Registration Dossier

Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2010-01-19 to 2010-08-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
other: draft report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650, Combined repeated dose Toxicity study with the Reproduction/ developmental Toxicity screening Test
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): POLYRAM SL
- Chemical Name: 2-Propanol, 1,1'-[[3-[(3aminopropyl)amino]propyl]imino] bis-, N-tallow alkyl derivatives
- Physical state: yellow viscous liquid
- Composition of test material, percentage of components: Fatty amine alkoxylated: 88.2%, Amines, tallow alkyl: 4.8 %, N-(tallow alkyl) triproplylenetetramine: 2.5%, Non amine: 2.5%, water content: 2.1%
- Purity test date: 12/01/2010
- Lot/batch No.: 96102715
- Expiration date of the lot/batch: 15/10/2011
- Storage condition of test material: at room temperature, protected from light

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: 8-9 weeks.
- Weight at study initiation: Females: 162-200 g, (mean: 177.58 g, ± 20%= 35.52 g), Males: 246-303 g, (mean: 276.95 g, ± 20%= 55.39 g)
- Fasting period before study: no
- Housing: individually in IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding, except for mating where 1 male and one female were cohoused
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 55 ± 10 %
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- Except for density (d= 0.910), no correction factor was used when weighing the test item. Based on available stability data (BSL 2010,study n°100107) The test item formulation was prepared on a daily basis: the test item was dispersed in sterile water and formulates were kept under magnetic stirring until the end of daily administration. Homogeneity of the aqueous dispersion was checked by visual inspection.

VEHICLE
- Justification for use and choice of vehicle: the vehicle was chosen due to solubility of the test item.
- Concentration in vehicle: 2.4, 4.8 and 9.6 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no. (if required): 7494 A 191, Provider: Braun
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: until mating occurred or 14 days had elapsed
- Proof of pregnancy: vaginal plug or sperm in vaginal smear . The day of confirmed mating was designed day 0 post-coitum.
- After successful mating each pregnant female was housed individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Principle and validation of method used for chemical analysis of dosage forms:
Analysis of the test item in aqueous formulation samples was performed using LC MS/MS detection. Validation of the method was based on the ICH Q2(R1) guideline adopted in October 1994 and accordingly the following parameters were checked: specificity, precision and accuracy, repeatability, linearity, Sensitivity Evaluation Test (SET) and stability of the test item in working solutions. The validation of the analytical method was conducted in BSL/Study No. 100107 and precise details are documented in this report.

- Determination of test item concentration in dosage forms:
The dosage form samples were assayed using the above validated method. Samples for the nominal concentration verification were taken in study week 1 (first week of pre mating period), 3 (first week of mating), 5 (gestation) and 7 (gestation/lactation)
Acceptance criterion was measured concentration = nominal concentration ± 15%

- Determination of dosage form stability:
Suitability of the proposed preparation process was confirmed by analysis of the stability, as described in BSL/Study No. 100107. Results of this study indicated satisfactory stability of the dosage forms for at least 6 hours at room temperature and 12 weeks at -20°C.
Homogeneity of the test item in the vehicle was analyzed for the low and high dose concentrations.Samples for homogeneity were taken from the top, middle and bottom of the high dose and low dose preparations in study week 1 and 5.



Duration of treatment / exposure:
- Males: 14 days before mating, during the mating period (up to 2 weeks) and until sacrifice (i.e. at least 4 weeks in total)
- Females: 14 days before mating, during the mating period (up to 2 weeks), during pregnancy, during lactation until day 3 post-partum inclusive.
Frequency of treatment:
Once daily
Details on study schedule:
- No F1 parental animals: only one generation mated
- Age at mating of the mated animals in the study: approximately 10 to 13 weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 12, 24, 48 mg/kg bw
Basis:
other: nominal
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on the dose range finding study "BSL 2010b/K1 SS/ Repeated-dose toxicity: oral, 14-day dose range finding study" reported in section 7.5.1 Repeated dose toxicity: oral.
- Rationale for animal assignment: assigned to the dose/control groups based on their body weight
- Rationale for selecting satellite groups: no satellite groups
Positive control:
no

