Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

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:
12 August 2009 - 09 October 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD 422 guidelines and GLP principles.
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
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:
Amines, N-(3-aminopropyl)-N’-[3-(C18 and C18-unsatd. alkyl amino)propyl]trimethylenedi and amines, N-(3-aminopropyl)-N’-(C18 and C18-unsatd. alkyl)trimethylenedi-
EC Number:
628-862-9
Cas Number:
1219458-11-3
Molecular formula:
UVCB - No molecular formula; Avg mw = 390.26 mw C16 Tetramine = 412.751 mw C18 Tetramine = 440.805
IUPAC Name:
Amines, N-(3-aminopropyl)-N’-[3-(C18 and C18-unsatd. alkyl amino)propyl]trimethylenedi and amines, N-(3-aminopropyl)-N’-(C18 and C18-unsatd. alkyl)trimethylenedi-
Test material form:
solid
Details on test material:
- Name of test material (as cited in study report): Tallow tripropylenetetramine
- Substance type: White paste
- Physical state: solid
- Analytical purity: (Certificate of Analysis is included in report)
- Impurities (identity and concentrations): Free Acrylonitrile < 4ppm and water 0,2%
- Composition of test material: see Certificate of Analysis.
- Purity test date: 05 February 2009
- Lot/batch No.: S001255
- Expiration date of the lot/batch: 23 January 2018
- Stability under test conditions: Yes: analytical verification of doses or concentrations performed
- Storage condition of test material: At room temperature in the dark under nitrogen.
- Other:
Density: 850 kg/m3 at 60°C
pH (1% in water, indicative range): 11.0
Stability at higher temperatures: Yes, maximum temperature: 80°C
Maximum duration: several hours
Solubility in Propylene glycol: Unknown

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): 19.8-21.6°C
- Humidity (%): 38-80%
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: 12 August 2009 To: 09 October 2009

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Remarks:
(specific gravity 1.036)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level by heating the formulation to 50°C for at least 10 minutes. Adjustment was made for specific gravity of the test substance and vehicle.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at NOTOX.
- Concentration in vehicle:6, 20, 60 mg/mL

Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated based on the latest individual body weight.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: A maximum of 13 instead of 14 days was allowed for mating. All females had mated within this period
- 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.
- 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 during the treatment phase , according to a validated method (NOTOX project 491257). 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 between 90% and 110% of the target concentration for Solutions (Group 2) or between 85-115% of the target concentration for suspensions (Groups 3 and 4). 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% for Group 2 and between 85% and 115% for Group 3 and Group 4. The formulation of Group 2 was a solution and the formulations of Group 3 and Group 4 were suspensions.

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.
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 42-54 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. Four females were not dosed during littering.
Details on study schedule:
- Age at mating of the mated animals in the study: 14 weeks.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 30, 100 and 300 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.
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 (NOTOX Project 491253). See Endpoint Study Record 7.5.1: Repeated dose toxicity: oral.NOTOX 491255
- Rationale for animal assignment (if not random): 5 animals/sex/group (main groups only) were randomly selected at allocation for functional observations, clinical pathology, macroscopic examination (full list), organ weights (full list) and histopathology (females with live offspring only).

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 onwards, between approximately 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. Arena observations were not performed when the animals were mating, or housed individually.

On a single day during post-coitum phase no clinical observations were conducted, and on another day of the post-coitum phase a clinical observation was also conducted immediately after dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. 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 the health status all Group 4 animals were also weighed on Day 10.


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 during lactation on Days 1 and 4 lactation. For one female (Group 3) no food consumption was determined over Days 11-14 post-coitum.

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

WATER CONSUMPTION : No. 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). No blood was collected from the control recovery animals from Group 1 or 4.
- 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.
- Animals fasted: Yes
- How many animals: 5 animals/sex/group (females with live offspring only). No blood was collected from the control recovery animals from Group 1 or 4.
- 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 selected males:
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:
PARAMETERS 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:
Sex was determined 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):
SACRIFICE
All animals were fasted overnight (with a maximum of approximately 21.5 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 6.
Female which failed to deliver: Post-coitum Day 25 (female with evidence of mating).
Males (Main): Following completion of the mating period (a minimum of 28 days of dose administration).
Males (Recovery): Control allocation: sacrificed together with the control Main allocation animals.
Group 4 allocation: killed in extremis on Day 10.

