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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on test type (migrated information)
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:
guideline study

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

Constituent 1
Chemical structure
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:
N-[3-(dimethylamino)propyl]-N,N',N'-trimethylpropane-1,3-diamine
Test material form:
gas under pressure: refrigerated liquefied gas

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Animals were selected and/or grouped based on stratified randomization by using body weights taken before treatment. Computerized statistical programme was used for randomization.

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.

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
Remarks:
Doses / Concentrations:

Basis:
nominal in water
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
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.


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):
All the animals were subjected to gross examination.
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.

- All the data was checked for normality with Shapiro-Wilk W test .
- Data for each group of animals were subjected to analysis of variance (ANOVA). Values are given as mean +- standard deviation (SD)
- t- test was used to compare the difference between treated and control groups. Statistical significances of differences were calculated with one-way analyses of variance.
- Additionally, Non parametric "Mann-Wihtney ´U´test" was used to compare the differences between treated and control group.

P <= 0.05 (5% level of significance) was considered to represent the significance in the respective paraments, P > 0.05 was considered not signigicant
Reproductive indices:
The folllwoing 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 sings were observed in the control group and low dose group male and female animals.
In males, piloerection was observed in the intermediate dose group (100 mg/kg bw /day) from treatment Day 38 to Day 42. Piloerection and dullness was observed in the high dose group (200 mg/kg bw/day) on treatment Day 14 and continued to exhibit the gins till the last observation period (Day 42).
In females, piloerection was observed in the intermediate dose group (100 mg/kg bw /day) towards end of gestation and lactation period. In addition, cannibalism (eating its own pups on Day1 observation) and no lactation was noted in one animal.
In the high dose group (200 mg/kg bw/day), piloerection was observed in all the animals at the end of premating period and the gestation and lactation periods., in addition dullness was observed during gestation and lactation periods in two animals. In lactation phase one animal from high dose showed cannibalism.
The high dose satellite group also showed piloerection in all the animals. In addition, dullness was observed in two animals during the treatment and recovery period.
Mortality:
no mortality observed
Description (incidence):
There were no mortalities observed in the study. All animals survived the scheduled treatment or recovery periods.
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 of animals of both sexes when compared with the control group.
In males, intermediate dose group when 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 (%) was observed in the female animals of the intermediate dose group when compared with control group.
In male, the body weights and body weight gain (%) were significantly decreased in the high dose group when compared with control animals. The same observed in the high dose satellite group (recovery group) when compared with respective satellite control group.
In females, the body weights and the body weigh gain (%) were significantly decreased in the high dose group when compared with the 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 the respective satellite control group.
The body weight and body weight gain (%) was significantly decreased in the high dose animals of both sexes and intermediate dose group male animals when compared with the control group and this effect was not reversed in the satellite group 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 of few occasions. No significant changes in the feed consumption where observed in the female animals of the intermediate dose group when compared with control group during the gestation and lactation 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.
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
Treament with the test item had no effect on both sexes of all the groups with respect to the hematological parameters like RBC, WBC, Hb, PCV, PT. However, activated partial thromboplastin time was significantly descreased in low dose and high dose treated males when compared with control group. This decrease was also observed in high dose when comppared with low dose. Since, the change was not dose dependent was not observed in females animals of any group, it is considered to be not attributable to the test item. Significant increase in platelet count and decrease in the eosinophill count of male high dose satellite group when comparedto respective control group was not considered treatment related, the values lies in the normal biological range.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical biochemistry parameters revealed no significant changes in the test item treated and recovery male and female animals with an exception of creatinine in males and sodium in females. In males, creatinine levels decreased in low dose group, intermediate dose group and high dose group when compared with control group. Whereas, these levels were increased in high dose group when compared with intermediate group. In femaels, sodium levell were increased in high dose group when compared with intermediate and decreased in intermediate when compared with low dose group. Alkaline phosphatase, alanine transaminase, tryglycerides, sodium levels were decreased in the high dose satellite group (recovery group) females when compared with the respective control. The significance of these differences is not attributed to treatment due to marginal increase or decrease in individual control values. Hence, test item had no effect on the biochemistry parameters of treated animals.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment with test item had no effect on urinary parameters of the both sexes with an exception of decreased epithelial cells in low dose group when compared with control and increased epithelial cells in intermediate dose group in compasion with low dose group in males. In the high dose satellite group (recovery group), urinary volume increased in males and decreased in females when compared with respective control. In addition, decreased leukocyte count in females animals were observed. However, this significance is solely of individual variation not to represent any biological significance.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Mild to moderate treatment-related effects: centrilobular and/or arterial vacuolation in liver, vacuolation of glomerular tuft in kidneys; vacuolation in urinary bladder, thymus, arteries, skeletal muscle, choroid plexus, ovaries.
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)

No treatment-related effects were detected on mating performance. All the female animals of contro, low, intermediate and high dose group showed positive evidence of mating. However, one animal in low dose group did not conceive during the first 14 Days of cohabitation period with respective male and was remated with the proven make from the same group and mating was confirmed. No significant difference was observed in the pre-cital interval of females animals between control, low, intermediate and high dose group. Mating index for all groups is 100% (as confirmed by presence of sperm in the vaginal smear). In the control group 10/10, low and intermediate group 9 /10 animals and high dose 8 /10 animals achieved pregnancy following the positive evidence of mating in all 10 animals in each group. One animal in low dose did not achieve pregnancy which is correlated with findings of moderate atrophy of testes, azoospermia and/or necrotic debris in epdidymides and mild atrophy of seminal vesicles and prostate in the co-habitated male. One animal in the intermediate dose group did not achieved pregnancy. There were no histopathological correlated in the reproductive organs to elucidate the cause of the non-pregnancy in either the paired female or male partner which did not produce a pregnancy. For one animal in high dose group, corpora lutea of pregnancy and implant were evidenced however, no parturation or dead fetus were found, this may be due to early resorption. Remaining two animals did not achieve pregnancy and no gross or histological correlation in paired male and females was noted. The pregnany index was 100% in control, 90% in low and intermediate dose group and 80% in high dose group animals.

