Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-03-26 - 2012-05-2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-conform OECD Guideline study.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA, Health Effects Test Guidelines; OPPTS 870.3650: Combined Repeated Dose Toxicity Study With the Reproduction/Developmental Toxicity Screening Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
- Age at supplied: 10-12 weeks (11 - 13 weeks old at the beginning of treatment)
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Weight at study initiation: Males: 298.3 g - 335.9 g, Females 213.1 g - 236.9 g
- Fasting period before study: no
- Housing: individually in Makrolon type M III cages;
- Exceptions:
- During overnight matings, male and female mating partners were housed together in Makrolon type M III cages
- Pregnant animals and their litters were housed together until PND 4 (end of lactation).
- The cages with the test animals were arranged on the racks in such a way that uniform experimental conditions (ventilation and light) were ensured.
- Diet, ad libitum: Ground Kliba maintenance diet mouse-rat “GLP” meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland
- Water, ad libitum: drinking water
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24°C
- Humidity: 30-70%
- Air changes (per hr): 15
- Photoperiod: 12-hour light/12-hour dark cycle

IN-LIFE DATES: From: 2012-04-02 To: 2012-05-20
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance solutions in drinking water were prepared at the beginning of the administration period and thereafter in intervals, which took into
account the analytical results of the stability verification.
For the preparation of the administration solutions the test substance was weighed in a graduated flask depending on the dose group, topped up
with drinking water and subsequently thoroughly mixed by shaking until it was completely dissolved.

VEHICLE
- Justification for use and choice of vehicle: solubility
- Concentration in vehicle:
50.00 mg/100 mL (5 mg/kg bw/d), 150.00 mg/100 mL (15 mg/kg bw/d), 500.00 mg/100 mL (50 mg/kg bw/d)
- Amount of vehicle: 10 mL/kg body weight
Details on mating procedure:
Pairing of F0 generation parental animals
In general, each of the male and female animals was mated overnight in a 1:1 ratio for a maximum of 2 weeks. Throughout the mating period, each female animal was paired with a predetermined male animal from the same dose group.
A vaginal smear was prepared after each mating and examined for the presence of sperm. If sperm was detected, pairing of the animals was discontinued. The day on which sperm were detected was denoted "GD 0" and the following day "GD 1".
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical verifications of the stability of the test substance in drinking water for a period of 7 days at room temperature were carried out in a comparable batch prior to the start of the study. Samples of the test substance solutions were sent to the analytical laboratory once at the beginning of the study for verification of the concentrations. The analytical results confirmed the stability and expected concentrations of the test substance solutions. Due to the fact that the test substance preparations were true solutions, the prove of homogeneity by analytical methods was not necessary.
Duration of treatment / exposure:
Males were exposed for 30 days (prior to mating, during mating, and up to termination) and females 49 days (prior to mating, during mating, during post-coitum, and at least 4 days of lactation).
Frequency of treatment:
Daily
Details on study schedule:
- Dose selection rationale: Dose levels were selected by request of the sponsor
- Route of administration: The oral route was selected since administration by gavage has been proven to be appropriate for the detection of a toxicological hazard.
Remarks:
Doses / Concentrations:
5, 15 and 50 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
10 animals
Control animals:
yes, concurrent vehicle
Parental animals: Observations and examinations:
MORTALITY / CAGE SIDE OBSERVATIONS: Yes
A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity.
The littering and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams.
On weekdays (except public Saturday, Sunday and public holidays) the parturition behavior of the dams was inspected in the afternoons in addition
to the evaluations in the mornings.

DETAILED CLINICAL OBSERVATIONS: Yes
Detailed clinical observations (DCO) were performed in all animals prior to the administration period and thereafter at weekly intervals. The animals were transferred to a standard arena (50 × 37.5 cm with sides of 25 cm high).
The following parameters were examined: abnormal behavior when handled, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmus, feces (appearance/consistency), urine, pupil size

BODY WEIGHT: Yes
In general, the body weight of the male and female parental animals was determined once a week at the same time of the day (in the morning) until sacrifice.
The body weight change of the animals was calculated from these results.
The following exceptions are notable for the female animals:
- During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
- Females with litter were weighed on the day of parturition (PND 0) and on PND 4.
- Females without a litter and without positive evidence of sperm in the vaginal smear were weighed weekly. These body weight data were solely used for the calculations of the dose volume.

FOOD CONSUMPTION:
Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
- Food consumption was not determined after the second premating week (male parental
animals) and during the mating period (male and female F0 animals)
- Food consumption of the F0 females with evidence of sperm was determined on GD 0-7, 7-14, 14-20.
- Food consumption of F0 females, which gave birth to a litter, was determined for PND 1-4.
Food consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel).

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: In the morning towards the end of the administrative period
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group
- Parameters examined: see Table 1 in 'Any other information on materials and methods incl. tables'.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: In the morning towards the end of the administrative period
- Animals fasted: Yes
- How many animals: first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group
- Parameters examined: see Table 2 in 'Any other information on materials and methods incl. tables'.

