Registration Dossier

Diss Factsheets

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
18 August 2009-12 October 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
(See also Category polyamines in support of Oleyl dipropylenetriamine attached to Ch.13)
Structurally, the two linear-alkyl dipropylene triamines Oleyl dipropylene triamine and Coco (C12-18) dipropylenetriamine are very similar: a linear alkyl chain and a primary amine at the end, with 2 secondary amines in between separated by a propyl group. Consequently, they share the same chemical reactivity and their physico-chemical properties are very similar from which a comparable toxicological profile can be expected.
Within a specific chemical structure, the variability of the alkyl chain length is considered to have a possible modifying activity, which is related to modification of the physiological properties of the molecule by the increase or shortening of the apolar alkyl chain part. This is suspected to influence aspects related to bioavailability, but not aspects of chemical reactivity and metabolism pathways, aspects that could have an impact on specific mechanisms of toxicity. In series of substances that are chemically identical but differ in length of alkyl chains, those that have shorter chain length are likely to be more bioavailable compared to those with longer chain lengths. Therefore, results from the shortest chain length, in this case Coco (C12-C18 alkyl) dipropylene triamine, can be considered a worst-case approach for the longer chain lengths, specifically Oleyl (C18, unsaturated) dipropylene triamine.
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
N-(3-aminopropyl)-N- (C12-18 even numbered) alkyl-propane-1,3-diamine
Cas Number:
1219458-12-4
Molecular formula:
No molecular formula
IUPAC Name:
N-(3-aminopropyl)-N- (C12-18 even numbered) alkyl-propane-1,3-diamine
Test material form:
liquid: viscous
Details on test material:
- Name of test material (as cited in study report): Coco dipropylene triamine
- Substance type: Yellow clear to turbid liquid with flakes
- Physical state: Liquid
- Stability under test conditions: Stable
- Storage condition of test material: At room temperature in the dark under Nitrogen


Test animals

Species:
rat
Strain:
other: Crl:WI(Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Approximately 12 weeks.
At start treatment, animals were approximately 12 weeks old instead of approximately 10 weeks. A slight deviation in age does not affect the study integrity. Mating started shortly after the animals had attained full sexual maturity according to the OECD 422 guideline.
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages (MIV type, height 18 cm). This also accounts for the Recovery males for the complete treatment period.
Mating: Females were caged together with Main males on a one-to-one-basis in Macrolon cages (MIII type, height 18 cm).
Post-mating: Main males were housed in their home cage (Macrolon cages, MIV type, height 18 cm) with a maximum of 5 animals/sex/cage. Females were individually housed in Macrolon cages (MIII type, height 18 cm).
Lactation: Pups were kept with the dam until termination in Macrolon cages (MIII type, height 18 cm).
General: Sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) was supplied. Certificates of analysis were examined and then retained in the NOTOX archives. During activity monitoring, animals were housed individually in Macrolon cages (MIII type; height 15 cm) with sterilised sawdust as bedding material. No cage-enrichment was provided during overnight activity monitoring.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: At least 5 days prior to start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.7 – 21.9°C
- Humidity (%): 35 - 79%
Temporary deviations from the minimum level of relative humidity occurred in the animal room. Laboratory historical data do not indicate an effect of the deviations.
- Air changes (per hr): approximately 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours artificial light and 12 hours darkness per day. Temporary fluctuations from the light/dark cycle (with a maximum of 1 hour) occurred due to performance of functional observations in the room.


IN-LIFE DATES: From: 18 August 2009 To: 12 October 2009

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Remarks:
specific gravity 1.036
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Based on trial formulations performed at NOTOX. Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the test substance and the vehicle.
Formulations were kept on a magnetic stirrer and in a waterbath of 40°C (maximum) as much as possible before dosing.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at NOTOX and on information provided by the sponsor.
- Concentration in vehicle: 2, 6 and 20 mg/mL

Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
Details on mating procedure:
- M/F ratio per cage: Females were caged together with males on a one-to-one-basis in Macrolon cages (MIII type, height 18 cm).
- Length of cohabitation: Following a minimum of 14 days of exposure for the Main males and Main females, one Main female was cohabitated with one Main male of the same treatment group, avoiding sibling mating (Charles River will supply non-litter mates).

