Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

A NOAEL for reproduction/developmental toxicity of 100 mg/kg/day was established from a combined repeated dose/reproduction toxicity study with Tallow tripropylenetetraamine.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
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.
Reason / purpose for cross-reference:
reference to same study
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
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
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.
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
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
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: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
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.


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).
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
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.
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).
Reproductive effects observed:
not specified
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.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Consistent results from all studies within the whole group of Polyamines, indicating a no concerns for reproduction toxicity. The indicated NOAEL of 100 mg/kg is based on high maternal toxicity and mortality at the next higher dose level of 300 mg/kg bw/day.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Quality of whole database:
Physical-chemical properties of Tallow tripropylenetetraamine indicate a low likelihood for exposure via inhalation. The paste has a melting point of 31 °C, a boiling point > 300 °C and a low vapour pressure (4.7 x 10-5 Pa at 20°C for the coco dipropylene triamine, with the shortest average alkyl chain length representing the highest vapour pressure for the group of polyamines). Its use is limited to industrial and professional users and does not involve the forming of aerosols, particles or droplets of an inhalable size. So exposure to humans via the inhalation route will be unlikely to occur. Furthermore, as the substance is classified as corrosive, such testing should normally not be conducted.
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Quality of whole database:
Manufacture and use are highly controlled. Its use is limited to industrial and professional users where following its severe corrosive properties will provide for sufficient protection measures to prevent exposure.
Additional information

A combined repeated dose/reproduction screening toxicity study according to OECD 422 has been performed with Tallow tripropylenetetraamine in rats. Dose levels consisted of 0, 30, 100 and 300 mg/kgbw/day. The highest dose group of 300 mg/kgbw/day was terminated on day 10 due to high toxicity (Ulceration of the stomach). Parental effects for this study are summarised in the chapter on repeated dose toxicity. 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.

Cross-reading and category approach:

Within the category of the Polyamine there are further studies available that clearly demonstrate thesame toxicological profile for the various substances over this category, and a clear lack for concern for reproduction toxicity from the range of available relevant studies.

(See the document for the category justification for the polyamines that is attached to this dossier).

A combined repeated dose/reproduction screening toxicity study according to OECD 422 has been performed with Coco dipropylene triamine. Dose levels consisted 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).

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. 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 NOAEL could not be determined.

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

A full two-generation study on C12-Y-triamine has also shown no indication of concern for reproductive toxicity:

An oral two-generation reproductive toxicity study with rats, performed in accordance with OECD Guideline 416 (version of 1983) and under GLP is available (Inveresk, 1995). Parental animals received the test substance by gavage at dose levels of 0, 3, 9 and 27 mg/kg bw/day pure substance 10 weeks prior to mating until weaning of the F1 generation. The treatment of F1 animals commenced from day 25 after birth and continued to weaning of the F2 generation. The F1 animals were mated 11 weeks after weaning.

At the high dose level most animals of both generations showed signs of reaction to treatment, consisting of dyspnea, piloerection and hunched posture, and many animals also had episodes of post-dosing salivation. For occasional animals, the outline of the spine was prominent. A total of 8 animals died or were killed after showing marked signs of reaction, and a 9th death may also have been related to treatment. In both generations, at the highest dose level, mean weight gain was markedly lower than controls; this effect was apparent for males, and for females in the premating period and during gestation. Food consumption at the high dose level in both generations was slightly lower than in controls. Mating performance, fertility, duration of gestation, litter size and pup survival were considered to be similar in all groups.

Mean seminal vesicle weights in the high dose groups of both generations were significantly lower than controls, however, it was considered that this reduction was an indirect effect of the lower body weights, rather than a direct effect on the seminal vesicles. Mean absolute epidymides and testes weights in the same groups were lower than control, with the value for epidymides for F0 animals and the value for testis for F1 animals attaining statistical significance. However, after adjustment for bodyweight (covariance analysis) these effects were not apparent. Mean prostate weights were essentially similar in all groups of both generations.

In the F2 generation, slightly reduced pup and litter weights and observed clinical signs (2 pups with body tremors) were observed at 27 mg/kg bw/day.

