Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

The NOAEL for fertility is set at 100 mg/kg bw/day.

Link to relevant study records
Reference
Endpoint:
one-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
Jan-June 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Read-Across Justification: Read-Across is claimed between this substance (CAS 33329-35-0) and a structurally-similar aliphatic tertiary amine (CAS 3855-32-1). This study was performed using CAS 3855-32-1 (HH - N-[3-(dimethylamino)propyl]-N,N',N'-trimethylpropane-1,3-diamine). Structurally, CAS 33329-35-0 differs only in the presence of an additional (dimethylamino)propyl group linked to the central nitrogen. Both substances are water-soluble liquids with very similar partition coefficients. Both substances are demonstrated to be corrosive to skin, are of comparable acute toxicity and are negative in the Ames test. Both substances are predicted to be bioavailable. Negative response was seen in a Maximisation test performed with CAS 3855-32-1, whereas a negative response is reported for CAS 33329-35-0. Tests for skin sensitisation are of low sensitivity for corrosive substances as the predominant irritant response will limit the test concentrations used and there also is an increased chance of a false positive result due to an irritant response. The slight discrepancy between these two substances does not therefore indicate any fundamental difference in toxicological profile. It is notable that QSAR modelling (OECD QSAR Toolbox v4) does not indicate any protein binding potential for either substance (OECD or OASIS models). OECD QSAR Toolbox identifies an alert for DNA binding for both CAS 3855-32-1 and CAS 33329-35-0 based on the possible formation of an iminium ion from the aliphatic tertiary amine group present in the molecules. However, endpoint-specific predictions for both substances do not indicate any activity in the Ames test (ISS) or the in vivo micronucleus assay (ISS), DNA alerts for the Ames test, micronucleus and chromosomal aberration assay (OASIS) or for genotoxic carcinogenicity (ISS). Based on the identical predictions and the negative Ames tests available for both substances, meeting the additional genotoxicity data requirements for CAS 3855-32-1 by read-across to the studies (mouse lymphoma assay, micronucleus assay in human lymphocytes) for CAS 33329-35-0 is therefore considered to be scientifically justified. The available toxicological data for CAS 3855-32-1 and CAS 33329-35-0 indicate irritation/corrosivity as the predominant effect. The 28-day toxicity study performed with CAS 33329-35-0 identifies a NOAEL of 200 mg/kg bw/d based on effects at the highest dose level of 400 mg/kg bw/d. Findings in this study were limited to local effects on the gastrointestinal tract (or secondary findings) and are consistent with the corrosive nature of the substance. The results of a range-finding study performed with CAS 3855-32-1 showed no effects at a dose level of 100 mg/kg bw/d, marked toxicity and local effects on the stomach at 600 mg/kg bw/d and less marked effects at 300 mg/kg bw/d. A comparable level of toxicity and a similar predominance of local effects and an absence of systemic toxicity can be predicted for CAS 3855-32-1. The oral toxicity of the target substance N,N-bis[3-(dimethylamino)propyl]-N’,N’-dimethylpropane-1,3-diamine, CAS 33329-35-0, in rats, following daily oral administration for 13 consecutive weeks and recovery from any treatment-related effects during a period of 2 weeks, were investigated in another study according to OECD 408. Overall, some treatment-related effects (including mortality) were observed at 300/200mg/kg/day. However, these effects were ascribed to a strong local effect of the test item due to its chemical nature (strong base) to the gastrointestinal and respiratory tracts, following initial 4-day dosing at 300mg/kg/day. No treatment-related systemic effect which is considered to be adverse was observed following dosing at 200mg/kg for at least 87 days or at 100 and 30mg/kg/day. Therefore, it can be concluded that the high dose level of 200mg/kg/day may be considered as the No Observed Adverse Effect Level (NOAEL) for this study, under the reported experimental conditions. A 90 -day repeated dose oral study according to OECD TG 408 was performed with the source substance N-[3-(Dimethylamino)propyl]-N,N’,N’-trimethylpropan-1,3-diamine, CAS 3855-32-1 as well. Also in this case, all effects reported from oral repeated dose toxicity studies are considered to be secondary (decreased bodyweight and food consumption, degeneration in lungs and traches, lymphoid depletion as consequence of stress i.e. poor nutrition) induced by irritant properties of the substance or its trapping in cells and cellular compartments (a mechanism known for this substance group). In a OECD 422 study conducted with source substance N-[3-(Dimethylamino)propyl]-N,N’,N’-trimethylpropan-1,3-diamine, CAS 3855-32-1. No mortality was observed in the study. The oral administration of the test substance resulted in treatment-related clinical signs, changes in the body weights, feed consumption and microscopic effects in male and female animals treated with 100 mg/kg/day 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 where noted in the satellite group. The mechanism vacuolization is discussed in the literature and is induced by amines. In case of the physiological function of the affected tissues/organs is not adversely changed, the vacuolation can be considered as morphological change without toxic/adverse consequences. Relevant reproduction data (corpora lutea, number of implantation sites, pre-and post-implantation losses and number of foetuses at termination) did not indicated any test item-relate effect. Thus, occurrence of vacuoles in cells per se is not considered adverse, the “No Observed Adverse Effect Level” (NOAEL) for reproductive toxicity is considered to be 200 mg/kg/day. 28 and 90-day repeated dose studies conducted with source and target substances did not show any adverse effects on the reproductive organ of both genders. Based on the above consideration and read-across approach, overall, it can be assumed that the target substance does not have the potential to be toxic to reproduction. A full read-across justification is attached to this record.
Justification for type of information:
see attached justification
Reason / purpose for cross-reference:
read-across source
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
Specific details on test material used for the study:
Target substance: 1,3-Propanediamine, N,N-bis[3-(dimethylamino)propyl]-N',N'-dimethyl; 1,3-Propanediamine, N1,N1-bis(3-(dimethylamino)propyl)-N3,N3-dimethyl- / 33329-35-0 / 251-459-0; Polycat 9; Tris(3-Dimethylamino)Propylamine