URINALYSIS: Yes
- Time schedule for collection of urine: overnight towards the end of the administrative period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: see Table 3 in 'Any other information on materials and methods incl. tables'.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Functional observational battery (FOB) was performed in five male and 5 female animals (with litter) per group at the end of the administration
period
- Dose groups that were examined: all animals
- Battery of functions tested:
Home cage observations:
Attention was paid to: Posture, Tremors, Convulsions, Abnormal movements, Impairment of gait.
Open field observations:
Behavior when removed from cage, fur, skin, salivation, nose discharge, lacrimation, eyes/pupil size, posture, palpebral closure, respiration,
tremors, convulsions, abnormal movements/stereotypy, impairment of gait, activity/arousal level, feces excreted within two minutes, urine excreted within two minutes, number of rearings within two minutes.
Sensorimotor tests/reflexes:
Approach response, touch response, vision ("visual placing response"), pupillary reflex, pinna reflex, audition ("startle response"), coordination of
movements ("righting response"), behavior during "handling", vocalization, pain perception ("tail pinch"), grip strength of forelimbs and hindlimbs,
landing foot-splay test, other findings.
Motor activity (MA) measured on the same day as the FOB was performed.
Oestrous cyclicity (parental animals):
No.
Sperm parameters (parental animals):
Parameters examined in male parental generation:
- testis weight
- epididymis weight
- stages of spermatogenesis
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- number and sex of pups
- stillbirths
- live births
- postnatal mortality
- presence of gross anomalies
- weight gain, physical or behavioural abnormalities

BODY WEIGHT
- Pups were weighed on the day after birth (PND 1) and on PND 4

CLINICAL OBSERVATIONS
- Live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no; pups were scheduled sacrifice on PND 4
Postmortem examinations (parental animals):
SACRIFICE /GROSS NECROPSY
All parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs.

HISTOPATHOLOGY / ORGAN WEIGHTS
Organ weights:
- The following weights were determined in all animals: Anesthetized animals, Epididymides, Testes.
- The following weights were determined in 5 animals/sex and test group (females with litters, same animals as used for clinical pathology
examinations): Adrenal glands, Brain, Heart, Kidneys, Liver, Spleen, Thymus
- The following organs/ tissues were preserved in 4% buffered formaldehyde or in modified Davidson’s solution: Adrenal glands, All gross lesions, Aorta, Bone marrow (femur), Brain, Cecum, Cervix, Coagulating glands, Colon, Duodenum, Eyes with optic nerve, Esophagus, Extraorbital lacrimal gland, Epididymides (modified Davidson’s solution), Femur with knee joint, Heart, Ileum, Jejunum (with Peyer’s patches), Kidneys, Larynx, Liver, Lungs, Lymph nodes (axillary and mesenteric), Mammary gland (male and female), Nose (nasal cavity), Ovaries (modified Davidson’s solution), Oviducts, Pancreas, Parathyroid glands, Pharynx, Pituitary gland, Prostate gland, Rectum, Salivary glands, (mandibular and sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, Spinal cord (cervical, thoracic and lumbar cord), Spleen, Sternum with marrow, Stomach (forestomach and glandular stomach), Target organs,Testes (modified Davidson’s solution), Thymus, Thyroid glands, Trachea, Urinary bladder, Uterus, Vagina.

For further evaluation proceeding, see Table 4 in 'Any other information on materials and methods incl. tables'.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring was scheduled sacrifice on PND 4 under isoflurane anesthesia with CO2.
- These animals were subjected to postmortem examinations as follows: All pups were examined externally and eviscerated; their organs were assessed macroscopically. All stillborn pups and all pups that died before PND 4 were examined externally, eviscerated and their organs were assessed macroscopically.
All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding noted.

GROSS EXAMINATION OF DEAD PUPS: yes, Gross necropsy consisted of external examination
All pups with scheduled sacrifice on PND 4 were sacrificed under isoflurane anesthesia with CO2.
All pups were examined externally and eviscerated; their organs were assessed macroscopically.
All stillborn pups and all pups that died before PND 4 were examined externally, eviscerated and their organs were assessed macroscopically.
All pups without notable findings or abnormalities were discarded after their macroscopic evaluation.
Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding noted.