In the current version of the OECD 416 guideline, histopathology of the pups is part of the standard procedure. This study was performed in accordance with the 1983 version of the guideline in which these investigations were not required. However in the subchronic 90-day study in rats histopathological examinations were performed. The lack of effects on reproduction in the 2-generation study along with the supporting data from the 90-day study confidently allows the conclusion that the substance is not a selective reproductive toxicant. The NOAEL for systemic toxicity was set at 9 mg/kg bw/day, while the NOAEL for reproductive toxicity was considered to exceed 27 mg/kg bw/day.

Available data from reproduction toxicity studies in rat on Polyamines:

Sub-group

Alkyl chain

Study

Results NOAEL (in mg/kg bw/d)

Diamines

Oleyl

Development
(OECD 414)

No effects on pre/post implantation rate, late/early resorptions, corpora lutea or number of live fetuses were seen in this study.

There was a slight increase in visceral and skeletal variations. No malformations were observed.

NOAEL development: 1.25 mg

Triamines

Coco

OECD 422

NOAEL reproduction and development: 30 mg (highest dose since all animals were sacrificed at 100 mg/kg/day, and hence no potential effect on reproductive/developmental parameters could be determined at this dose level.)

Tallow

Development
(OECD 414)

NOAEL maternal = 30 mg/kg based on signs of toxicity and lower BW seen from 60 mg/kg.

NOAEL development: 30 mg/kg, based on the observation of pale adrenals in 7 foetuses, and signs of retarded skeletal ossification seen at 60 and 120 mg/kg.

Y-triamines

C12

Development
(OECD 414)

NOAEL maternal = 7.5 mg

NOAEL teratology > 60 mgNOAEL development = 22.5 mg, based on secondary effects due to maternal toxicity observed in the foetuses (increase in incidence of early embryonic deaths and slightly decreased mean foetal weight).

Reproduction
(OECD 416)

NOAEL maternal, F1 and F2: 9 mg. Based on reduced body weight gain, clinical signs of reaction to treatment at higher levels. There were no obvious adverse effects on mating and littering performance at any of the levels tested.

Tetraamines

Tallow

OECD 422

NOAEL reproduction and development: 100 mg (highest dose 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.)

 

Additionally, no adverse effects on reproductive organs were identified in any of the available repeated dose studies on Polyamines.

 

In addition the low likelihood of exposure can be considered as these substances are only applied in professional or industrial setting in asphalt applications, applying adequate PPE. Usage results to the inclusion into or onto a matrix. Consumers/general population will not be exposed. For corrosive substances, the use of protective gloves and other equipment, such as face shields, aprons and good work practices are mandatory. As a result, direct dermal contact occurs only occasionally. Therefore, repeated substantial daily dermal exposure is unlikely. Likelihood of exposures via inhalation is also low considering the high boiling point (> 300 °C) and very low vapour pressure (< 4.7 x 10-5 Pa at 20°C).

Conclusion: In view of the total lack of effects on reproduction in all these reproduction toxicity studies, a further 2-generation study is not considered to provide useful additional information for the evaluation of reproduction toxcity Tallow tripropylenetetraamine.

Effects on developmental toxicity

Description of key information
No developmental toxicity was observed in an OECD 422 screening study with Tallow tripropylenetetraamine.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.

Cross-reading to a prenatal developmental toxicity study on Tallow dipropylenetriamine resulted to a maternal NOAEL of 30 mg/kg. Based on the observation of pale adrenals, and signs of retarded skeletal ossification seen at 60 and 120 mg/kg, a developmental NOAEL of 30 mg/kg was selected.

