for un-mated and non-pregnant females and females which did not deliver).

Frequency of treatment:

Once daily, 7 days a week

Doses / concentrations

Remarks:

Doses / Concentrations:
Males: 725, 2175 and 6550 ppm; Females: 650, 1950 and 5900 ppm [target dose levels 50, 150 and 450 mg/kg bw/day] of active content
Basis:
nominal in diet

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Dose levels were selected on the basis of the results of 4-week toxicity study (Study No. 31265 TSR) in which the test item was administered by dietary admixture at the target dose-levels of 150, 450 and 1000 mg/kg bw/day.
- Rationale for animal assignment: During the acclimation period, the required number of animals (40 males and 40 females) was selected according to body weight and/or clinical condition. They were then allocated to groups (by sex), using a stratification procedure based on body weight.

Positive control:

Not applicable

Examinations

Parental animals: Observations and examinations:

CAGE SIDE AND CLINICAL OBSERVATIONS: Yes
Time schedule:
- Morbidity and mortality: Once a day during the acclimation period and at least twice a day during the treatment period.
- Clinical signs: Once daily

BODY WEIGHT: Yes
Time schedule for examinations:
- Male: Once before group allocation, on the first day of treatment (Day 1), then once a week until sacrifice.
- Female: Once before group allocation, on the first day of treatment (Day 1), then once a week until mated (or until sacrifice) and on Days 0, 7, 14 and 20 p.c. and Days 1 and 5 post-partum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Time schedule:
- Male: Once a week, over a 7-day period, from the first day of treatment until sacrifice.
- Female: Once a week, over a 7-day period, from the first day of treatment through gestation (Days 0-7, 7-14 and 14-20 p.c. intervals) and lactation (Days 1-5 post-partum interval) until sacrifice.
- During the mating period, the food consumption was noted for neither males nor females.
Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
- Achieved intake of the test item (in terms of mg/kg bw/day) was calculated on a weekly basis for each test item-treated group as follows:
D = C x FC/BW; where, D = achieved dosage (mg/kg bw/day), C = nominal concentration of active test item (ppm: mg test item/10^6mg food), FC = mean food consumption (g/animal/day), BW = mean body weight (g)

PARTURITION:
- Females were allowed to litter normally and rear their progeny until Day 5 post-partum inclusive. Any sign of a difficult or prolonged parturition was recorded. The day of completed parturition was designated Day 1 post-partum. The length of gestation was calculated.

Oestrous cyclicity (parental animals):

Estrous cycle stage was recorded daily until the female was positive for mating.

Sperm parameters (parental animals):

Parameters examined in male animals:
- Testes and epididymis (total and cauda weight) weight were recorded.

Litter observations:

LITTER SIZE
- Total litter size and numbers of pups of each sex were recorded as soon as possible after birth.
- Litters were observed daily for recording of the number of live, dead and cannibalized pups.
- External examination of each pup was carried out and any external malformation was noted.

CLINICAL SIGNS
- Pups were observed daily for clinical signs or abnormal behavior.

BODY WEIGHT
- Weight of each pup was recorded on Days 1 and 5 post-partum.

Postmortem examinations (parental animals):

SACRIFICE: On completion of the treatment period, all animals were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and sacrificed by exsanguination.
- Male animals: All surviving animals were sacrificed after delivery of the majority of the females per group.
- Maternal animals: All surviving animals [on Day 5 post-partum, 24-26 days after the last day of the mating period for the females which did not mate, on or after Day 25 p.c for the females which had not delivered by Day 25 p.c.]

GROSS NECROPSY
- Macroscopic post-mortem examination of the external surfaces, all orifices, cranial cavity, external surfaces of the brain, thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues.
- In all females, the number of implantation sites and corpora lutea was recorded.
- In the females which did not mate or were apparently non-pregnant, the presence of implantation sites on the uterine horns was checked using ammonium sulphide staining technique.

ORGAN WEIGTHS
- Epididymides (total and cauda weight), ovaries (with oviducts), prostate, seminal vesicles (with coagulating glands and their fluids) and testes were weighed.
- Ratio of organ weight to body weight (recorded immediately before sacrifice) was calculated.