HISTOPATHOLOGY / ORGAN WEIGTHS
Not performed
Statistics:
Please refer to 'Any other information on materials and methods incl. tables'.
Reproductive indices:
Male reproduction data
For the males, mating and fertility indices were calculated:

Male mating index (%) = (number of males with confirmed mating* / number of males placed with females) x 100
*defined by a female with vaginal sperm or with implants in utero

Male fertility index (%) = (number of males proving their fertility* / number of males placed with females x 100
*defined by a female with implants in utero


Female reproduction and delivery data
The number of mating days until vaginal sperm were detected, and gestational status were recorded for F0 females.
For the females, mating, fertility and gestation indices were calculated:

Female mating index (%) = (number of females mated* / number of females placed with males) x 100
*defined as the number of females with vaginal sperm or with implants in utero

Female fertility index (%) = (number of females pregnant* / number of females mated**) x 100
*defined as the number of females with implants in utero
**defined as the number of females with vaginal sperm or with implants in utero

Gestation index (%) = (number of females with live pups on the day of birth / number of females pregnant*) x 100
*defined as the number of females with implants in utero
The total number of pups delivered and the number of liveborn and stillborn pups were noted, and the live birth index was calculated for
F1 litters.

Live birth index (%) = (number of liveborn pups at birth / total number of pups born) x 100

The implantations were counted* and the postimplantation loss (in %) was calculated.
Postimplantation loss (%) = ((number of implantations – number of pups delivered) / number of implantations) x 100

*) apparently non-pregnant uteri were stained according to the method of SALEWSKI (1964)
Offspring viability indices:
Pup number and status at delivery
All pups delivered from the F0 parents (F1 litter) were examined on the day of birth to determine the total number of pups, the sex and the number of liveborn and stillborn pups in each litter. At the same time, the pups were also being examined for macroscopically evident changes. Pups, which died before this initial examination, were defined as stillborn pups.

Pup viability/mortality:
The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1 – 4 (lactation period) were determined. Pups which died accidentally or were sacrificed due to maternal death were not included in thesecalculations. The number of live pups/litter was calculated on the day after birth, and on lactation day 4.

Viability index (%) = (number of live pups on day 4 after birth / number of live pups on the day of birth) x 100

Sex ratio:
On the day of birth (PND 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle; normally, the anogenital distance is considerably greater in male than in female pups. The sex of the pups was finally confirmed at necropsy. The sex ratio was calculated at day 0 and day 4 after birth.

Sex ratio = (number of live male or female pups on day 0/4 / number of live male and female pups on day 0/4) x 100
CLINICAL SIGNS AND MORTALITY (all groups)
Parental animals:
There were no test substance-related or spontaneous mortalities in any of the groups. No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any male or female animals during the whole study.
Detailed clinical observations: Male and female animals of all dose groups (50, 15 and 5 mg/kg bw/d) did not show any abnormalities.

BODY WEIGHT AND WEIGHT GAIN
High-dose females had statistically significantly lower body weights on PND 7 (about 7% below control) and statistically significantly lower body weight change during GD 7 – 14 (about 29% below control). Mean body weights and mean body weight change of the male animals in all test substance-treated groups were comparable to the concurrent control group during the entire study period. Mean body weights of the high-dose
females were comparable to the concurrent control group during premating and gestation period. Additionally, the mean body weight change of the high-dosed females was comparable to the concurrent control group during premating and lactation period. Neither mean body weights nor mean body weight change of the females in the mid and low-dose groups were influenced by the test substance during the whole treatment period.

FOOD CONSUMPTION AND COMPOUND INTAKE
Food consumption of the high-dose F0 females (50 mg/kg bw/d) was statistically significantly below control during GD 14 - 20 (about 13%). Food consumption of the male F0 rats in all dose groups (50, 15 and 5 mg/kg bw/d) was comparable to the concurrent control throughout the entire study. Mid and low-dose females (15 and 5 mg/kg bw/d) did not show any test substance-related changes in food consumption during the whole treatment period.

HAEMATOLOGY
In high dose male rats (50 mg/kg bw/d) total white blood cell (WBC) counts were decreased, which was due to a decrease of the absolute cell counts of lymphocytes, monocytes and eosinophils.

CLINICAL CHEMISTRY
No treatment-related, adverse changes among clinical chemistry parameters were observed.

URINALYSIS
No treatment-related, adverse changes among urinalysis parameters were observed.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS) –

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS) --

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No alterations to reproductive performance or fertility were identified at any dose in the parental rats. However, two sperm positive high-dose females (50 mg/kg bw/d), one sperm positive mid-dose female (15 mg/kg bw/d) and one sperm positive low-dose female (5 mg/kg bw/d) did not deliver F1 pups.

ORGAN WEIGHTS
The liver weight increase and the thymus weight decrease in males of high dose group (50 mg/kg bw/d) were considered to be treatment-related.

GROSS PATHOLOGY
In 5 out of 10 males of the high dose group (50 mg/kg bw/d), the thymus was found to be reduced in size. All other gross findings noted at necropsy are regarded as incidental and spontaneous in nature and are not related to treatment.
Fertility: The five female animals, which were not pregnant as well as the male mating partners did not show relevant gross lesions.