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice per day
- Cage side observations comprised: behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity, including mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before the first exposure, and at least once a week thereafter
- Detailed clinical observations (outside the home cage in a standard arena) comprised: spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoe, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), and piloerection and pupil size. In addition changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypies, difficult or prolonged parturition or bizarre behaviour were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: males and females: at randomisation, on the first day of dosing and weekly thereafter, and at termination
females during gestation: gestation days 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum), and day 4 post-partum

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: On corresponding day of body weight measurement after beginning of the dose administration. Food consumption was not measured during mating period in males and females and post mating period in males.

OTHER:
FUNCTIONAL OBSERVATION BATTERY (FOB) and LABORATORY INVESTIGATIONS were also performed (see section 7.5.1 : BSL 2010a / K1 KS/ Repeated dose toxicity: oral)
Oestrous cyclicity (parental animals):
- When vaginal smear of a particular female was not found sperm positive, actual stage of the estrus cycle on that day was documented.
Sperm parameters (parental animals):
Parameters examined in male parental generation (F0):
epididymis and testis weights
Litter observations:
STANDARDISATION OF LITTERS: Not applicable.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- number and sex of pups,
- postnatal mortality and cannibalized pups,
- presence of gross malformations,
- clinical signs,
- body weight (recorded on days 1 and 4 post-partum)

GROSS EXAMINATION OF DEAD PUPS: yes
Postmortem examinations (parental animals):
SACRIFICE
- Male animals were sacrificed on day 29 and 30 (the latter were used for urinalysis, and measurement of haematological and clinical chemical parameters.
- Females were sacrificed on respective post natal day 4.
Non pregnant females were sacrificed on the respective day 26 after the sperm positive vaginal smear as an evidence of mating.

GROSS NECROPSY
- Gross necropsy consisted of careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents. Special attention was paid to the organs of the reproductive system, stomach and intestine due to the irritant/corrosive properties of the test item.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively (See table 1 in results and discussion free-text for details).
Postmortem examinations (offspring):
SACRIFICE
Surviving pups were sacrificed on day 4 post-partum, without fasting, by an intraperitoneal injection of sodium pentobarbital

Statistics:
Parameters like body weight change and food consumption was calculated for each animal as the difference in weight measured from one week to the next. The relative organ weights were calculated in relation to the body weight (measured at necropsy) and presented as percentage.
For statistical analysis one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test was carried out to reveal any differences between control and test groups. Statistical analysis performed with GraphPad Prism V.x software (p<0.05 was considered as statistical significant).
Reproductive indices:
Pre-implantation loss:
Number of corpora lutea - Number of implantation sites
_____________________________________________ x 100
Number of corpora lutea

Post-implantation loss:
Number of implantation sites - Number of live concepti
_____________________________________________ x 100
Number of implantations

Mating index:
Number of mated animals
_____________________ x 100
Number of paired animals

Fertility index:
Number of pregnant female partners
_______________________________ x 100
Number of mated pairs

Gestation index:
Number of females with live born pups
________________________________ x 100
Number of pregnant females

Live birth index:
Number of live born pups
_____________________ x 100
Number of delivered pups
Offspring viability indices:
- Viability Index: number of live offspring at day 4 / number of live offspring at birth X100

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)

Mortality:
There were no treatment-related deaths but five premature deaths were observed. One female of each dose- group died: on day 14 for the low-dose group, on day 5 for the mid-dose group and on day 8 for the high-dose group. Two males from the mid-dose group died on days 10 and 6 respectively. The macroscopic examinations of the five decedents together with the histopatological findings confirmed that the death was due to misgavaging or regurgitation/aspiration of the test item (see table 2 in remarks on results for details).