A maximum of 13 instead of 14 days was allowed for mating. One additional day of mating would probably not have resulted in successful mating of male no. 4 with female no. 54, and male no. 29 with female no. 74. In addition, sufficient litters were obtained in the dose group for an adequate evaluation of the study results.

- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion the treatment phase, according to a validated method (NOTOX project 491240). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration).

The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration for solutions. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

Results:
The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).

The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%). Formulations at the entire range were stable when stored at room temperature for at least 6 hours.

The analyzed concentrations of the test samples were corrected with the mean recovery of the procedural recovery samples, because the procedural recovery samples had recoveries of 116% and 124% and the analysed concentrations of the test samples had a similar tendency. With the correction procedure it was possible to obtain adequate results for the test samples.
Duration of treatment / exposure:
Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for at least 42 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to scheduled necropsy (Group 4 animals were not dosed on Day 9, and were sacrificed for humane reasons on Day 9).
Details on study schedule:
- Age at mating of the mated animals in the study: Approximately 14 weeks.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 10, 30 and 100 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
10, and an extra 5 males for Group 1 and 4. The study included a recovery phase for males only. These animals were not mated and, consequently, were not used for the assessment of reproduction/developmental toxicity. The control recovery allocation animals did not undergo a recovery period, but were sacrificed together with the control main allocation animals and the Group 4 allocation animals did not undergo a recovery period, but died spontaneously or were killed in extremis on Day 9.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected based on the results of the dose range finding/MTD study with Coco dipropylene triamine (NOTOX Project 491236). See End point Study Record 7.5.1: Repeated Dose Toxicity: oral.rat_NOTOX 491236.
Positive control:
no

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily (early morning/late afternoon)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from start of treatment onwards, between approxi-mately 1 and 2 hours after dosing detailed clinical observations were made in all animals. Once prior to start of treatment and at weekly intervals this was also performed outside the home cage in a standard arena. At weekly intervals this was also performed outside the home cage in a standard arena. Arena observations were not performed when the animals were mating, or housed individually.
No arena observations were conducted prior to dosing on Day 1 of the premating phase. Based on the conducted clinical observations on Day 1 it is considered that no significant abnormalities would have been detected at this predose arena observation.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter (except inadvertently for males on Day 14 of the mating period). Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum, and during lactation on Days 1 and 4. In order to monitor their health status, all Group 4 animals were also weighed on Day 6.
At the end of the last week of the mating period, no body weights were determined for males. Sufficient body weight measurements were conducted (including body weight measurements at termination) to allow an adequate interpretation of the study results.

FOOD CONSUMPTION: Yes
Weekly, for males and females. Food consumption was not recorded during the mating period. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

FOOD EFFICIENCY:
(Average food consumption (per animal per day)/average body weight per cage) X 1000

WATER CONSUMPTION : Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.00 and 10.30 a.m.
- Anaesthetic used for blood collection: Yes, iso-flurane
- Animals fasted: Yes, but water was available
- How many animals: 5 animals/sex/group (females with live offspring only).
- Parameters examined were: White blood cells, Differential leucocyte count (neutrophils, lymphocytes, monocytes,eosinophils, basophils), Red blood cells, Reticulocytes, Red blood cell distribution width, Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelets, Prothrombin time, Activated Partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.00 and 10.30 a.m.
- Anaesthetic used for blood collection: Yes, iso-flurane
- Animals fasted: Yes, but water was available
- How many animals: 5 animals/sex/group (females with live offspring only).
- Parameters examined were: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Total Protein, Albumin, Total Bilirubin, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate, Bile acids.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females were tested during lactation (all before blood sampling).
- Dose groups that were examined: Group 1, 2 and 3
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, grip strength and motor activity.
Oestrous cyclicity (parental animals):
not determined
Sperm parameters (parental animals):
Parameters examined in all male parental animals:
testis weight, epididymis weight.
For 5 males of the control and high dose group, slides of the testes were prepared to examine staging of spermatogenesis.

