N-[3-(dimethylamino)propyl]-N,N',N'-trimethylpropane-1,3-diamine

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

other: OECD422

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jan-June 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was performed according to OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test” (adopted 22 March 1996). The study was conducted in compliance with OECD Principles of Good Laboratory Practice, as revised in 1997 and adopted November 26th, 1997 by decision of the OECD Council [C(97)186/Final]. A seperate dose range finding study (DRF) was conducted to confirm the doses for the main study. Study procedures were periodically inspected with the exception of solubility test and dose range finding study. The study plan and report were audited by the Quality Assurance. Overall, the study is considered a high-quality, a performed to guidelines, and producing interpretable results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N-[3-(dimethylamino)propyl]-N,N',N'-trimethylpropane-1,3-diamine

EC Number:

223-362-3

EC Name:

N-[3-(dimethylamino)propyl]-N,N',N'-trimethylpropane-1,3-diamine

Cas Number:

3855-32-1

Molecular formula:

C11H27N3

IUPAC Name:

(3-{[3-(dimethylamino)propyl](methyl)amino}propyl)dimethylamine

Test material form:

gas under pressure: refrigerated liquefied gas

Specific details on test material used for the study:

Name of the Test Item: POLYCAT® 77 Catalyst
Chemical Name: N-[3-dimethylamino)propyl]-N,N,N'-trimethylpropane-1,3 diamine
CAS No: 3855-32-1
Description: Clear Colourless liquid
Purity (By GC): 96.9%
Batch No.: 1226246
Manufacture Date :October 27, 2011
Expiry Date: October 27, 2014
Stability of test item: Stable

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Total number of animals used: 120 (60 male, 60 Female)
Age on first treatment: 9-10 weeks
Body weight when treated: For Allocation A and B Male : 210.7 to 239.6 g Female : 182.1 to 197.1 g (Nulliparous and Non-pregnant (Weight variation was within the 20% of mean body weight of each sex))
Identification: By unique cage number and individual animal numbers marked with indelible marker pen on the base of the tail. The animals were marked before the start of test item administration and weekly thereafter. The animals were marked with the temporary animal numbers on the tip of the tail at start of acclimatization.
Acclimatization: 7 and 8 Days for step I and II animals of Allocation A and B under laboratory conditions, after veterinary examination. Only animals without any visible signs of illness were used for the study


Animals were selected and/or grouped based on stratified randomization by using body weights taken before treatment. Computerized statistical programme was used for randomization.
Daily dose levels:
Group 1: 0 mg/kg body weight
Group 2: 50 mg/kg body weight
Group 3: 100 mg/kg body weight
Group 4: 200 mg/kg body weight
Group 1S: 0mg/kg body weight
Group 4S: 200 mg/kg body weight

The animal room was air-conditioned with adequate air changes per hour (at least 10 air changes). The experimental room was continuously monitored for temperature and relative humidity. The ranges for room temperature and relative humidity were 20.7°C to 22.8°C and 52 to 65%, respectively. The animals were provided with a light cycle of 12 hours light and 12 hours dark. Initially (acclimatization and randomization period), all animals were housed in groups of two/ three in polycarbonate cages (approximate internal dimensions of 365 mm x 202 mm x 180 mm height) with corn cob bedding. After randomization, males and females were housed individually. During the mating phase, animals were housed on one male: one female basis within each dose group. After successful mating, the females were returned to their original cages and housed individually during gestation and lactation. Results of analyses for contaminants of corn cob will be archived at RCC Laboratories India Pvt. Ltd.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Daily dose levels
Group 1 : 0 mg/kg body weight
Group 2 : 50 mg/kg body weight
Group 3 : 100 mg/kg body weight
Group 4 : 200 mg/kg body weight
Group 1S : 0 mg/kg body weight
Group 4S : 200 mg/kg body weight

Details on mating procedure:

Animals were paired on a one male: one female basis within each dose group, for a maximum period of fourteen Days. Each female was examined for the presence of a copulation plug in the vagina. The presence of sperm in the vaginal smear was taken as positive evidence of mating (Day 0 of gestation).
Each pregnant female was observed twice a Day around the period of expected parturition (Gestation Day 19 – Day 23).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the dose formulations were taken immediately after dilution of the concentrates with the diluent (vehicle) on treatment start date (Day 1 – Premating i.e., 19/02/2013) and towards treatment end date (After first dam sacrifice i.e., 03/04/2013) for homogeneity (mean of homogeneity were given as dose concentration) analysis. On week 5, samples of all dose formulations was analysed for dose concentrations by analysing triplicate samples.