See Analogue Justification in Chapter 13.2
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The strain is recommended by guidelines.
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.
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)
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.
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
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.
Water consumption and compound intake (if drinking water study):
not examined
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: 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.
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%.
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
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
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
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

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.

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 the No Observed Effect Level (NOEL) for reproductive effects was determined as 100 mg/kg/day.
Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
200 mg/kg bw/day
Study duration:
subacute
Experimental exposure time per week (hours/week):
0
Species:
rat
Quality of whole database:
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.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Short description of key information:


In a combined Repeat Dose Toxicity Study With Reproduction / Developmental Toxicity Screening Test (according to OECD TG 422 and US EPA Health Effects Test Guideline OCSPP (Office of chemical safety and pollution prevention) 870.3650 2000 and under GLP conditions) with structural related N-[3-dimethylamino)propyl]-N,N´,N´-trimethylpropane-1,3-diamine, CAS 3855-32-1, the systemic toxicity and potential adverse effects of the test item on reproduction (including offspring development) were investigated in rats.


 


The test item (formulated in distilled water) was administered by gavage to three groups of rats (10 males and 10 females for each group) for 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. A control group of 10 males and ten females were dosed with vehicle control. In addition, ten males and 10 females were doses were allocated to satellite group to represent recovery groups for control, and high dose.


Satellite group were treated along with main group animals and dosing was stopped on the day of the first dam sacrifice. Satellite animals were then observed for a further 14-day treatment-free period.


 


Males were sacrificed on Day 43, followed by the sacrifice for all females on Day 5 post-partum. Pups were sacrificed on Day 4 postpartum.


No mortality was observed in the study. The oral administration of the test substance resulted in treatment-related clinical signs, changes in the body weights, feed consumption and microscopic effects in male and female animals treated with 100 mg/kg/day 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 where noted in the satellite group. Thus, the “No Observed Adverse Effect Level” (NOAEL) for reproductive toxicity is considered to be 200 mg/kg/day.


Furthermore, 28 and 90-day repeated dose studies conducted with the substance object of registration did not show any adverse effects on the reproductive organ of both genders.




Justification for selection of Effect on fertility via oral route:
This study was performed according to OECD Guidelines (No. 422/GLP) and is considered of good quality. 