OTHER:
General reproduction data:
Male number paired with, mating date, confirmation of pregnancy, and delivery day was recorded.
Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of these females was examined to detect signs of abortion or premature birth.
Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.
Litter observations:
SPARAMETERS EXAMINED
Each litter was examined to determine the following, if practically possible:

Mortality / Viability:
The numbers of live and dead pups at the First Litter Check (= check at Day 1 of lactation) and daily thereafter were determined. If possible, defects or cause of death were evaluated.

Clinical signs:
At least once daily, detailed clinical observations were made in all animals.

Body weights:
Live pups were weighed on Days 1 and 4 of lactation.

Sex:
SDetermined for all pups on Days 1 and 4 of lactation (by assessment of the ano-genital distance).

GROSS EXAMINATION OF DEAD PUPS: Yes
All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated.
Postmortem examinations (parental animals):
All animals were fasted overnight (with a maximum of approximately 23.5 hours) prior to planned necropsy, but water was provided. Animals surviving to scheduled necropsy and animals killed in extremis were anaesthetised using iso-flurane (Abbott Laboratories Ltd., Hoofddorp, The Netherlands) and subsequently exsanguinated.

Necropsy was conducted on the following days:
Females which delivered: Lactation Day 5 and 7
Females which failed to deliver: Post-coitum Day 25-27 (females with evidence of mating) or approximately 21 days after the last day of the mating period (females without evidence of mating).
Males: Following completion of the mating period (a minimum of 28 days of dose administration).

Several animals were necropsied later than after a maximum of 20 hours fasting, i.e. with a maximum of approximately 3.5 hours. The fasting period was only slightly longer and was considered not to have adversely affected the clinical laboratory, macroscopic or microscopic findings.

GROSS PATHOLOGY: Yes
After sacrifice or death all parental animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. No macroscopic examination was conducted on control recovery animals. Descriptions of all macroscopic abnormalities were recorded.

Samples of the following tissues and organs were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands):
Selected 5 animals/sex/group and all animals that were killed in extremis (except for Group 1 recovery animals): Identification marks: not processed, Ovaries. Adrenal glands, Pancreas, Aorta, Peyer's patches (jejunum, ileum) if detectable, Brain (cerebellum, mid-brain, cortex), Pituitary gland, Caecum, Preputial gland, Cervix, Prostate gland, Clitoral gland, Rectum, Colon, (Salivary glands - mandibular, sublingual), Duodenum, Sciatic nerve, Epididymides*, Seminal vesicles including coagulating gland, Eyes with optic nerve (if detectable) and Harderian gland*, Skeletal muscle, (Skin), (Female mammary gland area), Spinal cord (cervical, midthoracic, lumbar), Femur including joint, Spleen, Heart, Sternum with bone marrow, Ileum, Stomach, Jejunum, Testes*, Kidneys, Thymus, (Lacrimal gland, exorbital), Thyroid including parathyroid (if detectable), (Larynx), (Tongue), Liver, Trachea, Lung, infused with formalin, Urinary bladder, Lymph nodes (mandibular, mesenteric), Uterus, (Nasopharynx), Vagina, Oesophagus, All gross lesions.

All remaining animals and females which failed to deliver$:
Identification marks: not processed, Prostate gland, Cervix, Seminal vesicles including coagulating glands, Clitoral gland, Testes*, Epididymides*, Uterus, Ovaries, Vagina, Preputial gland, All gross lesions.

* Fixed in modified Davidson's solution (prepared at NOTOX using Formaldehyde 37-40%, Ethanol, Acetic acid (glacial)(all Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)) and transferred to formalin after fixation for at least 24 hours.
$ In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique (Salewski, 1964) in order to detect any former implantation sites (Salewski staining prepared at NOTOX using Ammoniumsulfide-solution 20% (Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)).

Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.

ORGAN WEIGHTS: Yes
The following organ weights (and terminal body weight) were recorded from the surviving animals:

Selected 5 animals/sex/group: Adrenal glands, Spleen, Brain, Testes, Epididymides, Thymus, Heart, Uterus (including cervix) , Kidneys, Prostate*, Liver, Seminal vesicles including coagulating glands*, Ovaries, Thyroid including parathyroid*.
* weighed when fixed for at least 24 hours.

From all remaining Main males: Epididymides, Testes.

No organ weights were determined from Control Recovery males and from Recovery Group 4 males.

For two animals no thyroid weight was determined. Sufficient thyroid weight data were collected for adequate interpretation.

HISTOTECHNOLOGY: Yes
All organ and tissue samples, as defined under Histopathology (following), were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands).

Of the selected 5 males/group of Group 1 and 3, additional slides of the testes were prepared to examine staging of spermatogenesis. The testes were processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin (Klinipath, Duiven, The Netherlands).

Organ and tissue samples collected from Group 4 animals other than gross macroscopic lesions were not processed into histological slides.

HISTOPATHOLOGY: Yes
The following slides were examined by a pathologist:
- The preserved organs and tissues of the selected Main animals/sex of Groups 1 and 3.
- The additional slides of the testes of the selected 5 Main males of Groups 1 and 3 to examine staging of spermatogenesis.
- The preserved organs and tissues of the animals of all dose groups which died spontaneously or were killed in extremis, except for the Group 4 animals in which only tissues with macroscopic findings were processed.
- The reproductive organs* of animals that failed to mate, conceive, sire or deliver healthy pups:
-Group 1: One male and one female (failed to mate)
-Group 3: One male and one female (failed to mate), One male (his mated female died shortly after mating. As there was no proof that the male generated a pregnancy, because the selected female was found dead 2 days after mating, this animal was also examined for any signs of infertility)
- Ileum, jejunum and mesenteric lymph nodes of all Group 2 animals, and mesenteric lymph nodes of all Group 4 animals.
- All gross lesions of all animals (all dose groups).

* Reproductive organs included the cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testis, uterus, and vagina.

One clitoral gland, ovaries from two animals and bone marrow from one animal were not found during processing for histopathology. Sufficient data was available for evaluation

All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.
Postmortem examinations (offspring):
SACRIFICE
Pups were killed by decapitation on Day 5 or 7 of lactation.

GROSS NECROPSY
All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated.

HISTOPATHOLOGY / ORGAN WEIGTHS
no.
Statistics:
The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Dunnett, 1955) (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (Miller, 1981) (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Fisher, 1950) was applied to frequency data.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.

Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may be rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
Reproductive indices:
For each group the following calculations were performed:

Percentage mating = Number of females mated/Number of females paired x 100

Fertility index = Number of pregnant females/Number of females paired x 100

Conception rate = Number of pregnant females/Number of females mated x 100

Gestation index = Number of females bearing live pups/Number of pregnant females x 100

Duration of gestation = Number of days between confirmation of mating and the beginning of parturition
Offspring viability indices:
Percentage live males at First Litter Check = Number of live male pups at First Litter Check/Number of live pups at First Litter Check x 100

Percentage live females at First Litter Check = Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100

Percentage of postnatal loss Days 0-4 lactation = Number of dead pups on Day 4 lactation/Number of live pups at First Litter Check x 100

Viability index = Number of live pups on Day 4 lactation/Number of pups born alive x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
At 100 mg/kg/day clinical signs primarily consisted of hunched posture and piloerection, and at a lower incidence, lethargy, laboured respiration, rales, ptosis, pale appearance and diarrhoea with brown staining of the genital region. At 30 mg/kg, hunched posture, piloerection and salivation were noted for female no. 72 who died spontaneously and for one male for a few days during the mating period only.

No treatment-related clinical signs were seen at 10 mg/kg.