Duration of treatment / exposure:

Up to 42 days for males, up to two weeks premating phase, two weeks pairing, three weeks gestation and early lactation for females, at dose levels of 50, 100 and 200 mg/kg/day.

Frequency of treatment:

Allocation A (Males were administered with test item daily up to 42 Days; Females were administered with test item daily during premating, mating, gestation periods and up to Day 4 post partum)

Allocation B (Satellite groups was dosed continuously without mating and dosing was stopped on first schedule sacrifice of dam)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

50 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

200 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

0 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Group1-Control: 10 males, 10 females
Group 2-Low dose: 10 males, 10 females
Group 3-Intermediate dose: 10 males, 10 females
Group 4 - High dose: 10 males, 10 females

Control animals:

yes

Details on study design:

Ten males and ten females were allocated to satellite group Group-1S, Group-4S, to represent recovery groups for control (0 mg/kg bw/Day), high dose (test item at 200 mg/kg bw/Day) respectively. Satellite group animals were treated along with main group (Allocation A) animals and dosing was stopped on the Day of first dam sacrifice. Satellite group animals were observed for a further 14-day recovery period.

Positive control:

None.

Examinations

Parental animals: Observations and examinations:

Recorded Observations:

Twice daily - Viability / Mortality
Clinical Signs : Daily cage-side clinical observations

Clinical Signs:
- during Acclimatization Period (daily)
- during Treatment Period: Twice daily on initial 3 Days after treatment; once daily thereafter
- during Recovery Period: Once daily

Feed Consumption during Treatment Period:
Feed weights were recorded weekly for males until termination (Day 8, Day 15, Day 22, Day 29, Day 36 and Day 42)
Females: feed weights recorded weekly at the following Days
Premating- Day 8 and Day 15
Gestation - Day 7, Day 14, Day 20
Lactation - Day 4

Body weights
- Males once weekly
- Females:
during Premating - Day 1, Day 8 and Day 15
during Gestation - Day 0, Day 7, Day 14 and Day 20
during Lactat0ion Day 1, Day 4

During Recovery Period, all observations recorded once Weekly.

Oestrous cyclicity (parental animals):

Not recorded.

Sperm parameters (parental animals):

The presence of sperm in the vaginal smear was taken as positive evidence of mating (Day 0 of gestation)

Litter observations:

For each litter the following were recorded:
- Number of offspring born
- Number of offspring alive were recorded daily and reported on Days 1 and 4 postpartum
- Sex of offspring on Days 1 post partum
- Clinical condition of offspring from birth to Day 4 post partum
- Individual offspring body weights on Days 1 and 4 post partum

Postmortem examinations (parental animals):

The male animals were sacrificed on Day 43 and female animals were sacrificed Day 5 post partum. All the animals were subjected to gross examination.

The uteri of females were examined for the presence and number of implantation sites and the number of corpora lutea in the ovaries were determined.

All animals were weighed and sent to the necropsy. Descriptions of all macroscopic abnormalities were recorded. All surviving animals were sacrificed under carbon dioxide asphyxiation.

The following organs were weighed:
Testes and epididymides of all adult males, ovaries of all females.

The following organ weights were recorded on the scheduled dates of necropsy for five males and five females selected for repeated dose toxicity screening and all the animals of satellite group. Wet weight was taken as soon as possible after dissection to avoid drying.
Thymus
Liver
Spleeen
Kidney
Brain
Adrenals
Heart

Samples of the following tissues and organs weer taken at necropsy and fixed in 10% Neutral Buffered Formalin solution (NBF) except testes and epididymides were fixed in modified Davidson’s fixative for 24 hours and transferred to NBF:

Adrenal glands
Aorta
Bone marrow (Sternum)
Brain (3 levels)
Cecum
Colon
Duodenum
Epididymides
Heart
Ileum, with Peyer’s patches
Jejunum
Kidneys
Liver
Lungs (inflated with NBF at necropsy)
Lymph nodes (mesenteric, axillary)
Oesophagus
Ovaries
Pancreas
Rectum
Skeletal muscle
Spinal cord (cervical, mid thoracic, lumbar)
Spleen
Stomach
Testes
Thymus
Thyroid
Trachea
Urinary bladder (inflated with NBF at necropsy)
Uterus
All Gross lesions

Postmortem examinations (offspring):

Dead pups were subjected to gross examination and live pups were sacrificed on Day 4 post partum and gross examination was performed.

