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Administrative data

Description of key information

NOAEL (parental systemic toxicity): 15 mg/kg bw/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-03-26 - 2012-05-20
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 oral 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 (if gavage): 10 mL/kg body weight
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
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
Details on study design:
- 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.
Observations and examinations performed and frequency:
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.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
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.
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

HISTOPATHOLOGY: Yes
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'.
Other examinations:
The supplier assayed the food used in the study for chemical and microbiological contaminants.
The drinking water is regularly assayed for chemical contaminants.
The bedding and the enrichment are regularly assayed for contaminants (chlorinated hydrocarbons and heavy metals).
Statistics:
Please refer to 'Any other information on materials and methods incl. tables'.
Details on results:
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.
Reproductive Performance: 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.

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
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.

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.

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.

OTHER FINDINGS
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
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
15 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Key study is GLP compliant and of high quality (Klimisch 1).

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
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). A reason for this alteration could not be found. 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. Please refer to section “toxicity for reproduction” for results regarding F1 generation.

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.

 

In a non-GLP and non-guidance study (Schmidt et al., 1974), the test substance was administered to groups of 0, 20, 40, 80, and 160 mg/kg bw/d by oral gavage. 40 daily treatments were performed over 54 days. The subsequent post-exposure period lasted 18 days. At 160 mg/kg bw/d all animals died until the 23rd study day. Body weight development was statistically decreased in the 80 mg/kg bw/d dose group. No further information on results is given.

 


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Most reliable study.

Repeated dose toxicity: via oral route - systemic effects (target organ) cardiovascular / hematological: thymus

Justification for classification or non-classification

Based on the results of an OECD 422 study, it is concluded that the classification and labelling criteria according to Directive 67/548/EEC and Regulation 1272/2008/EC are met (R48/22; STOT RE cat. 2).