Incidental clinical signs seen among control and treated animals included scabbing and alopecia of various body parts. These findings were within the range considered normal for animals of this age and strain. Salivation incidentally seen after dosing at 30 and 100 mg/kg/day was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing).

Several males at 100 mg/kg/day (animal nos. 38, 39, 41 and 44) died spontaneously before their scheduled necropsies. For two of these animals, marked ulceration of the stomach was the most likely cause of death; no cause of death was determined for the other animals. Due to the number of spontaneous deaths and the moribund condition of many of the animals at 100 mg/kg/day, it was decided to euthanize the remainder of the main animals at 100 mg/kg/day (of both sexes) and recovery animals for humane reasons.

A single female at 30 mg/kg (animal no. 72) died spontaneously before her scheduled necropsy, and no definitive cause was determined for her death. No further mortality occurred at 30 mg/kg/day, and no mortality occurred at 10 mg/kg/day or among control group animals.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
At 100 mg/kg/day, a statistically significant lower absolute body weight and body weight gain was observed for both sexes on Day 8 of the pre-mating period. Weight loss (up to 15%) or no weight gain was observed for all animals before death or euthanasia on Day 9.

No toxicologically relevant changes in body weight (gain) were observed at 10 and 30 mg/kg/day.

The statistically significant lower body weight gain of males at 30 mg/kg/day on Day 8 of the mating period was considered to be of no toxicological relevance since this change was slight in nature and within the range considered normal for rats of this age and strain.

REPRODUCTIVE PERFORMANCE:
No toxicologically relevant effects on reproductive parameters, fertility index and conception rate were noted (see table above).

No toxicologically relevant changes in precoital time, number of corpora lutea and implantation sites were noted. A statistically significant lower number of implantation sites was noted at 30 mg/kg/day. Mean implantation site numbers remained within the historical control range (95% confidence interval: 6.0-16.0, mean 12.5, n=227), and control values were considered to be slightly high compared to this control range. Also, since no further changes were noted in reproductive parameters, this was not considered to be of toxicological relevance.

DEVELOPMENTAL DATA:
No toxicologically significant effects on the number of females with live pups, gestation index, duration of gestation and early postnatal pup development (body weight, clinical signs, viability index and external macroscopy) were noted.

No deficiencies in maternal care were observed. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No signs of difficult or prolonged parturition were noted among the pregnant females.

At 30 mg/kg/day, a total of seven females were pregnant. Whilst this number is lower than the recommended number of pregnant females of eight as specified in the OECD 422 guideline, it was considered that the results obtained at this dose level allowed a meaningful evaluation of reproduction and developmental data.

Two litters in the control group had pups that were born dead or were missing before the scheduled necropsy. For litter 57, two pups were found dead at the first litter check, and one other pup was missing on lactation Day 3. For litter 59, two were dead at the first litter check. Macroscopy of these animals revealed autolysis, absence of milk in the stomach and/or cannibalism. No mortality occurred among pups delivered at 10 and 30 mg/kg/day. No clinical signs were noted among surviving pups.

ORGAN WEIGHTS (PARENTAL ANIMALS)
At 30 mg/kg, a statistically significant lower absolute thymus weight and thymus to body weight ratio occurred for males.

Organ weights and organ to body weight ratios at 10 mg/kg/day were similar to control levels.

The statistically significant higher brain and adrenal to body weight ratios for males at 10 and/or 30 mg/kg/day were considered to be of no toxicological relevance since these occurred in the absence of a dose-related trend and histopathological correlates, remained within the range considered normal for rats of this age and strain, and/or were related to slightly lower terminal body weights.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Macroscopic abnormalities among animals at 100 mg/kg/day were primarily confined to changes in the gastrointestinal tract and consisted of irregular surface of the forestomach and dilation of the small intestines and gelatinous/yellowish contents of the small intestines. Other, less frequent, changes in the gastrointestinal tract consisted of dilation of the caecum and red foci on the glandular mucosa of the stomach.