Statistics:

Statistical methods were used to analyze the Body weight, feed consumption, hematological, biochemical parameters and organ weight data, Pre-coital interval, gestation length, litter size and litter weights, sex ratio, corpora lutea and implantation sites, Implantation losses, viability indices, offspring body weight and body weight change.

Reproductive indices:

The follloing were observed
-Mating performance and fertility (Pre-coital interval, mating index and pregnancy index)
-Gestation and parturition Data (Gestation length and parturition index)
- Litter Responses (Pre implantation loss, Live/birth index, Sex ratio)

Offspring viability indices:

Number of offspring alive on Day 4/ Number of offspring alive on Day 1 x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No clinical signs were observed in the control group (0 mg/kg bodyweight) and low dose group (50 mg/kg body weight) male and female animals.

In males, piloerection was observed in the intermediate dose group (100 mg/kg body weight) from treatment Day 38 to Day 42. Piloerection and dullness was observed in the high dose group (200 mg/kg body weight) on treatment Day 14 and continued to exhibit the signs till the last observation period (Day 42).
In females, piloerection was observed in the intermediate dose group (100 mg/kg body weight) towards end of gestation and lactation period. In addition, cannibalism (eating its own pups on Day I observation) and no lactation was noted in one animal.
In high dose group (200 mg/kg bodyweight), piloerection was observed in all the animals at the end of premating period and throughout the gestation and lactation periods, in addition dullness was observed during gestation and lactation periods in animal number 74 & 79. In lactation phase one animal from high dose showed cannibalism (eating its own pups).
The high dose satellite group also showed piloerection in all the animals. In addition dullness was observed in two animals (animal number 111 and 118) during the treatment and recovery period.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no mortalities observed in the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No significance in the body weight and body weight gain (%) were observed in low dose group animals of both sexes when compared with control group.

In males, intermediate dose group showed decreased body weight and body weight gain (%) when compared with control group on few occasions. However, no significant changes in the body weight and body weight gain (%) were observed in the female animals of intermediate dose group when compared with control group.
In males, the body weights and body weight gain (%) were significantly decreased in the high dose group when compared with control group animals. The same was observed in the high dose satellite group (recovery group) when compared with respective satellite control group.

In females, the body weights and body weight gain (%) were significantly decreased in the high dose group when compared with control group animals at premating, gestation and lactation periods. The high dose satellite group (recovery group) females also showed decreased body weight and body weight gain (%) when compared with respective satellite control group.

The body weight and body weight gain (%) was significantly decreased in high dose animals of both sexes and intermediate dose group male animals when compared with control group and this effect was not reversed in the satellite group animals and hence these are considered to be treatment related.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

No significance was observed in the feed consumption in low dose group animals of both sexes when compared with control group.

Intermediate dose group male and female animals showed decreased feed consumption when compared with control group on few occasions. No significant changes in the feed consumption were observed in the female animals of the intermediate dose group when compared with control group during the gestation and lactation periods.
In males, the feed consumption was significantly decreased in the high dose group when compared with control group animals. Similarly, decreased feed consumption was observed in the high dose satellite group (recovery group) when compared with respective satellite control group during the treatment and recovery period.
In females, the feed consumption was significantly decreased in the high dose group when compared with control group animals during premating and gestation periods. No significant variation in the feed consumption was observed during the lactation period. The high dose satellite group (recovery group) also showed decreased feed consumption when compared with respective satellite control group during the treatment and recovery period.

This change of feed consumption in high dose group male and female animals can be considered as treatment effect. Since, decrease of body weight and body weight gain (%) is correlated with decreased feed consumption in high dose group animals.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No treatment related significance was observed in any of the haematology parameters.

However, activated partial thromboplastin time was significantly decreased in low and high dose males when compared with control group. Since the change was not dose dependent and was not observed in female animals of any group, it is considered to be not attributable to the test item.
Significant increase in the platelet count, decrease in the RBC and eosinophil count of male high dose satellite group was observed when compared to respective control group and was not considered treatment related since the values lies in the normal biological range.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No treatment related significance was observed in any of the biochemistry parameters.