Other available developmental toxicity studies on similar substances in the category of Polyamines showed no specific concerns for developmental or teratogenic properties.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
04 February - 14 March 2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles. However, interpretation of skeletal evaluations is disputable.
Justification for type of information:
(See also Category polyamines in support of Tetraamine T - 20120420.pdf attached to Ch.13)
Longer chain polyamines, i. e. increasing number of DP-groups (1,3-diamine propane), result to lower toxicity. As a consequence, results from a triamine can be considered as a worst case approach for a tetramine with same alkyl chain.
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
other: Wistar (Han)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
Females were nulliparous, nonpregnant and untreated at initiation of the study.
- Age at delivery: Females were approximately 11 weeks.
- Weight at study initiation: mean weight at start of treatment was 230 gr
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages.
Mating: Females were caged together with stock males on a one-to-one-basis in Macrolon cages.
Post-mating: Females were individually housed in Macrolon cages. Pups were kept with the dam until termination
General: Sterilised sawdust as bedding material and paper as cage enrichment were supplied.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

IN-LIFE DATES
From: 04 February - 14 March 2013
Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
- Method of formulation: Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenized to a visually acceptable level. In order to obtain homogeneity, the test substance (formulations) were heated in a water bath up to 60°C for a maximum of 25 minutes. The test substance formulations were allowed to cool down to a temperature of maximally 40ºC prior to dosing. Adjustment was made for density of the test substance and specific gravity of the vehicle. No correction was made for the purity/composition of the test substance.
- Storage conditions of formulations: At ambient temperature.
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at WIL Research Europe and on information provided by the sponsor.
- Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The delegated phase was performed by the Principal Investigator for Formulation Analysis. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 5 hours at room temperature under normal laboratory light conditions 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. 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%.

Details on mating procedure:
- M/F ratio per cage: 1/1 (one female was cohabitated with one stock male)
- Age at start of mating of the females in the study: Approximately 12 weeks
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage and/or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
- After successful mating each pregnant female was caged individually in Macrolon cages (MIII type, height 18 cm).
- Any other deviations from standard protocol: no
Duration of treatment / exposure:
Females were dosed from Day 6 to Day 19 post-coitum, inclusive.
Frequency of treatment:
Once daily for 7 d/w.
Duration of test:
Duration of treatment: From Days 6 to 19 post-coitum, inclusive.
No. of animals per sex per dose:
22
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based on results of the dose range finding study (Project 501612)
- Rationale for animal assignment: Upon detection of mating (Day 0 post-coitum), the females were distributed in a random sequence over the test groups. Females which were mated on the same day were classified in the same subgroup.
Maternal examinations:
CAGE SIDE OBSERVATIONS
- Time schedule: At least twice daily. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7). The circumstance of any death was recorded in detail.

DETAILED CLINICAL OBSERVATIONS
- Time schedule: At least once daily from Day 0 post-coitum onwards.
The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity.

BODY WEIGHT
- Time schedule for examinations: Days 0, 3, 6, 9, 12, 15, 17 and 20 post-coitum.

FOOD CONSUMPTION
- Days 0-3, 3-6, 6-9, 9-12, 12-15, 15-17 and 17-20 post-coitum.

FOOD EFFICIENCY: yes

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

OPHTHALMOSCOPIC EXAMINATION
No

HAEMATOLOGY
No

CLINICAL CHEMISTRY
No

URINALYSIS
No

NEUROBEHAVIOURAL EXAMINATION
No

GENERAL REPRODUCTION DATA
- Mating date and confirmation of pregnancy 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.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight.
- Number of corpora lutea.
- The number and distribution of live and dead fetuses.
- The number and distribution of embryo-fetal deaths.
- The weight of each fetus.
- The sex of each fetus from the ano-genital distance (during necropsy) and also from gonadal inspections (during further fetal examination).
- Externally visible macroscopic fetal abnormalities.
Fetal examinations:
External, visceral and skeletal fetal findings were recorded as developmental variations or malformations.
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (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 (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.