HISTOPATHOLOGY
In 9 out of 10 males of the high dose group (50 mg/kg bw/d), the thymus showed a reduced cellularity in the cortex and medulla. This change ranged from minimal to severe and was characterized by a decrease of the cellular density in the cortex and medulla and loss of the corticomedullary demarcation.
No histopathological correlate was found for the relative liver weight increase in males of the high dose group (50 mg/kg bw/d).
All other findings were either single observations, or were biologically equally distributed between controls and treated rats. All of them were considered to be incidental and/or spontaneous in origin.
Fertility: No histopathological findings were observed in 3 out of 5 non-pregnant females and their corresponding mating males that can explain the lack of offspring. In test groups 1 and 2 (5 and 15 mg/kg bw/d), no histopathology was performed in the other non-pregnant females and their respective mating males.

NEUROBEHAVIOUR
Home cage observations: No test substance-related effects were observed.
Open field observations: No test substance-related effects were observed.
Sensorimotor tests/reflexes: No test substance-related effects were observed.
Quantitative Parameters: No test substance-related effects were observed.
Motor activity measurement: There were no significant deviations concerning the overall motor activity (summation of all intervals) and regarding the single intervals, the statistically significantly decreased number of interrupted beams in mid-dose males (15 mg/kg bw/d) in one interval was considered spontaneous in nature and not treatment related.

OTHER FINDINGS (PARENTAL ANIMALS)
Findings which occurred either individually or were biologically equally distributed over control and treatment groups, were considered to be incidental or spontaneous in origin and without any relation to treatment.
Dose descriptor:
NOAEL
Remarks:
parental systemic toxicity
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Lymphopenia and/or thymus pathology at 50 mg/kg bw/d
Dose descriptor:
NOAEL
Remarks:
parental systemic toxicity
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Changes to body weight parameters at 50 mg/kg bw/d dose in females.
Dose descriptor:
NOAEL
Remarks:
parental reproductive toxicity - fertility
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No alterations to reproductive performance or fertility were identified at any dose
VIABILITY (OFFSPRING)
The viability index indicating pup mortality during lactation (PND 0 - 4) varied between 98.2% (50 mg/kg bw/d dose), 99.2 (15 mg/kg bw/d dose), 98.6 (5 mg/kg bw/d dose) and 99.1% (control) without showing any association to the treatment.

CLINICAL SIGNS (OFFSPRING)
There were no test substance-related adverse clinical signs observed in any of the F1 generation pups of the different test groups.

BODY WEIGHT (OFFSPRING)
The pup body weights were reduced at the 50 mg/kg bw/d dose. Both male and female pup body weights were approximately 12-13% low on PND 1, a difference which became more evident by PND 4 when the body weights were nearly 22% below controls. Pup body weight gain was similarly decreased by as much as a third over this period. The reduction in these body weight parameters was substantial enough to be considered to be a general sign of developmental toxicity. No differences in pup body weight parameters were noted at either the low- (5 mg/kg bw/d) or mid-doses (15 mg/kg bw/d). Neither were any specific substance-dependent developmental toxic effects identified at any dose.

GROSS PATHOLOGY (OFFSPRING)
A few pups showed spontaneous findings at gross necropsy, such as hydroureter, reddish discolored testis, hydronephrosis and post mortem autolysis. All these findings were not considered to be associated to the test substance.

OTHER FINDINGS (OFFSPRING)
The sex distribution and sex ratios of live F1 pups on the day of birth and PND 4 did not show substantial differences between the control and the test substance-treated groups.
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
15 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reduced postnatal offspring weight and weight gain at 50 mg/kg bw/d
Reproductive effects observed:
not specified
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In an OECD 422 study the test substance Dipropylene triamine was administered as an aqueous solution at 0, 5, 15 and 50 mg/kg bw/d daily by gavage to groups of 10 male and 10 female Wistar rats to test for repeated dose toxicity and screen for potential reproductive and developmental effects. After a two-week premating period, these parental animals were mated and the females were allowed to give birth and suckle the offspring until sacrifice on PND 4. Males were treated for 30 days, females for 49 days. Food consumption and body weights of F0 parents were determined. The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PND 1 and on PND 4. Their viability was recorded. At necropsy on PND 4, all pups were sacrificed and examined macroscopically for external and visceral findings. Clinico-chemical and hematological examinations as well as urinalyses were performed in 5 animals per sex and group towards the end of the administration period. At the end of the administration period a FOB was performed and motor activity was measured in 5 parental males and females per group. After sacrifice all F0 parental animals were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed.