Several animals were necropsied later than after a maximum of 20 hours fasting, i.e. with a maximum of 1 hour and 20 minutes. 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 following animals on the scheduled day of necropsy:

Selected 5 animals/sex/group (Group 1-3): 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.

All remaining males (Group 1-3): Epididymides, Testes.
No organ weights were determined from Control Recovery males and from Recovery Group 4 males.

Inadvertently, the thyroid of one group 3 female and bodyweight of one group 1 female were not weighed at necropsy.
Sufficient body weight data were available for evaluation. The thyroid weight of the female would not have been used for interpretation.

HISTOPATHOLOGY: Yes
The following slides were examined by a pathologist:
- The preserved organs and tissues of the selected 5 Main animals 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 Group 4 animals for which only tissues with macroscopic findings were processed (and other organs as specified below).
- The reproductive organs (cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testis, uterus, and vagina) of animals that failed to mate, conceive, sire or deliver healthy pups:
Group 3: one male and one female which failed to conceive, and one male of which the cohabitated female during mating was killed in extremis on Day 21 post-coitum. Therefore, there was no proof that this male generated a pregnancy with live offspring.
- Stomach, duodenum, jejunum, ileum, caecum of the selected animals of Groups 2 and all animals of Group 4.
- Thymus and mesenteric lymph node of the selected animals of Groups 2, and mesenteric lymph node of all animals of Group 4.
- All gross lesions of all animals (all dose groups).

The thymus of two males (group 4) and the mesenteric lymph node of one male were not collected at necropsy (not found during trimming). The thymus/mesenteric lymph node of Group 4 males were not intended to be examined histopathologically.


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 6 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.

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 mated: Number of females mated/Number of females paired x 100

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

Conception index: 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 of lactation:
Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check x 100

Viability index (%):
Number of live pups on Day 4 of 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
All animals (including recovery males) at 300 mg/kg were killed in extremis on Day 10 of treatment. Microscopic findings in the stomach (including ulcers and erosions) were considered to have contributed to the moribundity.

At 100 mg/kg, one male and one female were killed in extremis on Day 27 and 38 of treatment, respectively. No clear cause of moribundity was established histopathologically for the male, but for the female microscopic findings in the stomach (including an ulcer) were considered to have contributed to the moribundity.

No further mortality occurred among the dose groups.

At 300 mg/kg/day, clinical signs primarily included hunched posture and piloerection, and at lower frequency also laboured or shallow respiration, rales, lethargy, diarrhoea, faeces containing mucus and ptosis.

At 100 mg/kg/day, clinical signs primarily consisted of piloerection among females, and at lower frequency hunched posture, rales, laboured respiration and ptosis were incidentally observed among the animals.

No toxicologically relevant clinical signs were observed at 30 mg/kg/day. Piloerection was only incidentally observed for a single female of this dose group during lactation and hence considered to be of no toxicological relevance.

Incidental clinical signs seen among control and treated animals included alopecia of various body parts, chromodacryorrhoea, watery discharge from the eye and salivation. At the incidence observed these findings were within the range considered normal for animals of this age and strain. No clinical signs were noted among control males.

BODY WEIGHT AND WEIGHT GAIN
At 300 mg/kg/day, all animals showed weight loss (up to 13%) between Days 1-8 of the pre-mating period.

At 100 mg/kg/day, lower absolute body weights and body weight gain were recorded during the Repro period for males and females, achieving a level of statistical significance on most occasions. Females also showed a statistically significant lower body weight during post-coitum and lactation.

At 30 mg/kg/day, a statistically significant lower body weight gain was recorded for males during the Repro period, resulting in a 3% lower body weight compared to the control group at the end of the study. Body weight (gain) of females at this dose level remained similar to controls throughout the observation period.

FOOD CONSUMPTION
At 300 mg/kg/day, absolute and relative food consumption were notably lower than controls for males and females over Days 1-8 of the pre-mating period.

At 100 mg/kg/day, absolute and relative food consumption of females was lower than controls during the premating phase, and remained lower during the post-coitum period (with statistical significance on most occasions). No apparent treatment-related change in (relative) food consumption was observed among females during lactation.