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.
No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss. Dead fetuses, early and late resporptions and pre- and post-implantation loss were compared using the litter as the statistical unit.
Indices:
For each litter the following calculations were performed:
Pre-implantation loss (%) = (number of corpora lutea - number of implantation sites) / number of corpora lutea x 100
Post-implantation loss (%) = (number of implantation sites - number of live fetuses) / number of implantation sites x 100
The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison, calculating the number of affected fetuses as a mean litter proportion on a total group basis, where: Viable fetuses affected / litter (%) = number of viable fetuses affected / litter x 100
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical signs noted for the females at 60 and 120 mg/kg that died before the scheduled necropsy day included hunched posture, rales, laboured respiration, piloerection and/or salivation. These were seen 1-4 days before their deaths.
Clinical signs noted for surviving animals at 120 mg/kg included hunched posture, rales, piloerection, salivation, lean appearance, and to a lesser extent labored respiration, pale feces and lethargy were also seen. Most of these clinical signs were also noted for surviving animals at 60 mg/kg.
Rales was noted for 3 animals and piloerection was seen for one animal at 30 mg/kg on individual occasions. These were not considered to be toxicologically relevant at this dose level since the signs were only seen for a limited number of animals.
No other clinical signs were noted during the treatment period.
Mortality:
mortality observed, treatment-related
Description (incidence):
One female at 120 mg/kg (no. 84) died spontaneously on Day 13 post coitum and two females at 60 mg/kg died before the scheduled necropsy period. Animal no. 56 died spontaneously and no. 64 was killed in extremis on Days 18 and 12 of the post coitum period, respectively.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Absolute body weights and body weight gains were significantly lower for females at 120 and 60 mg/kg. Corrected terminal body weight and weight gain were also significantly lower than controls for these females.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Absolute and relative food consumption were significantly lower for animals at 60 and 120 mg/kg from post coitum Days 6-20.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Female no. 84 that died spontaneously at 120 mg/kg was noted with beginning autolysis and reddish foci on the thymus at the macroscopic examination. There were no macroscopic findings seen for the two females at 60 mg/kg that died before the scheduled necropsy.
For surviving animals at 120 mg/kg, treatment related macroscopic findings included emaciated appearance, enlarged adrenal glands, the duodenum, jejunum, ileum, caecum and/or colon distended with gas, thickened large and small intestines, and thymus reduced in size. Emaciated appearance, reduced size and discoloration of the thymus, and enlarged and irregular surface of the spleen were noted for a few animals at 60 mg/kg.
Alopecia and fluid in the uterus were incidental findings noted at the macroscopic examination and were in no way related to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Description (incidence and severity):
The percentage of early resorptions, total resorptions and post-implantation loss was relatively higher (not statistically significant) for females at 120 mg/kg than controls. This was attributable to female nos. 83 and 87 who both had 100% post-implantation loss, consisting of early resorptions.
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
Details on maternal toxic effects:
MORTALITY: One female at 120 mg/kg died spontaneously on Day 13 post coitum and two females at 60 mg/kg died before the scheduled necropsy period. One female died spontaneously and the other female was killed in extremis on Days 18 and 12 of the post coitum period, respectively.
CLINICAL SIGNS: Clinical signs noted for the females at 60 and 120 mg/kg that died before the scheduled necropsy day included hunched posture, rales, laboured respiration, piloerection and/or salivation. These were seen 1-4 days before their deaths. Clinical signs noted for surviving animals at 120 mg/kg included hunched posture, rales, piloerection, salivation, lean appearance, and to a lesser extent labored respiration, pale feces and lethargy were also seen. Most of these clinical signs were also noted for surviving animals at 60 mg/kg. Rales was noted for 3 animals and piloerection was seen for one animal at 30 mg/kg on individual occasions. These were not considered to be toxicologically relevant at this dose level since the signs were only seen for a limited number of animals. No other clinical signs were noted during the treatment period.