Food consumption was mildly reduced in the parental females of the high-dose group (50 mg/kg bw/d) during late-gestation (GD 14-20). This lower food consumption corresponded to slightly decreased parental female body weights beginning during this same time period, but only becoming statistically significant on PND 7. Similarly, there was a statistically significant decrease in body weight gain between GD 14 and GD 20. While there does seem to be an overall downward trend in the body weight parameters of the rats of the high dose group that may indicate the beginnings of systemic maternal toxicity, this reduction was small (<8%). No alterations to either parental female or male body weight parameters were observed during the premating or mating phases. No changes in either food consumption or body weight parameters were noted at either the low- (5 mg/kg bw/d) or mid-doses (15 mg/kg bw/d). Regarding clinical pathology, a slight lymphopenia was noted in males of the high-dose group (50 mg/kg bw/d). No deviations from normal were observed in parental females. Regarding pathology, the thymus of 9 out of 10 males (dose group 50 mg/kg bw/d) showed minimal to severe decreased cellularity in the cortex and medulla. This change, which is regarded as treatment-related and adverse was associated with significantly decreased mean absolute and relative thymus weight and reduced organ size in 5 of 10 male animals. A minimal but significant increase of the mean relative liver weight in males of the high dose group 3 (50 mg/kg bw/d) occurred without any histopathological correlate. Therefore, this change is regarded as treatment-related but not adverse. No alterations to reproductive performance or fertility were identified at any dose in the parental rats. The pup body weights were reduced at the 50 mg/kg bw/d dose. Both male and female pup body weights were approximately 12-13% low on PND 1, a difference which became more evident by PND 4 when the body weights were nearly 22% below controls. Pup body weight gain was similarly decreased by as much as a third over this period. No differences in pup body weight parameters were noted at either the low- (5 mg/kg bw/d) or mid-doses (15 mg/kg bw/d). Neither were any specific substance-dependent developmental toxic effects identified at any dose. All other findings recorded were considered to be incidental in nature and not related to treatment.

Thus, under the conditions of this reproduction/developmental toxicity screening test the NOAEL (no observed adverse effect level) for reproductive performance and fertility is 50 mg/kg body weight/day for the F0 parental rats. The NOAEL for general, systemic toxicity of the test substance is 15 mg/kg bw/d for the F0 parental males based on the lymphopenia and/or thymus pathology identified at 50 mg/kg bw/d. The NOAEL for F0 parental females was found to be 15 mg/kg bw/d based on the changes to body weight parameters at 50 mg/kg bw/d. The NOAEL for developmental toxicity was 15 mg/kg bw/d, based on reduced postnatal offspring weight and weight gain at 50 mg/kg bw/d.


Effects on developmental toxicity

Description of key information
NOAEL (maternal): 50 mg/kg bw/day
NOAEL (developmental): 50 mg/kg bw/day
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
Purity: 99.1%; clear colourless to yellowish liquid
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: Females were approximately 10 to 14 weeks (age at maiting)
- Weight at study initiation: 188-191 g (mean)
- Housing: Females were individually housed in Macrolon plastic cages
- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water (e.g. ad libitum): Free access to tap-water
- Acclimation period: At least 5 days prior to treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24°C
- Humidity (%): 40 - 70%
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made
for the specific gravity/density of the test substance. No correction was made for the purity/composition of the test substance.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of dose preparations were taken at the test facility on a single occasion during the treatment period (formulations were prepared and sampled on 24 August 2015). The samples were dispatched on dry ice to ABL where they were analyzed to assess accuracy of preparation (all groups), homogeneity (lowest and highest concentration) and stability in vehicle over 5 hours at room temperature (lowest and highest concentrations).
Details on mating procedure:
Untreated females were mated at the Supplier, and were at Day 0 or 1 post-coitum on arrival at the Test Facility (Day 0 post-coitum was the day of successful mating; confirmed by vaginal plug).
Duration of treatment / exposure:
From Days 6 to 20 post-coitum, inclusive. Females no. 33 and 77: from Days 6 to 19 post-coitum, inclusive.
Frequency of treatment:
once daily
Duration of test:
On day 21 pos-coitum all survived animals were sacrificed.
Females with early delivery on day 21 (nos. 7, 10, 28, 29, 30, 31 and 73): Within 24 hours of early delivery.
Females with early delivery on Day 20 (nos. 33 and 77): Within 24 hours of early delivery
Remarks:
Doses: 0, 5, 15 and 50 mg/kg bw
Dose selection rational: see "Details on study design".