At 30 mg/kg, no macroscopic findings were noted that were considered to be related to treatment, except for beginning autolysis and reddish discoloration of the small intestines for one female (no. 72) that died spontaneously.

No toxicologically relevant macroscopic findings were noted at 10 mg/kg.

Advanced autolysis and cannibalism of several organs were noted for one male at 100 mg/kg/day (no. 39) that died spontaneously.

Incidental findings among control and treated animals included a reduced size of the thymus and red foci on the thymus, red foci on the forestomach, thickened limiting ridge of the stomach, gray-white foci on the forestomach, red foci on the stomach glandular mucosa, reddish discoloration of the jejunum, gastrointestinal tract distended with gas, nodules on the epididymides, clitoral glands or uterine adipose tissue, red-brown foci on the clitoral glands, a reddish focus on the left lateral lobe of the liver, a yellowish focus on the tongue, reduced size of the clitoral gland (left side), reduced size of the testes and epididymides and alopecia or scabbing. The incidence of these findings was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. These necropsy findings were therefore considered to be of no toxicological significance.

HISTOPATHOLOGY (PARENTAL ANIMALS)
The following microscopic findings were considered to be related to treatment:
- Jejunum: foamy macrophage infiltrate in 1/5 males at 10 mg/kg/day (minimal), 3/5 males and 3/6 females at 30 mg/kg/day (minimal or slight), and 13/14 males and 10/10 females at 100 mg/kg/day (minimal to moderate).
- Ileum: foamy macrophage infiltrate in 3/5 males and 4/5 females at 10 mg/kg/day (minimal), 4/4 males and 6/6 females at 30 mg/kg/day (minimal to slight) and 13/14 males and 10/10 females at 100 mg/kg/day (minimal to moderate).
- Mesenteric lymph node: foamy macrophage foci in 4/5 males and 4/5 females at 10 mg/kg/day (minimal to slight), 5/5 males and 6/6 females at 30 mg/kg/day (minimal to marked), and in 15/15 males and 10/10 females at 100 mg/kg/day (minimal to marked).
- Stomach (100 mg/kg/day):
- Hyperplasia of the squamous epithelium of the forestomach in 13/14 males and 8/10 females (minimal to marked).
- Forestomach inflammation in 13/14 males and 9/10 females (minimal to marked).
- Diffuse hyperkeratosis in 13/14 males and 3/10 females (minimal to moderate).
- Ulcer in the forestomach in 8/14 males and 6/10 females.

There was no microscopic correlate to the dilation and gelatinous and/or yellowish contents of the small intestine and caecum.

All other microscopic findings recorded were considered to be within the normal range of background pathology encountered in Wistar rats of this age and strain.

One selected male rat at 30 mg/kg/day (no. 29) had a bilateral grade 5 oligospermia which accounted for its infertility and prohibited staging of the spermatogenesis for this animal. This was considered to be a spontaneous developmental abnormality with no likely relationship to treatment. No other abnormalities were seen in the reproductive organs of non-fertile animals which could account for infertility.

The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis which could be related to the test item.

Effect levels (P0)

Dose descriptor:
NOEL
Remarks:
not identified
Effect level:
< 10 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on the presence of foamy macrophage infiltration in the ileum and jejunum and foamy macrophage foci in the mesenteric lymph nodes at 10 mg/kg/day, a parental No Observed Effect Level (NOEL) could not be determined.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined

Details on results (F1)

REPRODUCTIVE DATA:
No toxicologically relevant effects on reproductive parameters, fertility index and conception rate were noted (see table above).

No toxicologically relevant changes in precoital time, number of corpora lutea and implantation sites were noted. A statistically significant lower number of implantation sites was noted at 30 mg/kg/day. Mean implantation site numbers remained within the historical control range (95% confidence interval: 6.0-16.0, mean 12.5, n=227), and control values were considered to be slightly high compared to this control range. Also, since no further changes were noted in reproductive parameters, this was not considered to be of toxicological relevance.