BODY WEIGHTS: Absolute body weights and body weight gains were significantly lower for females at 120 and 60 mg/kg beginning Days 12 and Day 17 of the post coitum period, respectively, and persisted through the remaining duration of the treatment period. Corrected terminal body weight and weight gain were also significantly lower than controls for these females.
FOOD CONSUMPTION: Absolute and relative food consumption were significantly lower for animals at 60 and 120 mg/kg from post coitum Days 6-20.
MACROSCOPIC EXAMINATION: One female that died spontaneously at 120 mg/kg was noted with beginning autolysis and reddish foci on the thymus at the macroscopic examination. There were no macroscopic findings seen for the two females at 60 mg/kg that died before the scheduled necropsy. For surviving animals at 120 mg/kg, treatment related macroscopic findings included emaciated appearance, enlarged adrenal glands, the duodenum, jejunum, ileum, caecum and/or colon distended with gas, thickened large and small intestines, and thymus reduced in size. Emaciated appearance, reduced size and discoloration of the thymus, and enlarged and irregular surface of the spleen were noted for a few animals at 60 mg/kg. Alopecia and fluid in the uterus were incidental findings noted at the macroscopic examination and were in no way related to treatment.
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
< 30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Fetal body weights were significantly lower at 120 mg/kg compared to controls, which was secondary to the lower gains/maternal weight loss at this dose level.
Body weights of fetuses (sexes combined) were 3.4, 3.4, 3.4 and 2.8 grams for the control, 30, 60 and 120 mg/kg groups, respectively.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): Fetal body weights were significantly lower at 120 mg/kg compared to controls, which was secondary to the lower gains/maternal weight loss at this dose level.
Body weights of fetuses (sexes combined) were 3.4, 3.4, 3.4 and 2.8 grams for the control, 30, 60 and 120 mg/kg groups, respectively.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Skeletal evaluations included the observation of effects that that were interpreted as polydactyly and malpositioned metatarsals. This finding considerd an artefact is further discussed below.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Visceral malformations were observed in 4(3), 0(0), 4(4) and 1(1) fetuses (litters) in the control, 30, 60 and 120 mg/kg groups, respectively. None of the malformations noted at 60 and 120 were considered to be treatment related.
Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
Litter size: No treatment related effect on litter size was noted up to 120 mg/kg.The mean number of viable fetuses per litter was 12.3, 12.3, 12.4 and 10.9 in the control, 30, 60 and 120 mg/kg groups, respectively.
Sex ratio: There were no treatment-related effects on the sex ratio of the fetuses.
Fetal body weight: Fetal body weights were significantly lower at 120 mg/kg compared to controls, which was secondary to the lower gains/maternal weight loss at this dose level. Body weights of fetuses (sexes combined) were 3.4, 3.4, 3.4 and 2.8 grams for the control, 30, 60 and 120 mg/kg groups, respectively.
Visceral malformations were observed in 4(3), 0(0), 4(4) and 1(1) fetuses (litters) in the control, 30, 60 and 120 mg/kg groups, respectively. None of the malformations noted at 60 and 120 were considered to be treatment related.
The viscerally malformed fetus at 120 mg/kg had internal hydrocephaly which was considered to be spontaneous in origin due to its single occurrence. At 60 mg/kg, 3 fetuses from 3 litters either had an absent eye or a small eye. These eye anomalies only occurred at 60 mg/kg and therefore no dose relationship could be established. Another fetus in the mid dose group had situs inversus whereby all thoracic and abdominal organs were laterally transposed. The same finding was observed in 3 control fetuses and thus the occurrence at 60 mg/kg was not considered to be toxicologically relevant or treatment related.
Other visceral malformations in this study only affected control fetuses. Abnormal lobation of the lung was observed in two fetuses which also had situs inversus. In addition, one of these fetuses had multiple cardiovascular abnormalities (narrow pulmonary trunk, absent ductus arteriosus, malpositioned left subclavian and ventricular septum defect) and a split spleen. Another control fetus had abnormal lobation of the liver, absent lung lobe, transposition of the great vessels and ventricular septum defect.
Of the visceral variations, discolored adrenal glands were observed in 0(0), 0(0), 7(2) and 48(5) fetuses (litters) of the control, 30, 60 and 120 mg/kg groups, respectively.