No. of animals per sex per dose:
22 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based on data from a 14-day range finding study and an OECD 422 study. Within the range finding study with 3 Wistar rats per sex and dose, reduced food consumption (males: up to 22%; females: up to 33%), reduced body weight change in males (up to 51%) and body weight loss in females was observed after treatment by oral gavage at 100 mg/kg bw/day. At 200 mg/kg bw/day the animals showed tremors and piloerection and 1/3 males was found dead on day 1. On study day 3, 2/3 males and 3/3 females were sacrificed moribund. On the basis of the range finding study, doses of 5, 15 and 50 mg/kg bw were used for the subsequent OECD 422 study: decreased food consumption (females: up to 13% from gestation day 14-20), decreased body weight (females: up to 7%), decreased body weight gain (females: up to 29% from gestation day 7-14), decreased white blood cell count (males: 35%), decreased lymphocyte count (males: 38%), decreased monocyte count (males: 45%), decreased eosinophil count (males: 50%), decreased absolute and relative thymus weight (males: 44%/42%), decreased cellularity in cortex and medulla of thymus (males: 9/10) and increased relative liver weight (males: 11%) was observed in the animals of high dose. The body weight of pups was significantly decreased on postnatal day one (males: 13%, females: 12%) and four (males: 21%, females: 22%), the body weight gain of pups was significantly decreased on postnatal days 1-4 (males: 35%, females: 39%). The NOAEL was observed at 15 mg/kg bw.
Based on the findings observed in the range finding and subsequent OECD 422 study and due to an obviously steep dose response relationship, doses of 5, 15 and 50 mg/kg bw were selected for the OECD 414 study.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: al least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from Day 2 post-coitum onwards up to the day prior to necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: Days 2, 6, 9, 12, 15, 18 and 21 post-coitum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption: Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice: on day 21 post-coitum

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
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes: [half per litter]
Statistics:
Dunnett-test, Steel-test, Fischer Exact-test, Mann Whitney test, AVON test, Dunn`s test
Indices:
For each litter the following calculations were performed:
Pre-implantation loss (%) = (number of corpora lutea - number of implantation sites) / (number of corpora lutea) x 100

Post-implantation loss (%) = (number of implantation sites - number of live fetuses) / (number of implantation sites) x 100