Clinical signs:
In the high-dose group, respiratory sounds were observed in 7 out of 10 males generally for 1 to 3 consecutive days although in 1 male it was observed during 7 consecutive days. The number of male rats showing loud breathing increased from 3 to 7 between weeks 1 and 4. In females, respiratory sounds were observed in 8 out of 10 animals including one found dead on day 8 due to aspiration of the test item in the lungs. Respiratory sounds were observed for 1 to 2 days during the premating period in 5 females and throughout gestation in 5 females also.
In the mid-dose group, respiratory sounds were observed in 3 out of 10 males including one found dead on day 10 due to a gavaging error. The 2 remaining animals had respiratory sounds on day 9 and from day 23 until terminal sacrifice respectively. Respiratory sounds were occasionally observed in 1 female of this dose-group on GD 5.
In the Low-dose group, respiratory sounds were occasionally observed in 3 males during week 2 or 3 and in 1 female the day before its death causing by a gavaging error. In the control group, one male had occasional loud breathing during week 3.

Salivation was observed in all females of the high-dose group throughout gestation and during the premating period for 4 of them. In males, the number of animals showing ptyalism increased from 6 to 9 between weeks 1-2 and 4. Salivation was generally observed for 1 to 2 consecutive days in both sexes although in 2 males it was observed during the 7 consecutive days of the week 4.
In the mid-dose group, salivation was noted in 5 males for 1 to 4 days during weeks 3 or 4 and as early as day 9 in 1 of them. 2 females showed salivation throughout gestation and 1 female with successful mating but not pregnant showed salivation in week 2 of treatment.

Piloerection was observed in 9 out of 10 males during week 4 of treatment at high dose and was coupled with excessive salivation in 6 of them. Piloerection was also observed in week 1 for 1 male. In females, piloerection was noted in 4 out of 10 animals including one found dead on day 8 due to aspiration of the test item in the lungs. In the surviving animals, 2 had piloerection coupled with salivation during week 2 of gestation and 1 had only piloerection on PND1. In the mid-dose group, piloerection was noted in one male during the last week of treatment (week 4). (See table 4 in remarks on results).

BODY WEIGHT AND WEIGHT GAIN (PARENTAL ANIMALS)

No test item-related effects on body weight and body weight change in any dose group were observed.

FOOD CONSUMPTION (PARENTAL ANIMALS)

In males, statistical analysis of the food consumption data revealed no significant effect in treated groups when compared with controls.
In females, no effect on food consumption was observed in treated groups during premating and gestation period as compared to controls. However, a statistically significant decrease in food consumption in the high-dose group was observed during post natal days 0-4 when compared with controls (- 17.4% compared to controls). This statistically significant and dose dependent effect on food consumption could be considered as a treatment related effect.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)

- no effects

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- No treatment related effect was observed on precoital interval, duration of gestation when compared with controls and values were comparable between the groups. All pregnancies resulted in normal births.
- All males and females mated: the copulation index was 100% whatever the dose administered.
- Reduced fertility index was observed in Low (88.89%) and mid-dose groups (77.78%) as compared to control (100%).
One female in the low dose group was not pregnant and another was found dead on day 14 due to a gavaging error. In the mid-dose group, 2 females were found not pregnant but histopathological examinations revealed in one of them a regressing placentae that might have hint a pregnancy with a low number of pups and cannibalism before cage-side observation. In the high dose group, the fertility index was 100%.
(see table 3 in remarks on results for details).
- The mean number of corpora lutea and the mean number of implantations were both comparable with the controls for all groups. The number of Live pups Born on PND 0, the percentage of Pre and post Implantation Loss were similar to controls.

ORGAN WEIGHTS (PARENTAL ANIMALS)


There were no treatment-related effects on the absolute and relative organ weights.

GROSS PATHOLOGY (PARENTAL ANIMALS)


There were no treatment-related macroscopic findings.