At 30 mg/kg/day, (relative) food consumption was similar to control levels throughout the observation period.

HAEMATOLOGY
A statistically significant higher relative and absolute neutrophil count occurred for males at 100 mg/kg/day. Females at 30 and 100 mg/kg/day also showed higher relative neutrophil counts, but absolute neutrophil counts were not statistically significantly different from controls.

The statistically significant higher relative lymphocyte counts in males at 100 mg/kg/day and in females at 30 and 100 mg/kg/day occurred without a concurrent change in absolute lymphocyte numbers, and was therefore considered to be without toxicological relevance.

No further treatment-related changes in haematology parameters were noted among the dose groups. The statistically significant lower prothrombin time (PT) of males at 30 mg/kg/day occurred in the absence of a dose-related trend and remained within the range considered normal for rats of this age and strain. The notably higher partial thromboplastin time (APTT) of one female at 100 mg/kg/day was considered not to be related to treatment since other animals of this dose group showed normal values for this parameter.

CLINICAL CHEMISTRY
The following statistically significant changes in clinical biochemistry parameters distinguished animals at 100 mg/kg/day from control animals:
- Higher alanine aminotransferase activity (ALAT) in males and females,
- Higher aspartate aminotransferase activity (ASAT) in females
- Lower total protein level in males,
- Lower albumin level in males,
- Higher total bilirubin level in females,
- Higher urea level in females,
- Higher cholesterol level in females,
- Higher bile acid level in females.

No toxicologically relevant clinical biochemistry changes were noted at 30 mg/kg/day. The statistically significant lower calcium level of males at 30 mg/kg/day occurred in the absence of a dose-related trend and the mean remained within the range considered normal for rats of this age and strain. No toxicological relevance was therefore ascribed to this change.

NEUROBEHAVIOUR
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals.

The variation in motor activity did not indicate a relation with treatment. Two females at 100 mg/kg/day showed higher motor activity values recorded by the low sensors. Since a similar change was not observed among other animals of this dose group, no toxicological relevance was ascribed to these variations.

ORGAN WEIGHTS
The following (statistically significant) changes in organ weights and organ to body weight ratios distinguished animals at 100 mg/kg/day from control animals:
- Lower liver weight in males,
- Lower spleen weight in males and females (not statistically significant),
- Lower prostate weight (not statistically significant),
- Lower heart weight in males and females (not statistically significant for males),
- Lower thymus weight in males and females, and lower thymus to body weight ratio in males.

The statistically significant higher testes to body weight ratio at 100 mg/kg/day was considered to be related to the lower terminal body weights, since absolute testes weights were similar to control levels. The statistically significant lower brain weight of females at 30 mg/kg/day occurred in the absence of a dose-related trend and remained within the range considered normal for rats of this age and strain. No toxicological relevance was ascribed to these changes.

GROSS PATHOLOGY
At 300 mg/kg/day, all sacrificed animals showed yellowish contents of the gastro-intestinal tract, irregular surface of the forestomach, dilation of the small intestines or caecum and reduced size of the thymus.

At 100 mg/kg/day, the sacrificed male and female showed gelatinous contents or distension with gas of the gastro-intestinal tract, yellowish contents of the caecum and/or reduced size of the thymus or spleen.

No treatment-related necropsy findings were observed in the surviving animals at 100 mg/kg/day, and among animals at 30 mg/kg/day. Incidental findings among sacrificed and surviving animals included a red nodule on the left lateral lobe of the liver, a nodule on the epididymides and greenish discolouration of the kidneys, red or black-brown discolouration of the adrenal glands, enlarged adrenal glands, dilation of the oesophagus at the level of the lungs and an accentuated lobular pattern of the liver, yellowish foci on the epididymides, pelvic dilation of the kidneys, a watery-clear cyst on the ovaries, alopecia, red foci on the clitoral glands, The incidence of these findings was within the background range of findings that are encountered among rats of this age and strain, did not show a dose-related incidence trend and/or occurred in the absence of correlating treatment-related histopathology findings. These necropsy findings were therefore considered to be of no toxicological relevance.