The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison, calculating the number of affected fetuses as a
mean litter proportion on a total group basis, where:
Viable fetuses affected/litter (%) = (number of viable fetuses affected/litter / number of viable fetuses/litter) x 100
Historical control data:
This species and strain of rat has been recognized as appropriate for developmental toxicity studies. WIL Research Europe B.V. has historical data on the background incidence of fetal malformations and developmental variations in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of developmental toxicants.
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs were noted that were considered to be related to treatment with the test substance. One female (no.48) at 15 mg/kg bw/day showed alopecia from Day 13 of post coitum onwards. This remained within the range of background findings to be expected for rats of this age and strain.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean body weight, body weight gain and weight gain corrected for uterus weight remained within the same range as controls over the complete study period.
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Description (incidence and severity):
No necropsy findings were noted that were considered to be treatment related. Alopecia in the chest region was recorded in one female (no. 48) at 15 mg/kg bw/day and confirmed the in-life observation in this animal. This remained within the range of background findings to be expected for rats of this age and strain and was considered not to be of toxicological relevance.
Number of abortions:
no effects observed
Description (incidence and severity):
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There were no effects on the number of implantation sites or pre- or post-implantation loss noted with treatment up to 50 mg/kg bw/day.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
All females, including the females that delivered early, were pregnant with viable fetuses.
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
Mortality
No mortality occurred.
Nine females delivered early (two vehicle controls, five 5 mg/kg bw/day-treated females and two 50 mg/kg bw/day- treated females), on Day 20 or 21 post coitum. As these incidences did not show a relationship with the dose, they were considered not to be treatment related.
Clinical Signs
No clinical signs were noted that were considered to be related to treatment with the test substance.
One female (no.48) at 15 mg/kg bw/day showed alopecia from Day 13 of post coitum onwards. This remained within the range of background findings to be expected for rats of this age and strain.
Body Weights
Mean body weight, body weight gain and weight gain corrected for uterus weight remained within the same range as controls over the complete study period.
Food Consumption
No toxicologically relevant treatment related effect on food consumption was noted.
At 50 mg/kg bw/day absolute food consumption was slightly lower on Days 18-21 post coitum (statistically significant). As this was very minimal and food consumption corrected for body weight appeared unaffected by treatment, this change was not considered toxicologically relevant.
Macroscopic Examination
No necropsy findings were noted that were considered to be treatment related.
Alopecia in the chest region was recorded in one female (no. 48) at 15 mg/kg bw/day and confirmed the in-life observation in this animal. This remained within the range of background findings to be expected for rats of this age and strain and was considered not to be of toxicological relevance.
Maternal Pregnancy Data
There were no effects on the number of pregnant females, corpora lutea, implantation sites or pre- or post-implantation loss noted with treatment up to 50 mg/kg bw/day. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. All females, including the females that delivered early, were pregnant with viable fetuses.
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean combined (male and female) fetal body weights were slightly lower (6%) at 5 mg/kg bw/day and 50 mg/kg bw/day (not statistically significant) compared to the concurrent control. As this did not show a relation with dose and the difference was only slight, it was considered not to be treatment related. Mean combined (male and female) fetal body weights were 5.1, 4.8, 5.2 and 4.8 grams for the vehicle control, 5, 15 and 50 mg/kg bw/day, respectively.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
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
External malformations:
no effects observed
Description (incidence and severity):
The numbers of fetuses (litters) available for morphological examination were 226 (22), 232 (22), 224 (22) and 232 (22) in Groups 1, 2, 3, and 4, respectively. External examination was done for all fetuses. There were no external developmental malformations or variations for any of the fetuses following treatment up to 50 mg/kg/day.
Skeletal malformations:
no effects observed
Description (incidence and severity):
There were no treatment related effects on skeletal morphology following treatment up to 50 mg/kg bw/day. Skeletal malformations were noted in one Group 3 fetus and two control fetuses. The Group 3 fetus
(no. A046-04) had bent scapulae and the control fetuses either had fused sternebrae (no. A020-06) or a vertebral centra anomaly (absence of a cervical hemicentrum; no. A007-05). The latter fetus was delivered early. As these malformations were limited to individual fetuses and/or occurred in control fetuses, these were considered chance findings and thus not considered treatment related. The variation of unossified metacarpals and/or metatarsals occurred in 6.3%, 29.4%, 12.3% and 21.6% of fetuses per litter in Groups 1, 2, 3 and 4, respectively. This group distribution would indicate retarded skeletal ossification in Groups 2 and 4, which corresponds with the slightly lower fetal weights in these groups (fetal weights were 5.1, 4.8, 5.2 and 4.8 grams in Groups 1, 2, 3 and 4, respectively). However, retarded ossification in Groups 2 and 4 was not unambiguous substantiated by other ossification parameters such as reduced ossification of the skull and unossified sternebrae nos. 5 and 6. Mean litter percentages for these respective findings were 6.1%, 4.3%, 11.6% and 6.5%, and 0.6%, 5.9%, 0.0% and 0.8% per litter in Groups 1, 2, 3 and 4, respectively. Therefore, and due to absence of a dose-relationship, the finding unossified metacarpals and/or metatarsals was not considered to be related to treatment. The variation of 14th full ribs was observed in 11.7%, 10.8%, 10.0% and 3.5% of fetuses per litter in Groups 1, 2, 3 and 4, respectively. The mean litter proportion in Group 4 was lower (not statistically significant) than in the other groups, but this was not accompanied by a higher incidence for 14th rudimentary ribs in this group (viz.. 56.0%, 70.4%, 70.1% and 68.5% of fetuses per litter in Groups 1, 2, 3 and 4, respectively). Therefore, and due to absence of statistical significance, the lower litter incidence of 14 full ribs in Group 4 was considered to be a change finding and not treatment related. Remaining skeletal variations were not considered treatment related as they occurred infrequently or were observed in control fetuses only.
Visceral malformations:
no effects observed
Description (incidence and severity):
There were no treatment related effects on visceral morphology following treatment up to 50 mg/kg bw/day. Visceral malformations were not observed for any of the fetuses and the variations that were noted, namely small supernumerary lobe(s) or appendix of the liver and partially undescended thymus horn(s), occurred at low incidences and/or in the absence of a dose-related trend.
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Litter Size
There were no treatment-related effects on litter size for any group.
Mean litter sizes were 10.4, 10.4, 10.2 and 10.7 fetuses/litter for the vehicle control, 5, 15 and 50 mg/kg bw/day groups, respectively.
Sex Ratio
The male:female ratio was unaffected by treatment up to 50 mg/kg bw/day.
Fetal Body Weight
Mean combined (male and female) fetal body weights were slightly lower (6%) at 5 mg/kg bw/day and 50 mg/kg bw/day (not statistically significant) compared to the concurrent control. As this did not show a relation with dose and the difference was only slight, it was considered not to be treatment related. Mean combined (male and female) fetal body weights were 5.1, 4.8, 5.2 and 4.8 grams for the vehicle control, 5, 15 and 50 mg/kg bw/day, respectively.
Placenta Weights
Mean placental weights were slightly lower at 50 mg/kg bw/day for both males and females (only statistically significant in males).
Mean male/female placenta weights were 0.47/0.44, 0.48/0.47, 0.47/0.43 and 0.42/0.40 grams for the vehicle control, 5, 15 and 50 mg/kg bw/day, respectively.
Fetal Morphological Examinations
Note: In order to enter animal numbers into WTDMSTM an adjustment in the numbering was made, for example: animal 1 was reassigned as animal A001, animal 2 as A002 etc. Also numbering of fetuses was changed; Fetus 1 of animal 1 was reassigned as A001-01 etc.
The numbers of fetuses (litters) available for morphological examination were 226 (22), 232 (22), 224 (22) and 232 (22) in Groups 1, 2, 3, and 4, respectively. External examination was done for all fetuses, visceral examination was done for approximately half of the fetuses of all groups, and skeletal examination was done for the other half of fetuses. Nine dams had an early delivery.
External Malformations and Variations
There were no external developmental malformations or variations for any of the fetuses following treatment up to 50 mg/kg/day.
Visceral Malformations and Variations
There were no treatment related effects on visceral morphology following treatment up to 50 mg/kg bw/day.
Visceral malformations were not observed for any of the fetuses and the variations that were noted, namely small supernumerary lobe(s) or appendix of the liver and partially undescended thymus horn(s), occurred at low incidences and/or in the absence of a dose-related trend.
Skeletal Malformations and Variations
There were no treatment related effects on skeletal morphology following treatment up to 50 mg/kg bw/day.
Skeletal malformations were noted in one Group 3 fetus and two control fetuses. The Group 3 fetus (no. A046-04) had bent scapulae and the control fetuses either had fused sternebrae (no. A020-06) or a vertebral centra anomaly (absence of a cervical hemicentrum; no. A007-05). The latter fetus was delivered early. As these malformations were limited to individual fetuses and/or occurred in control fetuses, these were considered chance findings and thus not considered treatment related.
The variation of unossified metacarpals and/or metatarsals occurred in 6.3%, 29.4%, 12.3% and 21.6% of fetuses per litter in Groups 1, 2, 3 and 4, respectively. This group distribution would indicate retarded skeletal ossification in Groups 2 and 4, which corresponds with the slightly lower fetal weights in these groups (fetal weights were 5.1, 4.8, 5.2 and 4.8 grams in Groups 1, 2, 3 and 4, respectively).
However, retarded ossification in Groups 2 and 4 was not unambiguous substantiated by other ossification parameters such as reduced ossification of the skull and unossified sternebrae nos. 5 and 6. Mean litter percentages for these respective findings were 6.1%, 4.3%, 11.6% and 6.5%, and 0.6%, 5.9%, 0.0% and 0.8% per litter in Groups 1, 2, 3 and 4, respectively. Therefore, and due to absence of a dose-relationship, the finding unossified metacarpals and/or metatarsals was not considered to be related to treatment.
The variation of 14th full ribs was observed in 11.7%, 10.8%, 10.0% and 3.5% of fetuses per litter in Groups 1, 2, 3 and 4, respectively. The mean litter proportion in Group 4 was lower (not statistically significant) than in the other groups, but this was not accompanied by a higher incidence for 14th rudimentary ribs in this group (viz.. 56.0%, 70.4%, 70.1% and 68.5% of fetuses per litter in Groups 1, 2, 3 and 4, respectively). Therefore, and due to absence of statistical significance, the lower litter incidence of 14 full ribs in Group 4 was considered to be a change finding and not treatment related. Remaining skeletal variations were not considered treatment related as they occurred infrequently or were observed in control fetuses only.
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day
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
Abnormalities:
no effects observed
Developmental effects observed:
not specified