HISTOPATHOLOGY (PARENTAL ANIMALS)

Test item-related histopathological changes were observed in the small intestine (duodenum, jejunum and ileum) and in the mesenteric lymph node:

In the duodenum, villous infiltration with histiocytes graded as minimal were observed in 3 out of 5 males of the high-dose group. No particular findings were found in the duodenum of females and mid-dose and low-dose males.
In the jejunum, villous infiltration with histiocytes generally graded as minimal or mild were observed in all males and all but one females of the high-dose group. No particular findings were found in the jejunum of the females treated with the mid-dose or the low-dose whereas 1 male in each of these dose groups showed a minimal infiltration with histiocytes.
In the ileum, villous infiltration with histiocytes graded as minimal to moderate were seen in all males and females of the high and mid-doses groups. 1 female and 2 males from the low-dose group showed a minimal or mild infiltration with histiocytes.
In the small intestine, histiocytic infiltration were observed with a dose-related trend at all dose-levels in the ileum of males and females and from the mid-dose level in the jejunum of males. In the absence of any degenerative changes, this observation was considered to be non adverse.

In the mesenteric lymph nodes, infiltration with histiocytes were observed in all males whatever the dose and in all females of the mid and high-doses groups and in 2 out 5 females of the low-dose group. The grade of the histiocytic infiltration increased in a dose-dependent manner in both sexes being minimal in 5 animals and mild in 2 animals of the low-dose level and mild in 2 animals and moderate in 8 animals of the high-dose level. In the absence of degenerative changes, this observation was considered to be non adverse.


OTHER FINDINGS (PARENTAL ANIMALS)
- Results based on other examinations (hematology, clinical chemistry, urinalysis, histology of all organs) are reported in section 7.5.1 "Repeated dose toxicity: oral"

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
24 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Changes in haematological parameters and decrease in food consumption during the post –natal period in the next higher dose (ie 48 mg/kg bw/day)
Dose descriptor:
NOEL
Remarks:
reproductive performance (mating and fertility)
Effect level:
48 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No relevant effect at maximal tested dose-level

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
- slightly reduced viablity index in mid-dose (98.81%) and high-dose (98.15%)
- 1 pup in the mid-dose group and 2 pups in the high-dose group (coming each one from 2 different litters) missing on post-natal days 1 and 3, probably cannibalized
- results deemed within normal biological range
- see table 3 for results

CLINICAL SIGNS (OFFSPRING)
there were no particular clinical signs.

BODY WEIGHT (OFFSPRING)
- slightly reduced pup mean body weight at birth in the high dose group, completely recouped on post-natal day 4, therefore deemed non-adverse
-see table 5 for details


GROSS PATHOLOGY (OFFSPRING)
No test item treatment-related gross external abnormalities were observed on pups from any groups.



OTHER FINDINGS (OFFSPRING)
there were no effects on the sex ratio

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
toxic effects on progeny
Generation:
F1
Effect level:
48 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Slightly reduced pup mean body weight at birth in the high dose group, completely recouped on post-natal day 4.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 2: Mortality Table

Group

Treatment period

Male

Female

C (0 mg/kg)

0/10

0/10

LD (12 mg/kg)

0/10

1/10

MD (24 mg/kg)

2/10

1/10

HD (48 mg/kg)

0/10

1/10

Animals affected / total number of animals

Table 3: Reproductive Indices

Index

Group

 

C
(0
mg/kg)

LD
(12
mg/kg)

MD
(24
mg/kg)

HD
(48
mg/kg)

Copulation Index

(%)

100

100

100

100

Fertility Index

(%)

100

88.89

77.78

100

Delivery Index

(%)

100

100

100

100

Viability Index

Mean

100.00

100.00

98.81

98.15

SD

0.00

0.00

3.15

3.77

N

10

8

7

9

Copulation Index (%)= (No. of rats copulated /No.of pairs)X 100

Fertility Index (%)= (No. of Females Pregant/No.of females copulated)x 100 Delivery Index(%)= (No. of dams with live newborns/ No.of pregnant dams)X 100

Viability Index (%)= (No. of live offspring at day 4/ No.of live offspring at birth)x 100 No. of live offspring at day 4/ No.of live offspring at birth)x 100