HISTOPATHOLOGY
- Microscopic examination was performed on the macroscopic lesions, ovaries, testes and epididymides (with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure) in all males and females of the control and high-dose groups.
- See table 7.8.1/1

Postmortem examinations (offspring):

SACRIFICE
- Pups were sacrificed on Day 5 post-partum, by intraperitoneal injection of thiopental sodium.

GROSS NECROPSY
- Macroscopic examination was performed for all pups, including those found dead.

Statistics:

- Data other than organ weights: Mean values were compared by one-way analysis of variance and the Dunnett test (mean values being considered as normally distributed and variances being considered as homogeneous). Percentage values were compared by the Fisher exact probability test.
- Organ weights: PathData software (version 6.2b5) was used for the statistical analyses of organ weight data with a level of significance of 0.05 or 0.01.

Reproductive indices:

- Pre-implantation loss: [(Number of corpora lutea - Number of implantation sites) / Number of corpora lutea] X 100
- Post-implantation loss: [(Number of implantation sites - Number of live concepti) / Number of implantations] X 100
- Mating index: [Number of mated animals / Number of paired animals] X 100
- Fertility index: [Number of pregnant female partners / Number of mated pairs] X 100
- Gestation index: [Number of females with live born pups / Number of pregnant females] X 100

Offspring viability indices:

- Live birth index: [Number of live born pups / Number of delivered pups] X 100
- Viability index on Day 4 post-partum: [Number of surviving pups on Day 4 post-partum / Number of live born pups] X 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- No mortality and no test item treatment-related clinical signs were noted during the study.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
- At 450 mg/kg bw/day, the mean body weight gain was lower than controls during the first 2 weeks (males; Days 1-8: +23 g vs. + 46 g; Days 8-15: +21 g vs. +29 g; not statistically significant) or the first week (females; +1 g versus +20 g, p<0.001) of the premating period; this was associated with a slightly low mean food consumption [(males; +26 g or +25 g versus +30 g or +29 g) and females (+15 g/animal/day versus +21 g/animal/day in controls, p<0.001)].
- At 450 mg/kg bw/day, the lower overall mean body weight gain in males (Days 1 to 43: +138 g vs. +160 g) and females [during the pregnancy (Days 0-20: +143 g vs. +173 g) and lactation periods (Days 1-5: +13 g vs.+23 g)] were observed.
- At 50 and 150 mg/kg bw/day, mean body weight, body weight gain and food consumption were unaffected by the test item treatment.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
- Mean achieved dosages increased in a nearly dose-proportional manner and mean values were very close to the targeted dose-levels of 50, 150 or 450 mg/kg bw/day.
- See table 7.8.1/2

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
- Estrous cycle was not affected by treatment with the test item at any dose level.
- At 50 mg/kg bw/day, one female which did not mate (spent 9 consecutive days in met-estrus followed by 5 days in diestrus); this was not related to the test item treatment since one control female also spent 9 days in met-estrus before mating.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
- At 450 mg/kg bw/day, low weights correlated to hypospermatogenesis in the testes and oligospermia in the epididymides. In the testes of this rat, there was bilateral minimal, multifocal hypospermatogenesis accompanied by minimal, multifocal vacuolation of the seminiferous tubules and spermatid retention, and by moderate oligospermia and minimal abnormal content (sloughed spermatids) in the epididymides.
- This isolated finding was considered to be spontaneous and unrelated to the test item since a control rat presented similar changes, although unilateral.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Mating data:
- Only one female treated at 50 mg/kg bw/day, did not mate.
- Mean number of days of pairing before mating was 4.4, 3.1, 3.6 and 2.8 days at the dose levels of 0, 50, 150 and 450 mg/kg bw/day, respectively and there was no test item treatment-related effects were noticed.

Fertility data:
- Mating index was 100, 90, 100 and 100 % at the dose-levels of 0, 50, 150 and 450 mg/kg bw/day, respectively. At 50 mg/kg bw/day, 1/10 females did not mate and it was sacrificed after the theoretical end of gestation (i.e. on Day 52).
- Fertility index was 100, 88.9, 90 and 90 % at the dose-levels of 0, 50, 150 and 450 mg/kg bw/day, respectively. 1/9, 1/10 and 1/10 mated females were not pregnant at 50, 150 and 450 mg/kg bw/day, respectively.
- None of these variations were considered to be related to treatment with the test item.