For result tables see "Attached background material"

Conclusions:
Based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) for 3,3’-iminodi(propylamine) was established as being at least 50 mg/kg bw/day.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
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

The prenatal developmental toxicity of 3,3’-iminodi(propylamine) was investigated in an OECD 414 study (BASF SE, 2016). Eighty-eight mated female Wistar Han rats were assigned to four dose groups. The test item was administered once daily by oral gavage from Days 6 to 20 post-coitum at doses of 5, 15, and 50 mg/kg bw/day. The rats of the control group received the vehicle, water, alone. Females were checked daily for the presence of clinical signs. Food consumption and body weight were determined at periodic intervals. Formulations prepared on one day during treatment were analyzed for accuracy, homogeneity and stability.

All animals surviving to Day 21 post-coitum were subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. A laparohysterectomy was performed on each surviving female of the groups. The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. The placentas were weighed. Gravid uterine weights were recorded, and corrected body weights (changes) were calculated. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations. All live fetuses were euthanized. One half of the fetuses in each litter (all groups) was examined in a fresh state for visceral anomalies. These fetuses were decapitated and the heads examined after fixation in Bouin’s fixative. The other half of the fetuses were subjected to skeletal examination after fixation in 96% aqueous ethanol and staining with Alizarin Red S. Accuracy, homogeneity and stability of formulations were demonstrated by analyses.

No maternal toxicity was observed up to 50 mg/kg bw/day. No developmental toxicity was observed up to 50 mg/kg bw/day.

Nine females delivered early (two vehicle controls, five 5 mg/kg bw/day-treated females and two 50 mg/kg bw/day- treated females), on Day 20 or 21 post coitum. As these incidences did not show a relationship with the dose, they were considered not to be treatment related. Additional fetal data collection and morphological examination was performed for the early delivered litters. Consequently, sufficient data was available for developmental toxicity evaluation. Mean placental weights were slightly lower at 50 mg/kg bw/day for both males and females (only statistically significant in males). As there were no treatment related effects on fetal weights and external, visceral or skeletal malformations or variations, the reduced placental weight at 50 mg/kg bw/day was considered to be of no toxicological relevance.

Based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) for 3,3’-iminodi(propylamine) was established as being at least 50 mg/kg bw/day.

Justification for classification or non-classification

The criteria for classification and labelling according to Directive 67/548/EEC and Regulation 1272/2008/EC with respect to effects on fertility and developmental toxicity are not met.