Table 4: Pre and Postnatal Data

Parameter

Group

 

C
(0
mg/kg)

LD
(12
mg/kg)

MD
(24
mg/kg)

HD
(48
mg/kg)

Corpora Lutea

Mean

13.20

13.25

13.00

12.89

SD

1.03

1.16

1.63

1.76

N

10

8

7

9

Implantation Sites

Mean

12.70

12.88

12.71

12.67

SD

0.82

1.13

1.11

1.41

N

10

8

7

9

Live Pups Born on PND 0

Mean

12.40

12.75

11.57

11.89

SD

1.51

1.39

0.98

1.90

N

10

8

7

9

% Pre Implantation Loss

Mean

2.00

2.68

1.79

1.39

SD

6.32

5.31

4.72

4.17

N

10

8

7

9

% Post Implantation Loss

Mean

2.31

1.14

8.85

6.38

SD

10.72

3.21

5.09

8.14

N

10

8

7

9

Table 5: Litter Data

Parameter

Group

 

C
(0
mg/kg)

LD
(12
mg/kg)

MD
(24
mg/kg)

HD
(48
mg/kg)

PND 0

Total No. of Pups Born

Mean

12.40

13.00

12.00

12.22

SD

1.51

1.07

1.53

1.48

N

10

8

7

9

No. of Male

Mean

6.90

6.50

5.29

5.78

SD

1.73

1.51

3.45

1.48

N

10

8

7

9

No. of Female

Mean

5.50

6.50

6.71

6.33

SD

1.43

1.69

2.63

1.73

N

10

8

7

9

Sex Ratio (m/f)

Mean

1.38

1.09

1.07

1.02

SD

0.59

0.43

0.91

0.52

N

10

8

7

9

Live Pups

Mean

12.40

12.75

11.57

11.89

SD

1.51

1.39

0.98

1.90

N

10

8

7

9

Still Birth

Mean

0.00

0.25

0.43

0.33

SD

0.00

0.46

0.79

0.71

N

10

8

7

9

Runt

Mean

0.00

0.00

0.00

0.00

SD

0.00

0.00

0.00

0.00

N

10

8

7

9

PND 4

No. of Male

Mean

6.90

6.25

5.00

5.44

SD

1.73

1.49

3.06

1.33

N

10

8

7

9

No. of Female

Mean

5.50

6.50

6.43

6.22

SD

1.43

1.69

2.94

1.79

N

10

8

7

9

Total No. of Live Pups on
PND 4

Mean

12.40

12.75

11.43

11.67

SD

1.51

1.39

0.98

1.87

N

10

8

7

9

Sex Ratio (m/f)

Mean

1.38

1.04

1.16

0.97

SD

0.59

0.39

1.02

0.45

N

10

8

7

9

Applicant's summary and conclusion

Conclusions:
Based on the data generated from this study the parental No Observed Adverse Effect Level (NOAEL) was considered to be 24 mg/kg/day.
The No Observed Effect Level (NOEL) for reproductive performance (mating and fertility) was considered to be 48 mg/kg/day.
The No Observed Adverse Effect Level (NOAEL) for toxic effects on progeny was 48 mg/kg/day since the lower body weight at birth at this dose-level was only slight and completely recouped on post-natal day 4.
Executive summary:

The objective of this study was to evaluate the potential toxic effects of the test item following daily oral administration (gavage) to male and female rats from before mating, through mating and, for the females, through gestation until day 3 post-partum.

This study provides initial information on possible toxicological effects likely to arise from repeated exposure over a relatively limited period of time and on male and female reproductive performance, such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

The study was conducted according to OECD Guideline 422 and EPA guideline OPPTS 870.3650 and was in compliance with the principles of Good Laboratory Practice regulations.

Three groups of 10 males and 10 females Wistar rats received the test substance daily, by gavage, before mating and through mating and, for the females, through gestation until day 3post-partum.The dose-levels were 12, 24 or 48 mg/kg bw/day. Another group of 10 males and 10 females received the vehicle, sterile water, alone, under the same experimental conditions and acted as a control group. The dosing volume was 5 mL/kg bw/day.