Creatinine levels decreased in all treated males when compared with control group animals.
In females, sodium levels were increased in high dose group when compared with intermediate and decreased in intermediate when compared with low dose group.
Alkaline phosphatase, alanine transaminase, triglycerides and sodium levels were decreased in the high dose satellite group females when compared with respective control.
The significance of these differences can't be attributed to treatment due to marginal increase or decrease in individual control values.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Treatment with test item had no effect on urinary parameters in both the sexes with an exception of decreased epithelial cells in low dose group when compared with control and increased epithelial cells in intermediate dose group when compared with low dose group in males. In the high dose satellite group, urinary volume increased in males and decreased in females when compared with respective control. In addition, decreased leukocyte count in female animals were observed. However, this significance is solely of individual variation not to represent any biological significance.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Moderate centrilobular vacuolation, vacuolation inkidney, bladder, arteries, parietal layer of stomach, testes. Apoptotic necrosis in thymus.

No abnormality was observed attributable to the test item at low dose group (G2).

Microscopic examination of high dose (G4) and intermediate dose (G3) group animals revealed test item related minimal to moderate centrilobular and/or arterial vacuolation in liver; minimal to moderate arterial vacuolation and/or minimal to mild vacuolation of glomerular tuft in kidneys, minimal to mild arterial vacuolation in urinary bladder; minimal to marked vacuolation of arteries, trabeculae, red and pulp in spleen; minimal to mild arterial vacuolation in thymus; minimal to moderate arterial and/or cortical vacuolation in axillary and/or mesenteric lymph nodes; minimal to moderate vacuolation of arteries, bronchiolar epithelium and/or muscularis in lungs; minimal to moderate epithelial vacuolation in trachea; minimal to moderate vacuolation of vasavasorum, aorta and/or pulmonary artery in heart; minimal to moderate arterial and/or exocrine vacuolation in pancreas, mild to moderate vacuolation of tunica muscularis, arterial and/or parietal layer of stomach; minimal to mild arterial vacuolation in skeletal muscle; minimal to marked vacuolation and necrosis of choroid plexus in brain in both sexes, minimal to mild arterial vacuolation in ovaries of female animals.

Microscopic examination of high dose satellite (G4S) group animals revealed test item related minimal to moderate centrilobular vacuolation, necrosis and/or fibrosis in liver; minimal to mild arterial vacuolation and/or minimal to mild vacuolation of glomerular tuft in kidneys; minimal to moderate arterial vacuolation and/or epithelial vacuolation in urinary bladder; minimal to marked vacuolation of arteries, trabeculae, red and/or white pulp in spleen; minimal to mild arterial vacuolation and/or increased incidence of apoptotic necrosis in thymus; minimal to moderate arterial and/or cortical vacuolation in axillary and/or mesenteric lymph nodes; mild to marked vacuolation of arteries, bronchiolar epithelium and/or muscularis in lungs; minimal to moderate epithelial vacuolation in trachea; minimal to mild vacuolation of vasavasorum, aorta and/or pulmonary artery in heart; minimal to marked arterial and/or exocrine vacuolation in pancreas; minimal to marked vacuolation of tunica muscularis, arterial and/or parietal layer of stomach; minimal to mild arterial vacuolation in skeletal muscle; mild to marked vacuolation of choroid plexus in brain in both sexes, mild arterial vacuolation in testes of male animals and minimal to mild arterial vacuolation in ovaries of female animals.
The lesions observed in high dose satellite (G4S) group did not reverse after recovery period and seems to be more severe.

Additionally, male and female animals from control and high dose groups and target organs from low dose, intermediate dose and recovery dose group animals showed lesions such as minimal to mild individual cell necrosis, lymphocytic infiltration and/or sinusoidal dilatation in liver; minimal to mild necrosis, basophilic tubules, vacuolation of tubular epithelium, tubular dilatation and/or proteinaceous material in kidneys; minimal to mild lymphocytic infiltration, alveolar histiocytosis, haemorrhages and/or presence of keratinized cyst in lungs; minimal to mild vacuolation, dilatation and/or presence of accessory adrenocortical tissue in adrenals; minimal to mild lymphoid necrosis in axillary lymph node; minimal lymphoid necrosis in spleen; minimal to mild necrosis or apoptotic necrosis in pancreas, minimal to mild lymphoid or apoptotic necrosis in thymus and presence of ectopic thymus or ultimobranchial cyst in thyroid in both sexes and mild oligospermia in epididymis of male animals.

The histopathological examination of organs showing macroscopic findings revealed mild sinusoidal dilatation in liver and mild congestion of stomach, minimal tubular hypertrophy of kidneys, moderate atrophy of testes, azoospermia and/or necrotic debris in epididymides and mild atrophy of seminal vesicles and prostate in one male animal of low dose (G2) group, whereas mild polymorphonuclear cells infiltration and luminal dilatation of uterus in one female animal of low dose (G2) group and two female animals of high dose satellite (G4S) group.