Effects on developmental toxicity

Description of key information

The NOAEL for maternal and developmental toxicity is set at 150 mg/kg bw/day.


Justification for selection of Effect on fertility via oral route:
This study was performed according to OECD Guidelines (No. 414/GLP) and is considered of good quality. 

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2020-04-30 to 2020-07-23
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
25 June 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
- Storage condition of test material: Room temperature, protected from light and oxidants (under
nitrogen)
- Stability under storage conditions: stable
- Stability under test conditions: The required amount of PU-2019-871 was dissolved/suspended in the vehicle. The formulation was prepared daily or weekly
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: The stability of the preparations at 1-50mg/mL was assessed at:
• up to 28 hours at room temperature
• after 8 days at 2-8°C
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy
- Age at study initiation: 9 weeks old (virgin females), 11 weeks old (males)
- Weight at study initiation: 203-223 g (virgin females); 340 g (males)

- Housing: Before and after the pairing period, the animals were housed no more than 5 of one sex in polysulfone solid bottomed cages, measuring 59.5×38×20cm. Nesting material was provided inside suitable bedding bags and changed at least twice a week. In addition, suitable nesting material was provided as necessary. Nesting material was changed at least 2 times a week. During the pairing period, the rats were housed on the basis of 1 male to 1 female in clear polysulfone cages measuring 42.5×26.6×18.5cm with a stainless-steel mesh lid and floor. Each cage tray held absorbent material which was inspected and changed daily.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: one week before the start of the treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C±2°C
- Humidity (%): 55%±15%
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2020-05-22 To: 2020-06-19
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was dissolved/suspended in the vehicle. The formulation was prepared daily or weekly according to stability data obtained from test laboratory study (concentrations of 3.75, 7.5 and 15mg/mL), unless specified otherwise.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Preparation of the test item:
The required amount of the test item was dissolved/suspended in the vehicle. The formulation was prepared daily or weekly according to stability data obtained from a separate study (concentrations of 3.75, 7.5 and 15mg/mL), unless specified otherwise.

Analysis of test item preparations:
Analysis was performed in a separate study in order to validate the analytical method and the preparation procedure and to verify the stability of the preparations. The stability of the preparations at 1-50mg/mL was assessed at:
– up to 28 hours at room temperature
– after 8 days at 2-8°C
Samples of the preparations prepared during the first and last week of treatment of the
current study were analyzed to check the concentration. Results of the analyses were within the acceptability limits stated in test laboratory SOPs for concentration of solutions (90-110%).
The validated software used for this activity was Analyst 1.6.2 (ABSciex).
Details on mating procedure:
Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: overnight
- Further matings after two unsuccessful attempts: yes. Vaginal smears were taken daily in the morning from the day after pairing until a positive identification of mating was made.
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear or vaginal plug referred to as day 0 of pregnancy
Duration of treatment / exposure:
All animals were dosed once a day from Day 3 through Day 19 post coitum.
Frequency of treatment:
daily
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Remarks:
High Level (Main Group 4)
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Remarks:
Medium Level (Main Group 3)
Dose / conc.:
37.5 mg/kg bw/day (actual dose received)
Remarks:
Low Level (Main Group 2)
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels of 37.5, 75 and 150 mg/kg/day were selected by the based on information from a preliminary non-GLP compliant study (ERBC Study no. Y0470). In thepreliminary study, the dose levels of 100, 200 and 400 mg/kg/day were used. The test item induced slight to moderate signs of maternal toxicity at all dose levels, in terms of clinical signs recorded and decreased body weight gain. These effects were more evident at the two highest dose levels in which unscheduled death and decrease in absolute weight gain were also noted.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Throughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day.


DETAILED CLINICAL OBSERVATIONS: Yes / No / No data
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on Days 0, 3, 6, 9, 12, 15, 18 and 20 post coitum.