DEVELOPMENTAL DATA:
No toxicologically significant effects on the number of females with live pups, gestation index, duration of gestation and early postnatal pup development (body weight, clinical signs, viability index and external macroscopy) were noted.

No deficiencies in maternal care were observed. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No signs of difficult or prolonged parturition were noted among the pregnant females.

At 30 mg/kg/day, a total of seven females were pregnant. Whilst this number is lower than the recommended number of pregnant females of eight as specified in the OECD 422 guideline, it was considered that the results obtained at this dose level allowed a meaningful evaluation of reproduction and developmental data.

Two litters in the control group had pups that were born dead or were missing before the scheduled necropsy. For litter 57, two pups were found dead at the first litter check, and one other pup was missing on lactation Day 3. For litter 59, two were dead at the first litter check. Macroscopy of these animals revealed autolysis, absence of milk in the stomach and/or cannibalism. No mortality occurred among pups delivered at 10 and 30 mg/kg/day. No clinical signs were noted among surviving pups.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 30 other: mg/kg/day
Sex:
male/female
Basis for effect level:
other: No reproductive/developmental toxicity was observed at any dose level.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
A reproduction/developmental NOAEL of 30 mg/kg/day was determined.
Executive summary:

Coco dipropylene triamine was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 0, 10, 30 and 100 mg/kg/day. The males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 28 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 42-55 days).

Formulation analysis showed that the formulations were prepared accurately, were homogeneous and were stable for at least 6 hours at room temperature.

Parental findings:

At 100 mg/kg/day, five males died spontaneously or were sacrificed over Days 6-9 of treatment; the remaining animals of both sexes (including the recovery animals) were euthanized in extremis on Day 9. Toxicologically-relevant clinical signs that were noted among the majority of animals of both sexes included hunched posture and piloerection, and at a lower incidence, lethargy, laboured respiration, rales, ptosis, pale appearance and diarrhoea with brown staining of the genital region. Significant weight loss was observed among these animals (up to 15%) along with notably lower food intake levels. Additionally, several macroscopic findings were noted in both sexes, and most commonly included irregular surface of the forestomach, and gelatinous/yellowish contents and dilation of the small intestines. The most prominent histopathological finding seen in most animals at 100 mg/kg/day consisted of ulceration of the stomach which was considered to be the most likely cause of death/moribundity for these animals. Other histopathological changes noted among all animals of this dose group included inflammation and diffuse hyperkeratosis of the stomach, hyperplasia of the squamous epithelium of the forestomach, and foamy macrophage infiltrate of the jejunum, ileum and mesenteric lymph nodes.

At 30 mg/kg/day, one female was found dead on Day 20 of treatment. Prior to death this female showed hunched posture, piloerection and salivation. Histopathological examination did not reveal an apparent cause of death. However, given that all animals at 100 mg/kg/day were found dead or sacrificed, it could not be excluded that this death was related to treatment. Motor activity at this dose level appeared slightly reduced but there were no supportive clinical signs (such as lethargy). Toxicologically relevant findings at 30 mg/kg/day included higher absolute and relative neutrophil counts and higher absolute white blood cell counts, and lower lymphocyte counts (males and/or females) along with a corresponding reduction in absolute and relative thymus weights (males). These lower thymus weights were not correlated histopathologically. Microscopic findings noted in most animals of this dose group included foamy macrophage infiltration of the jejunum and ileum and foamy macrophage foci in the mesenteric lymph node.

At 10 mg/kg, treatment-related findings were confined to microscopic findings that included foamy macrophage infiltration in the ileum and jejunum and foamy macrophage foci found in the mesenteric lymph nodes of most animals.

Reproductive/Developmental findings:

No reproductive/developmental toxicity was observed at any dose level.

Based on the presence of foamy macrophage infiltration in the ileum and jejunum and foamy macrophage foci in the mesenteric lymph nodes at 10 mg/kg/day, a parental No Observed Adverse Effect Level (NOAEL) could not be determined.

A reproduction/developmental NOAEL of 30 mg/kg/day was determined.