The changes observed other than the test item related lesions in various tissues during evaluation of control, low, intermediate, high dose and satellite dose group animals were comparable and hence considered incidental. These observed changes can usually be considered to be species, age, gender, congenital, physiological or mode of death related and are covered in background historical data of pathology

Histopathological findings: neoplastic:

not specified

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Test substance intake: Feed consumption was significantly decreased in the high dose group when compared with control group animals, and is consodered treatment-related.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No treatment-related effects were detected on mating performance. Mating index for all the groups is 100% (as confirmed by presence of sperm in the vaginal smear).

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Description (incidence and severity):

The pre and post implantation loss in all treated groups was comparable with control group.

Details on results (P0)

The treatment with test item resulted in no mortalities. All the animals survived to the scheduled sacrifice.
There were test item related clinical signs such as piloerection and dullness observed in the intermediate and high dose groups, significant decrease of food consumption and body weights in the intermediate and high dose group were correlated and considered to be treatment related. The clinical signs, body weight and feed consumption between low dose group and control group were comparable.

No treatment-related effects were observed for reproduction/ development such as precoital interval, mating index, fertility index, gestation length, pre-implantation loss, post-implantation loss, sex ratio and offspring growth and development at any dosage in the animals treated with 50 and 100 mg/kg/Day. However significant change in sex ratio (represented in the form of % male) of offspring was considered to be treatment related in the absence of this effect in the other dose groups including the control.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

for Reproduction / Developmental toxicity

Effect level:

> 100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: A change in sex ratio (represented in the form of % male) of offspring was considered to be treatment related in the absence of this effect in the other dose groups including the control

2

Dose descriptor:

NOAEL

Remarks:

for Systemic Toxicity

Effect level:

>= 50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Vacuolation detected in the various organs of high dose and intermediate dose group were considered to be treatment related and no recovery of microscopic changes were noted in the satellite group

Results: F1 generation

General toxicity (F1)

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Offspring mortalities were observed in the high dose group at Day 4 lactation when compared to Day 1.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There was significant decrease in the male offspring body weights at Day 1 and Day 4 in the intermediate and high dose group when compared with control and low dose group.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

Reddened testes was observed in one male pup each from two dams of low dose.

Histopathological findings:

not examined

Details on results (F1)

Offspring mortalities were observed in the high dose group at Day 4 lactation when compared to Day 1.

Sex ratio was calculated as percent (%) males. On Day 1, there was significant decrease of % male offspring in high dose group when compared with control group. However there was no statistical significance observed on Day 4. This may be due to the litter mortalities in the high dose group. This alteration in the sex ratio in the high dose group may be considered treatment related.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: See remark:

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

No toxicologically significant effects were detected in both sexes treated with 50 mg/kg/day. The vacuolation detected in the various organs of high dose and intermediate dose group were considered to be treatment related and no recovery of microscopic changes were noted in the satellite group. Hence, the 'No Observed Effect Level' (NOEL) for systemic toxicity is considered to be 50 mg/kg/day. 

No treatment-related effects were observed on reproduction/ development such as mating index, fertility index, gestation length, pre-implantation loss, post-implantation loss, sex ratio and offspring growth and development in the animals treated at 50 and 100 mg/kg/day therefore, a ‘No Observed Effect Level’ (NOEL) for reproductive toxicity is considered to be 100 mg/kg/day. The reported sex ratios are: Group1 Grpup2 Group3        Group4 Sex ratio % males at Day 1 post partum 52.88 43.53 50.24 36.06 Sex ratio % males at Day 4 post partum 52.88 43.53 50.29 39.39

Summary Tables of Reproductive parameters and organ weights are attached.

Applicant's summary and conclusion

Conclusions:

This study was performed according to OECD Guidelines (No. 422/GLP)

Oral administration of the test substance to Wistar rats by gavage (50, 100 and 200 mg/kg/day) resulted in treatment-related clinical signs, changes in the body weights, feed consumption and microscopic effects in male and female animals dosed at 100 and 200 mg/kg/day. No toxicologically significant effects were detected in both sex treated with 50 mg/kg/day.

Treatment-related reproduction / developmental toxicity effect of influencing the sex ratio (decrease in the % male offspring) were observed in the high dose group (200 mg/kg/day).

Test item related microscopic changes were observed in the testes and epididymides of male and ovaries of female animals in the high dose group (200 mg/kg/day).