GESTATION AND PARTURITION
No treatment-realted effects were detected in the leght of gestation for treated females when compared to controls. All animals showed gestation lenghts between 21 to 23 Days. Ony one of the control animals, gestation lenght was 24 Days.
The parturition index was 100% in control, low and intermediate dose group. Whereas, in the high dose group the parturition index was 88%.

Effect levels (P0)

Key result
Dose descriptor:
NOEL
Effect level:
ca. 100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance
Remarks on result:
other: 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 gro

Results: F1 generation

General toxicity (F1)

Clinical signs:
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
All animals survived the scheduled treatment or recovery periods.
Pre- and post implantation loss in all treated groups was comparable with control group.
Of the litters delivered, in all treatment groups, litter size at birth and subsequently on Days 1 and4 post-partum were comparable to controls. No significant difference in live birth index and vability index was noted between control and treated groups.

Sex ratio was calculated as percent (%) male. On Day 1, there was a significant decrease of % male offspring in high dose group when compared with control group. However, the was no statistical significance observed on Day4. This may be due to the litter mortalities in the high dose group. The significance in sex ration on Day 1 did not show any dose repsonse and also no related reproductive findings were noted and it therefore considered to have arised by chance or a biological varation.
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. In addition on Day 1, decrease if male offspring body weight was observed in high dose when compared with the intermediate dose group. In female offspring, decreased body weight was observed at Day 1 in the intermediate and high dose group when compared with control and low dose group. In addition on Day 1, decrease of female offspring body weight was observed in high dose when compared with intermediate dose group. At Day 4, this significance was observed in the high dose group females alone when compared wiith the respective low dose and intermediate dose group . However, no significant change in the body weight gain (%) of treated male and female offspring was noted when compared with control with an exception of decreased body weight gain (%) in intermediate dose when compared with control, low dose an d high dose.

Litter weights on Day 1 post-partum showed significant decrease in high dose group as comparted to control, low and intermediate groups. No significance was observed in the litter on Day 4 post partum between control and treated groups.
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
In main experiment male animals, there was a significant decrease in absolute weight of thymus in high dose and intermediate dose groups when compared with control group and also in high dose when compared with low dose group while in case of female animals there was significant decrease in absolute weight if thymus in high dose and intermediate dose when compared with low dose and significant increase absolute weight of thymus in low dose when compared with control group.
Whereas, there was significant increase in relative weight of liver and testes in high dose and intermediate dose groups when compared with control and low dose groups and significant increase in relative weight of brain in high dose and intermediate dose groups when compared with control group and also high dose when compared with low dose group in male animals. In case of female animals, significant decrease in relative weight of thymus in intermediate dose group when compared with low dose group while significant increase in relative weight of thymus in high dose group when compared with intermediate dose group and also in low dose group when compared with control group was observed. In addition, significant increase in relative weight of kidneys of female animals in high dose and intermediate dose groups when compared with control and low dose groups.
In the satellite group group, there was significant decrease in absolute weight of epididymides in male animals while significant increase in absolute wight if hearth, thymus and spleen in case of female animals of high dose group wen compared with the respective control satellite group. Whereas, significant increase in relative weight of adrenals, kidneys, liver, heart, spleen testes and brain in male animals and kidneys, liver, heart, thymus and spleen in females animals of high dose satellite group when compared with respective control satellite group was observed.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Necropsy performed at the end of the treatment period revealed no abnormality in any of the male and female animals of control, intermediate and high dose groups while fragile liver, enlarged kidney, reddened stomach and small sized testes, epididymis, seminal vesicles and prostate in one male animal and distended uterus with watery content in one female animal of low dose group was observed. While following recovery period, no abnormality was observed in male animals whereas, distended uterus with watery content was observed in teo female animals og high dose satellite group.
These findings were considered to be gender, congenital and/or physiology related and considered as biological variations.
In case of pups, reddened testes was observed in one male pup each from two dams of low dose groups while cannibalization, autolysis, weak and/or pups with empty stomach in male and female pups from two dams each of intermediate dose and high dose groups.
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
see Microscopic findings

Effect levels (F1)

Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
ca. 100 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other: 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

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

No abnormality was attributable to the test item at the low dose group.


Microscopic examination of the high dose and intermediate 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/or white 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, whereas mild arterial vacuolation in testes and/or epididymides of male animals and minimal to mild arterial vacuolation in ovaries of female animals.


Microscopic examination of high satellite 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 to 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 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 and7or 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 tests of male animals and minimal to mild arterial vacuolation in ovaries of female animals.


However, the lesions observed in high dose satellite 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 o mild individual cell necrosis, lymphocytic infiltration and/or sinusoidal dilation 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 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 tests, azoospermia and/or necrotic debris in epididymides and mild atrophy of seminal vesicles and prostate in one male animal of low dose group, whereas mild polymorphonuclear cells infiltration and luminal dilatation of uterus in one female animal of low dose group and two female animals of high dose satellite group.


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

Applicant's summary and conclusion

Conclusions:
This study was performed according to OECD Guidelines (No. 422/GLP) and is considered of good quality. Recorded results show a consistent treatment-related effect

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.

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.

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 animals being administered a corrosive/irritating substance. However, a No Observed Effect Level (NOEL) for systemic toxicity was derived at 50 mg/kg/day, whilst teh No Observed Effect Level (NOEL) for reproductive effects was determined as 100 mg/kg/day.

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