Delivery data:
- Gestation index was similar between the groups.
- Mean duration of gestation was slightly longer in the test item-treated groups (not dose-related) when compared to controls, but this was considered not to be related to the test item treatment since the duration for each female remained within the physiological range (i.e. 21 or 22 days after mating).
- At 50 and 450 mg/kg bw/day, the mean numbers of corpora lutea were lower than in the controls but the values were close and not dose-related, therefore this variation was considered not to be related to treatment with the test item. Mean number of implantation sites per litter was therefore slightly lower than controls as well as the mean number of pups delivered per litter and these effects were not related to the test item treatment.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Males:
- Absolute and/or relative testes and epididymides weights of control male and high-dose male were low but they were both considered to be incidental.
Females:
- Lower absolute ovary weights noted for high-dose females when compared to controls were considered to be a reflection of reduction in body weight and not due to test item-related organ toxicity.
- However, the absolute and relative ovary weights for a single high-dose female were both particularly low (0.156 g and 0.04864 versus mean weights of 0.2174 g and 0.06022 in control females, respectively for absolute and relative weights) and this correlated to a macroscopically reduced size. In the absence of microscopic correlates, these organ weight and gross findings were considered to be of no toxicological significance.

GROSS PATHOLOGY (PARENTAL ANIMALS)
- No macroscopic findings related to the test item treatment were observed.

HISTOPATHOLOGY (PARENTAL ANIMALS)
- No histopathological findings related to the test item treatment were observed.

Effect levels (P0)

Dose descriptor:

NOEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

VIABILITY (OFFSPRING)
- Live birth index was 100 % in each group and viability index on Day 4 post-partum was similar in the control, 50 and 450 mg/kg bw/day groups.
- At 150 mg/kg bw/day, viability index was slightly lower (88 % vs. 96 % in controls) as the number of pups found dead or cannibalized between Days 1 and 4 post-partum was statistically significantly higher than controls (13 vs. 6, p<0.05). In the absence of a dose-relationship, this variation was not attributed to treatment with the test item.

CLINICAL SIGNS (OFFSPRING)
- No test item treatment-related clinical signs were noted in pups.

BODY WEIGHT (OFFSPRING)
- Mean pup body weights and body weight gains were similar to those of the controls throughout the lactation period.

SEX RATIO (OFFSPRING)
- Mean percentage of male pups was between 45.0 and 48.3 % at birth and between 45.7 and 48.4 % on Day 5 post-partum.
- No effects on the sex ratio of the pups were observed at any dose-level.

GROSS PATHOLOGY (OFFSPRING)
- No macroscopic findings were observed in the pups sacrificed on Day 5 post-partum.

Effect levels (F1)

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

Table 7.8.1/2: Achieved dosages

Mean achieved dosages (mg/kg bw/day) in males
Concentration (ppm)	725	2175	6550
Target dose level (mg/kg bw/day)	50	150	450
Pre-mating (Days 1-15)	49.8	140.9	385.7
Comparison with target dose-levels (%)	0	-6	-14
Mean achieved dosages (mg/kg bw/day) in females
Concentration (ppm)	650	1950	5900
Target dose level (mg/kg bw/day)	50	150	450
Pre-mating (Days 1-15)	48.7	139.1	387.5
Gestation (GD 0-20)	49.6	148.8	457.4
Lactation (Days 1 -5 p.p.)	60	214	575
Overall mean (females)	48.8	150.2	433.5
Comparison with target dose-levels (%)	-2	0	-4

 

GD: Gestation Day, p.p.: post-partum

Table 7.8.1/3: Changes in mean body weight gain and body weight (g)

Dose-level (mg/kg bw/day)	0	50	150	450
Mean body weight gain (g) - male
Premating
Days 1 to 8	46	53	60	23
Days 8 to 15	29	35	38	21
Days 1 to 43	160	188	192	138
Mean body weight gain (g) - female
Premating
Days 1 to 8	20	21	17	1 #
Days 8 to 15	9	12	11	13
Days 1 to 15	30	33	28	14*
Pregnancy
Days 7 to 14 p.c.	47	43	40	37*
Days 14 to 20 p.c.	95	74*	85	70*
Days 0 to 20 p.c.	173	156	169	143
Lactation: Days 1 to 5 p.p.	23	23	29	13
Mean body weight (g) - male
Premating
Day 1	416	417	418	411
Day 15	491	505	516	455*
Day 29	537	559	564	510
Day 43	575	605	610	549
Mean body weight (g) - female
Premating
Day 1	266	262	265	262
Day 15	295	295	293	276*
Pregnancy
Day 0 p.c.	309	302	290	275 #
Day 20 p.c.	482	458	459	418 #
Lactation
Day 1 p.p.	352	349	331	322
Day 5 p.p.	375	372	360	335**

 

*: p<0.05; **: p<0.01; #: p<0.001.