No treatment-related effects were observed on reproduction/ development indices of animals in the 50 and 100 mg/kg/day dose groups. Mating index, fertility index, gestation length, pre-implantation loss, post-implantation loss, sex ratio and offspring growth and development were unaffected.

Some of the observed effects appear consistent with the maternal toxicity from administration of a corrosive/irritating substance. However, a No Observed Effect Level (NOEL) for systemic toxicity was derived at 50 mg/kg/day, whilst the No Observed Effect Level (NOEL) for reproductive effects was determined as 200 mg/kg/day.

The vacuolation detected in the various organs of high dose and intermediate dose group were considered to be treatment related and no recovery of microscopic changes were noted in the satellite group. Hence, the 'No Observed Effect Level' (NOEL) for systemic toxicity is considered to be 50 mg/kg/day.

Executive summary:

The test item, POLYCAT®77 (formulated in distilled water) was administered by gavage to three treatment groups, each of ten male and ten female Wistar rats, for up to 42 Days for males, up to two weeks premating phase, two weeks mating, three weeks gestation and early lactation for females, at dose levels of 50, 100 and 200 mg/kg/Day. A control group of ten males and ten females was dosed with vehicle alone (Distilled water). In addition, ten males and ten females were allocated to satellite group Group-1s, Group-4S, to represent recovery groups for control (0 mg/kg bw/Day), high dose (test item at 200 mg/kg bw/Day) respectively. Satellite group animals were treated along with main group (Allocation A) animals and dosing was stopped on the Day of first dam sacrifice. Satellite group animals were observed for a further 14-day recovery period.

The treatment with test item resulted in no mortalities. All the animals survived to the scheduled sacrifice. There were test item related clinical signs such as piloerection and dullness observed in the intermediate and high dose groups, significant decrease of food consumption and body weights in the intermediate and high dose group were correlated and considered to be treatment related. The clinical signs, body weight and feed consumption between low dose group and control group were comparable.

No test item effects were observed in any of the hematological, clinical biochemistry and urine parameters in the treated groups including the satellite group which represents the recovery.

The body weight and body weight gain (%) was significantly decreased in high dose animals of both sexes and intermediate dose group male animals when compared with control group and this effect was not reversed in the satellite group animals and hence these are considered to be treatment related.

In females, the body weights and body weight gain (%) were significantly decreased in the high dose group when compared with control group animals at premating, gestation and lactation periods. The high dose satellite group (recovery group) females also showed decreased body weight and body weight gain (%) when compared with respective satellite control group.

No treatment-related effects were observed for reproduction/ development parameters such as precoital interval, mating index, fertility index, gestation length, pre-implantation loss, post-implantation loss, sex ratio and offspring growth and development at any dosage in the animals treated with 50 and 100 mg/kg/Day. However significant change in sex ratio on day I did not show any dose response and also no related reproductive findings were noted and it is therefore considered to have arisen by chance or a biological variation.

In control group 10 out of 10, low and intermediate group 9 out of 10 animals and high dose 8 out of 10 animals achieved pregnancy following the positive evidence of mating in all 10 animals in each group.

One animal in low dose group did not achieve pregnancy which is correlated with findings of moderate atrophy of testes, azoospermia and/or necrotic debris in epididymides and mild atrophy of seminal vesicles and prostate in the co-habited male.

Parturition was observed in 10 females from control dose, 9 females in low dose, 9 females in the intermediate dose and 7 females in the high dose. Pregnancy loss in one animal of the high dose was due to early resorption.

Two females, one from intermediate and one from high dose showed cannibalism (eating its own pups) at lactation phase. Off spring body weight at Day 1 and Day 4 significantly differed in the high dose when compared with control in the absence of significance in the % body weight, this was not considered treatment effect.

Offspring mortalities were observed in the high dose group at Day 4 lactation when compared to Day 1. Even though, statistically there was no significance, the minor effects such as reduction in the litter size of three females and body weight decrease were observed in the high dose group, lack of significance may also be due to the lower number of dams i.e., seven animals in the high dose group.

Test item related microscopic change were seen in the testes and epididymides of male and ovaries of female animals at dose of 200 mg/kg body weight. The significant variation in sex ratio on day 1 in the high dose group (200 mg/ kg bw/day) may be associated with maternal toxicity moreover no such effects were found in intermediate dose group (100 mg/ kg bw/day) and low dose group (50 mg/ kg/ bw/day). Hence, considering the overall effects the reproductive and developmental NOEL (no observed effect level) is estimated as 200 mg/kg bw/day.