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # GD 20
- Organs examined: Thyroid gland

OTHER: Abnormalities
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes /
- Number of early resorptions: Yes: only placental remnants visible
- Number of late resorptions: Yes: placental and foetal remnants visible.
Fetal examinations:
- External examinations: Yes: half per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: all per litter
Statistics:
For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.
Statistical analysis of non-continuous variables was carried out by means of the Kruskal-
Wallis test and intergroup differences between the control and treated groups assessed by a
non-parametric version of the Williams test.
Indices:
-Pre, post- and totoal implantation loss were calculated as a percentage.
- Sex ratios of the foetuses were calculated as the percentage of males per litter.
- Anogenital distance (AGD): The AGD of each live foetus was measured on Day 20 post
coitum. The AGD was normalized to the cube root of body weight collected on Day 20 post
coitum. Individual and mean data were reported.
-
Clinical signs:
no effects observed
Description (incidence and severity):
The clinical signs (scabs or hairloss) recorded during the the study were not considered
treatment related since they were equally observed among the treated and control groups.
Piloerection was seen in one female receiving 150 mg/kg/day on Day 4 post coitum only.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths during the study. One Female receving 75 mg/kg/day was found not pregnat at necropsy. The remaining females sacrifices on Day 20 post-coitim were pregnant.
Pregnancy status: There were 25/25, 25/25, 24/25 and 25/25 pregnant females in control,
37.5, 75 and 150 mg/kg/day. All pregnant females had viable foetuses.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean body weight and mean body weight gain did not give any indication of test -Item related effect.
Food efficiency:
no effects observed
Description (incidence and severity):
No statistical significant differences in food consumption were observed amongst the control and the groups receiving the test item at the dose levels of 37.5, 75 and 150 mg/kg/day.
Water consumption and compound intake (if drinking water study):
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Endocrine findings:
no effects observed
Description (incidence and severity):
With regard Thyroid horme determination -Delegated phase
No deifferences between control and treated animals were recorded.
For more information see Results on Thyroid hormone measurments and maternal developmental toxicity attached in under in the "Attached background material" under "Overall Remarks"
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Terminal body weight, uterus weight and mean absolute weight gains (body weight at
necropsy minus gravid uterus weight, minus body weight at Day 0 of pregnancy) of treated
females were comparable to the control group. No treatment-related was seen in thyroid weight if treated females compared to the control. The decrease of relavtive (% to body weight) (approximately 18%) seen in females receiving 150 mg/kg/day, compared to controlm was not considered test item.related since no concurrent histopathological findings were noted.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Females that completed the treatment period did not show relevant macroscopic changes
that could be considered treatment-related.
No treatment-related changed were noted in control and treated females, following gross pathological examination. All observed changes were considered spontaneous and incidental, having a comparable incidence in control and treated groups and /or are characteristically seen in untreated Sprague Dawley SD rats of the same age.
Neuropathological findings:
not examined
Description (incidence and severity):
No treatment-related finsing were noted in the microscopic evaluation of the thyroid gland of treaed females, when compared to their controls.
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Description (incidence and severity):
No differences between control and treated animals were recorded.
Number of abortions:
no effects observed
Description (incidence and severity):
There were no statistically significant differences between the control and treated groups
in the number of corpora lutea, number of implantations, live foetuses and early and late
resorptions, nor in the pre- and post implantation loss or in the sex ratio of the foetuses.
Furthermore, no significant differences in the litter and mean foetal weights were observed
between control and treated groups
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Post-implantation losses, as percentage of implantation sites, were 2.5%, 1.8%, 2.2% and 1.9% in control and in females treated at 37.5, 75 and 150 mg/kg/day, respectively. Number of live fetuses corrrespond to 97.4%, 98.4%, 97.7% and 97.8% of implantation sites in control and in females treated at 37.5, 75 and 150 mg/kg/day, respectively.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
Litter mean of treated groups were similar to the control and therefore were not considered to be affected by the treatment with the test item.
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
No test item-related effects on the sex ratio of the fetuses were noted at any dose level. The
proportion of male foetuses was 52%, 50%, 50% and 52% in order of ascending dose levels.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
changes in number of pregnant
changes in pregnancy duration
dead fetuses
early or late resorptions
effects on pregnancy duration
maternal abnormalities
number of abortions
pre and post implantation loss
total litter losses by resorption
Key result
Dose descriptor:
NOAEL
Effect level:
>= 150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
clinical signs
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Mean fetal weights (combined or by sex) of treated groups were similar to the control and therefore were not considered affected by the treatment with the test item .
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Anogenital distance of all rodent fetuses:
no effects observed
Description (incidence and severity):
No changes were seen in AGD (anogenital distance) between control and treated groups. No differences were noted in the mean values of the AGD of the fetuses of both sexes, maternally exposed at all dose levels compared to the control group.
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
During the external examination of fetuses, abnormal findings were noted in the three fetuses of control groups, in one fetus at 37.5 mg/kg/day, in seven fetusus at 75 mg/kg/day and three fetuses at 150 mg/kg/day. Abnormal findings such as small fetusus (weight < 2.7 g), acaudia, imperforate anus and malrotated forelimbs were considered to be incidental due the the presence also in control group (acaudia and imperforate anus) and / or because they occured without dose-dependancy
Skeletal malformations:
no effects observed
Description (incidence and severity):
Abnormalities and variations noted during the skeletal examination were observed in control and treated groups wihtout of dose-dependency, thus they were not considered to be attributed to the treatment with the test item. During skeletal examination of the fetuses, findings were noted in:

- 90% examined fetuses (in 100% litters) in control group:;
- 88% examined fetuses (in 100% litters) at 37.5 mg/ kg bw/day:
- 90% examined fetuses (in 100% litters) at 75 mg/ kg bw/ day;
- 81% examined fetuses (in 100% litters) at 150 mg/kg bw/ day.

The distribution of these alteration among the groups, control and treated, did not indicate any test item related effects.
Major abnormality (pubis bone no ossification) were noted in one control fetus ( in 1 litter), 1 fetus maternally exposed at 37. 5 mg/ kg bw/ day (out of 1 litter) , in 6 fetuses (out of 2 litters) exposed at 75 mg/kg bw/ day and in 3 fetuses (out of 2 litter) exposed at 150 mg/kg bw/ day. Two additional fetuses, one maternaly exposed at 37.5 mg/kg bw / day and one at 150 mg/ bw/ day showed cleft palate and polydactyly, respectively. The distribution of these alterations among the groups, control and treated, did not indicate any test-tem related effects.
Visceral malformations:
no effects observed
Description (incidence and severity):
There was no visceral malformation associated with treatment with the test item.
Oveerall, fetal variations observed during the microdissection were commonly seen in rat foetuses in this age. Thus, these findings could be incidental and not attributed to the treatment with the test item . Consequently, under the conditions described for the present study, the test item did not reveal teratorgenic potential up to and including the dose level of 150 mg/ kg bw/ day
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
>= 150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
changes in postnatal survival
external malformations
skeletal malformations
visceral malformations
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Results in Tabular form about effects level on Maternal animals and fetuses  are available under in the "Attached background material" under "Overall Remarks"

Conclusions:
The test item was administered, by oral gavage, to mated female Sprague Dawley rats (25 females per dose group) from Days 3 to 19 post coitum.
The vehicle was softened water (by reverse osmosis) and the dose volume used was 10 mL/kg.
The dose levels were 0, 37.5, 75 and 150 mg/kg/day. On Day 20 post coitum the dams were killed and macroscopically examined.
Gravid uterus and thyroid were weighed and fetuses were examined after caesarian section. External, visceral and skeletal examinations were performed on fetuses of all groups.
No mortalities were observed. One female receiving 75 mg/kg/day was found not pregnant at necropsy.
Further, daily clinical observations during the gestation period did not reveal any remarkable findings in the animals’ appearance, general condition or behaviour amongst the dosing and control groups.
No significant differences in mean body weights, body weight gain and food consumption were observed amongst the control and treated groups.
There were no statistically significant differences between the control and treated groups in the number of corpora lutea, number of implantations, live fetuses and early and late resorptions, nor in the pre- and post-implantation loss or in the sex ratio of the fetuses.
Furthermore, no significant differences in the litter and mean fetal weights were observed between control and treated groups.
No remarkable differences in gravid uterus weight, terminal body weight or absolute weight gain were observed between the control group and the groups receiving the test item.
Fetal external observations, visceral and skeletal examination did not reveal any remarkable findings which could be related to treatment.
Based on the results obtained in the study, the NOAEL for maternal and developmental toxicity could be set at 150 mg/kg/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
This study was performed according to OECD Guidelines (No. 414/GLP) and is considered of good quality.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Justification for selection of Effect on developmental toxicity: via oral route:
This study was performed according to OECD Guidelines (No. 414, GLP) and is considered of good quality. 