Animals were examined daily for clinical signs and mortality and detailed clinical observations were performed once before the beginning of the treatment period and then once a week until the end of the study. Body weight was measured weekly, food consumption also except during the mating period. A Functional Observation Battery includingsensory reactivity to different stimuli, grip strength, motor activity assessments and other behaviour observations was performed in the week before treatment and at the end of the study. Blood and urine samples were taken for analysis of hematology, blood biochemistry and urine parameters at the end of the study from randomly selected five males and five females (except urine) from each group.

The animals were paired for mating after 2 weeks of treatment and the females were allowed to litter and rear their progeny until day 4 post-partum. The total litter sizes and numbers of pups of each sex were recorded after birth. Pups were observed daily for clinical signs and pup body weights were recorded on days 1 and 4 post-partum.

Males were sacrificedafter completion of the mating periodon treatment days 29 and 30and the females along with their pups were sacrificed onpost natal day 4. Non delivered females were sacrificed on the respective day 26 after the sperm positive vaginal smear as an evidence of mating.Dams were subjected to a complete macroscopic examination with particular attention paid to the reproductive organs. Designated organs were weighed and examined microscopically.

Pups sacrificed on postnatalday 4 and those found dead, were carefully examined for gross external abnormalities.

There were no treatment related deaths but 5 premature deaths (1 female, 1 female and 2 males and 1 female from the low, mid and high-dose groups respectively) were observed.The macroscopic examinations of the five decedents together with the histopatological findings confirmed that the death was due to misgavaging orregurgitation/aspiration of the test item.

Salivation was observed in all males and females of the high-dose group and in 5 males and 3 females of the mid-dose group. In addition, most of the animals treated at the highest dose had piloerection and respiratory sounds. In the mid-dose group, only 1 male had piloerection while respiratory sounds were noted in 3 males and 1 female respectively. In the low-dose group, respiratory sounds were occasionally observed in few animals as well as in one animal of the control group. No other clinical signs were observed at 12 mg/kg/day.

No test item related effects on body weight and body weight change wereobserved. In males, statistical analysis of the food consumption data revealed no significant effect in treated groups when compared with controls. In females, no effect on food consumption was observed in treated groups during premating and gestation period as compared to controls. However, statistically significant decrease in food consumption in the high-dose group was observed during post natal days 0-4 when compared with controls. There were no effects in females food consumption at 24 or 12 mg/kg/day.

There were no effects on mating, fertility or delivery at any dose-level. There were no increases in pup mortality in the test item-treated groups and there were no relevant clinical signs or gross abnormalities in the pups. Pup mean body weight at birth was slightly lower in the high-dose group when compared with controls.

However, the gain in body weight was recouped during the post natal period as the mean body weight of these pups on post-natal day 4 was in the normal biological range.

In the female and male reproductive organs, no histopathological lesions were found in treated groups which were considered to be test item related. However, test item-related histopathological changes were observed in small intestine and the mesenteric lymph node in all treated groups. Both males and females had infiltration with histiocytes that probably contained test-item lipid complexes which are poorly degraded by lysosomal enzymes. As no degenerative changes were associated, this effect was therefore considered to be non-adverse.

Based on the data generated from this study:

A No Observed Effect Level (NOEL) could not be established for the parental toxicity due to histopathological findings in small intestine and mesenteric lymph nodes in all treated groups. As no degenerative changes were associated, these findings were therefore considered to be non-adverse and the No Observed Adverse Effect Level (NOAEL) was considered to be 24 mg/kg/day.

The No Observed Effect Level (NOEL) for reproductive performance (mating and fertility) was considered to be 48 mg/kg/day.

The No Observed Adverse Effect Level (NOAEL) for toxic effects on progeny was 48 mg/kg/day since the lower body weight at birth at this dose-level was only slight and completely recouped on post-natal day 4.