Table 7.8.1/4: Delivery data

Dose-level (mg/kg bw/day)	0	50	150	450
Number of pregnant females	10	8	9	9
Number of females delivering	10	8	8	9
Gestation index (%)	100	100	88.9	100
Mean duration of gestation (days)	21.5	22.0**	22.0**	21.9*
Mean number of corpora lutea	16.8	13.5	15.8	14.2
Mean number of implantation sites per litter	16.5	12.9*	15.6	13.0*
Mean pre-implantation loss (%)	1.8	4.2	0.8	7.0

 

*: p<0.05; **: p<0.01.

Table 7.8.1/5: Summary of number of pups and pup survival

Dose-level (mg/kg bw/day)	0	50	150	450
Number of implantation sites	165	103	125	117
Number of pups delivered	149	87	108	105
Mean number of pups delivered per female	14.9	10.9	13.5	11.7
Live birth index (%)	100	100	100	100
Number of live pups on Day 4 post-partum	143	83	95*	105*
Viability index on Day 4 post-partum (%)	96.0	95.4	88.0	100.0
Post-implantation loss (%) (calculated manually)	9.7	15.5	13.6	10.3

 

*: p<0.05

Applicant's summary and conclusion

Conclusions:

Under the test conditions, the No Observed Effect Level (NOEL) of CHIMEXANE HB was considered to be 150 mg/kg bw/day (concentration of 2175 ppm in males and 1950 ppm in females) for parental, embryo-fetal and pup developmental toxicity up to day 5 post-partum in Sprague-Dawley rats.

Executive summary:

In a GLP-compliant reproduction/developmental toxicity screening study performed according to OECD Guideline 421, CHIMEXANE HB was administered by dietary admixture to groups of Sprague-Dawley, Rj Han: SD rats (10/sex/dose) at the dose levels of 0,725, 2175 and 6550 ppm active content for the males and 0, 650, 1950 and 5900 ppm active content for the females (equivalent to target dose-levels of 0, 50, 150 and 450 mg active content/kg bw/day) for 15 days before mating, through the mating and post-mating periods until sacrifice for the males, and through gestation and the beginning of the lactation period (until Day 4 post-partum (p.p.) inclusive) for the females. Clinical signs and mortality were checked daily. Body weight and food consumption were recorded at designated intervals throughout the study, except during the mating period when the food intake was not recorded. After 15 days of dosing, the males and females were paired and the females were allowed to litter normally and rear their progeny until Day 5 p.p. Males were sacrificed approximately 1 week after the end of the mating period, females on Day 5 p.p. The animals were sacrificed and subjected to a complete macroscopic examination and designated organs were weighed and examined microscopically. The pups were observed daily for clinical signs, sexed and weighed on Days 1 and 5 p.p and sacrificed on Day 5 p.p., and subjected for macroscopic examination.

 

No mortality and no clinical signs attributed to treatment with the test item were observed. At 450 mg/kg bw/day, males showed a lower mean body weight gain associated with lower mean food consumption during the first 2 weeks of dosing. The overall mean body weight gain was slightly lower than in controls. Females had a lower mean body weight gain than controls throughout the study and the mean food consumption was slightly low. Body weight and food consumption were considered to have been unaffected at the lower dose-levels. Mating and fertility parameters were not adversely affected by the test item treatment. The test item treatment had no effects on the clinical condition of the pups, body weight or sex ratio. Organ weights did not reveal the test item treatment related effect. Macroscopic and histopathological findings did not reveal any test item treatment-related effect.

 

Under the test conditions, the No Observed Effect Level (NOEL) of CHIMEXANE HB was considered to be 150 mg active content/kg bw/day (concentration of 2175 ppm active content in males and 1950 ppm active content in females) for parental, embryo-fetal and pup developmental toxicity up to day 5 post-partum in Sprague-Dawley rats.