Toxicity to reproduction: other studies

Additional information

In a combined Repeat Dose Toxicity Study With Reproduction / Developmental Toxicity Screening Test (according to OECD TG 422 and US EPA Health Effects Test Guideline OCSPP (Office of chemical safety and pollution prevention) 870.3650 2000 and under GLP conditions) with structural related N-[3-dimethylamino)propyl]-N,N´,N´-trimethylpropane-1,3-diamine, CAS 3855-32-1, the systemic toxicity and potential adverse effects of the test item on reproduction (including offspring development) were investigated in rats.


 


The test item (formulated in distilled water) was administered by gavage to three groups of rats (10 males and 10 females for each group) for 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. A control group of 10 males and ten females were dosed with vehicle control. In addition, ten males and 10 females were doses were allocated to satellite group to represent recovery groups for control, and high dose.


Satellite group were treated along with main group animals and dosing was stopped on the day of the first dam sacrifice. Satellite animals were then observed for a further 14-day treatment-free period.


 


Males were sacrificed on Day 43, followed by the sacrifice for all females on Day 5 post-partum. Pups were sacrificed on Day 4 postpartum.


No mortality was observed in the study. The oral administration of the test substance resulted in treatment-related clinical signs, changes in the body weights, feed consumption and microscopic effects in male and female animals treated with 100 mg/kg/day 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 where noted in the satellite group. Thus, the “No Observed Adverse Effect Level” (NOAEL) for reproductive toxicity is considered to be 200 mg/kg/day.


 


The effect of the substance N,N-bis[3-(dimethylamino)propyl]-N’,N’-dimethylpropane-1,3-diamine, CAS  33329-35-0, was investigated after oral administration in female rats during pregnancy and embryo-foetal development. Animals were administered during gestation period, with 37.5, 75 and 150 mg/kg bw/day, starting from Day 3 through Day 19 post coitum at the dosing volumes of 10 mL/kg.


There were no unscheduled deaths during the study. Pregnancy status: There were 25/25, 25/25, 24/25 and 25/25 pregnant females in control, 37.5, 75 and 150 mg/kg/day.


All pregnant females had viable fetuses. No treatment-related clinical signs were recorded. No significant changes in body weight or body weight gain were seen between groups throughout the study. Food consumption was comparable between groups. No differences between control and treated animals were recorded. Body wight at termination, uterus weight and the absolute weight gain of females were unaffected by treatment.


Relevant reproduction data (corpora lutea, number of implantation sites, pre-and post-implantation losses and number of fetuses at termination) did not indicated any test item-relate effect. Litter and mean foetal weights (combined or by sex) of treated groups were similar to the control and therefore were not considered to be affected by the treatment with the test item. No test item-related effects on the sex ration of the fetuses were noted at any dose level. No changes were seen in anogenital distance between control and treated groups. No relevant changes were observed in the absolute and relative thyroid weight of treated females, when compared to the control data. Females that completed the treatment period did not show relevant macroscopic changes that could be considered treatment-related. No treatment-related findings were noted at the microscopic evaluation of the thyroid gland of the treated females, when compared to their controls. External observations of the fetuses did not reveal any remarkable findings which could be related to treatment. Visceral examinations did not reveal teratogenic potential of the test substance up to and including the dose level of 150 mg/kg bw day.


No treatment-related findings were recorded at skeletal examination of fetuses.


Based on the results obtained in the study, the NOAEL for maternal and developmental toxicity could be set at 150 mg/kg bw/day.


 

Justification for classification or non-classification

Based on the available data on toxicity to reproduction and development do not meet the classification criteria according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.

Additional information

Categories Display