HISTOPATHOLOGY:
Treatment-related microscopic findings were present in the following organs:

300 mg/kg/day (animals sacrificed in extremis on Day 10):
- Forestomach:
- Lymphogranulocytic inflammation in 12/15 males and 8/10 females (minimal-moderate)
- Erosions in 1/15 males (slight) and 2/10 females (slight)
- Ulcers in 2/15 males (slight) and 5/10 females ((minimal-slight)
- Hyperplasia of the squamous epithelium in 11/15 males and 8/10 females (minimal-moderate)
- Duodenum: Foamy macrophages in the villi of 13/15 males and 10/10 females (minimal), hypertrophy of the epithelium in 7/15 males and in 4/10 females (minimal-slight).
- Jejunum: Foamy macrophages in the villi of 14/15 males and 8/10 females (minimal-slight), hypertrophy of the epithelium in 4/15 males and in 6/10 females (minimal-slight).
- Ileum: Foamy macrophages in the villi 13/15 males and 10/10 females (minimal-slight), hypertrophy of the epithelium in 1/15 males and 4/10 females (minimal).
- Caecum: Hypertrophy of the epithelium in 5/15 males (minimal-moderate) and 3/10 females (minimal).
- Thymus: Lymphoid atrophy in three males and seven females (minimal to marked).
- Mesenteric lymph nodes: Foamy macrophages were recorded in 14/14 males and 10/10 females (slight-moderate).

30 and 100 mg/kg/day:
- Stomach: lymphogranulocytic inflammation (moderate) and an ulcer (moderate), combined with hyperplasia of the squamous epithelium of the forestomach (minimal) in one female at 100 mg/kg/day killed moribund (animal no. 80).
- Duodenum: Foamy macrophages in the villi of males at 100 mg/kg/day (3/6: minimal, 1/6: slight). Hypertrophy of the epithelium in 1/6 males at 100 mg/kg/day (minimal).
- Jejunum: Foamy macrophages in the villi in males at 100 mg/kg/day (1/6: minimal, 5/6 moderate) and females (6/6: slight) and in 1/5 females (moderate) and 1/5 males (minimal) at 30 mg/kg/day.
- Ileum: Foamy macrophages in the villi in males at 100 mg/kg/day (2/6: minimal, 2/6: slight, 1/6: moderate) and females (1/6: minimal, 5/6: slight) and in males at 30 mg/kg/day (4/5: minimal, 1/5: slight) and females (1/5: minimal, 4/5: slight).
- Caecum: Hypertrophy of the epithelium in 1/6 females at 100 mg/kg/day (minimal).
- Mesenteric lymph node: Macrophage foci in males at 100 mg/kg/day (3/6: moderate, 3/6: marked) and females (4/6: moderate, 2/6: marked) and in males at 30 mg/kg/day (1/5: minimal, 4/5: slight) and females (5/5: slight). In one male and one female at 100 mg/kg/day this was accompanied by a minimal necrosis.
- Thymus: Lymphoid atrophy in 1/6 females (marked) at 100 mg/kg/day.

No abnormalities were seen in the reproductive organs of all animals that failed to mate, conceive, sire or deliver healthy pups which could account for infertility.

The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis.


Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No reproductive changes were seen at this level. The higher level (300 mg/kg) was terminated on day 10 due to high toxicity (Ulceration of the stomach).

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:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

Reproductive data:

No toxicologically significant effects on reproductive parameters were noted.

Percentage mating, fertility index, conception rate, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.

Developmental data:

No toxicologically significant changes in gestation index, duration of gestation, number of dead and living pups at first litter check, sex ratio, postnatal loss, viability index, and early postnatal pup development (mortality, clinical signs, body weight and external macroscopy) were noted.

Body weights of male and female pups at 100 mg/kg/day were slightly lower than controls on Day 4 of lactation. Since no level of statistical significance was obtained, the means remained well within the range considered normal, and the mean body weight gain over Days 1-4 between control pups and pups at 100 mg/kg was similar, this difference was considered not to be of an adverse nature. This slight change pup body weights may have occurred secondarily to the lower body weights of the females.

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.

There were five litters with a single pup found dead or missing at first litter check or before scheduled necropsy, i.e. one litter in the control group, three litters at 30 mg/kg/day and one litter at 100 mg/kg/day. Findings observed among these pups during lactation or macroscopy included small size, pale appearance and/or autolysis. No relationship with treatment was established for these findings.

Incidental clinical symptoms and macroscopic findings seen among surviving pups included small size, cold, absence of milk in the stomach, small tail and scabbing. The complete litter of one control group female was noted as having no milk in the stomach at macroscopic examination. No relationship with treatment was established for these observations and they were considered to be within the normal biological variation for rats of this age and strain.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Highest dose level that could be tested. The next higher dose group of 300 mg/kgbw/day was terminated on day 10 due to high toxicity (Ulceration of the stomach).

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Since all animals were sacrificed at 300 mg/kg/day, and hence no potential effect on reproductive/developmental parameters could be determined at this dose level, the reproductive and developmental NOAEL was established to be 100 mg/kg/day.
Executive summary:

Tallow Tripropylenetetramine was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 0, 30, 100 or 300 mg/kg/day. The males were exposed for 2 weeks prior to mating, during mating, and up to termination (for at least 28 days). The females were exposed for 42-54 days, i.e. 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation.

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

Parental results:

All animals at 300 mg/kg were killed in extremis on Day 10 of treatment. Microscopic findings in the stomach (including ulcers and erosions, corresponding to irregular surface of the forestomach at necropsy) were considered to have contributed to the moribundity. Prior to death these animals primarily showed hunched posture and piloerection, along with notable weight loss (up to 13%) and reduced food intake. At necropsy, all sacrificed animals showed yellowish contents of the gastro-intestinal tract, irregular surface of the forestomach, dilation of the small intestines or caecum and reduced size of the thymus. Histopathological changes other than stomach effects noted at 300 mg/kg/day included hypertrophy of the epithelium of the duodenum, jejunum, ileum and caecum with or without foamy macrophages in the villi, foamy macrophages in the mesenteric lymph nodes, and lymphoid atrophy of the thymus, corresponding to dilation of the small intestines or caecum and reduced size of the thymus at necropsy.

At 100 mg/kg, one male and one female were killed in extremis on Day 27 and 38 of treatment, respectively. For one of these animals findings in the stomach (including an ulcer) were considered to have contributed to the moribundity, whilst for the other animal no clear cause of moribundity was established histopathologically. Clinical signs at 100 mg/kg/day primarily consisted of piloerection among females. In addition, lower absolute body weights and body weight gain were recorded during the Repro period for males and females, and for females also during post-coitum and lactation. Also, absolute and relative food consumption of females was lower than controls during the premating phase, and remained lower during the post-coitum period, but not during lactation. Clinical biochemistry changes in males and/or females consisted of higher alanine and aspartate aminotransferase activity, lower total protein and albumin levels, and higher total bilirubin, urea, cholesterol and bile acid levels. Haematological changes were confined to a higher relative and/or absolute neutrophil count, and higher platelet counts. Necropsy revealed lower liver, spleen, prostate, heart and thymus weight in males and/or females, which may at least in part be explained by the lower terminal body weights. Histopathological changes among the surviving animals consisted of hypertrophy of the epithelium and/or foamy macrophages in the villi of the duodenum, jejunum, ileum and caecum, lymphoid atrophy of the thymus and macrophage foci in the mesenteric lymph node.

At 30 mg/kg/day, a lower body weight gain was recorded for males during the Repro period without changes in food intake. Haematology showed a higher relative neutrophil count in females, but absolute neutrophil counts remained similar to controls. Clinical biochemistry parameters remained unaffected. Histopathological changes consisted of foamy macrophages in the villi of the ileum and jejunum, hypertrophy of the epithelium of the caecum and macrophage foci in the mesenteric lymph node.

No treatment-related changes were noted during functional observation tests at any dose level.

Reproductive/Developmental results:

No toxicologically relevant differences in reproductive/developmental parameters occurred up to 100 mg/kg/day. No potential effect on reproductive/developmental parameters could be determined at 300 mg/kg since all animals were sacrificed at this dose level.

Since all animals were sacrificed at 300 mg/kg/day, and hence no potential effect on reproductive/developmental parameters could be determined at this dose level, the reproductive and developmental NOAEL was established to be 100 mg/kg/day.