Other variations noted in test substance treated groups were small supernumerary liver lobes, liver appendix, partially undescended thymus horns, convoluted ureter, dilated ureter and right subclavian originating from the aortic arch. These variations were not considered to be treatment related, because they occurred at similar frequencies in the control group, occurred infrequently, occurred without dose-relationship and/or occurred at frequencies within the historical control range.
Skeletal malformations were observed in 7(4), 13(6), 14(9) and 3(1) fetuses (litters) in the control, 30, 60 and 120 mg/kg groups, respectively.
Two malformations, polydactyly and malpositioned metatarsals, which are considered to be closely related, were observed unilaterally in the in 1(1), 6(3), 5(4) and 3(1) fetuses (litters) in the control, 30, 60 and 120 mg/kg groups, respectively, where one fetus in the 30 mg/kg group showed both polydactyly and malpositioned metatarsals in the left hind paw.
Another malformation observed was bent limb bones. It occurred in 4(3), 8(4) and 6(3) and 0(0) fetuses (litters) in the control, 30, 60 and 120 mg/kg groups, respectively, resulting in 1.5%, 2.9%, 3.0% and 0.0% of fetuses per litter in these same respective dose groups. In all these fetuses one or both scapulas were bent and additionally, the humerus and/or radius were involved in two fetuses of Group 2 and one of Group 3.
The incidences of bent limb bones in the 30 and 60 mg/kg groups (2.9% and 3.0% per litter, respectively) were higher than the historical control data range (0.0%-1.6% per litter) and concurrent control value (1.5% per litter), but as no cases occurred in the high dose group, a relation to treatment could not be established.
Remaining skeletal malformations observed in fetuses of test substance treated groups were sternoschisis (two fetuses in one litter of Group 3) and vertebral anomaly with or without associated rib anomaly (one fetus of group 3). Because these findings occurred at a low incidence, were seen in historical controls and, in the case of vertebral anomaly with or without associated rib anomaly, occurred in a concurrent control fetus they were not considered to be treatment related.
The only other skeletal malformation in this study was noted in one control fetus. This fetus had a vertebral centra anomaly.
Skeletal variations were observed in 87.7%, 84.1%, 83.3% and 80.1% of fetuses per litter in the control, 30, 60 and 120 mg/kg groups, respectively.
Ossification parameters observed at a statistically significantly higher incidence in the 120 mg/kg group compared to the control group were unossified sternebrae nos. 5 and/or 6 (36.5% versus 13.3% per litter), unossified vertebral centra (9.2% versus 0.8% per litter) and unossified metacarpals and/or metatarsals (9.5% versus 0.0% per litter). Additionally, higher (not statistically significant) mean litter incidences were noted at 120 mg/kg compared to controls for unossified hyoid (1.5% versus 0.7% per litter), unossified sternebrae nos. 1, 2, 3 and/or 4 (8.5% versus 0.4% per litter), entire sternum unossified (3.7% versus 0.0% per litter), reduced ossification of sternebrae (18.6% versus 1.3% per litter), reduced ossification of vertebral centra (11.8% versus 1.8% per litter), reduced ossification of vertebral arches (14.2% versus 2.5% per litter), unossified or reduced ossification of pubis (7.6% versus 1.2% per litter) and unossified or reduced ossification of ischium (1.9% versus 0.4% per litter). A statistically significantly decreased incidence was observed for the finding of reduced ossification of the skull at 120 mg/kg (9.7% per litter) compared to the control value (16.5% per litter).
At 60 mg/kg, a higher incidence (not statistically significant) was noted for unossified sternebrae nos. 1, 2, 3 and/or 4 (2.3% versus 0.4% per litter), reduced ossification of sternebrae (8.4% versus 1.3% per litter), reduced ossification of vertebral arches (7.7% versus 2.5% per litter), unossified or reduced ossification of pubis (4.5% versus 1.2% per litter) and unossified or reduced ossification of ischium (2.2% versus 0.4% per litter) compared to the control group.
Decreased mean litter incidences of ossification parameters at 60 mg/kg were observed for unossified sternebrae nos. 5 and/or 6 (8.3% versus 13.3% per litter, statistically significant) and reduced ossification of the skull (8.0% versus 16.5% per litter, not statistically significant).
At 30 mg/kg, mean litter incidences of ossification parameters listed in text table 2 were comparable to the control values.
Of the other skeletal variations, bent ribs were noted in 21.8%, 23.7%, 25.8% and 4.6 % of fetuses per litter in the control, 30, 60 and 120 mg/kg groups, respectively. The value at 120 mg/kg was statistically significantly decreased compared to the control value, but was within the historical control data range (1.6%-27.4% per litter). A reduced number of fetuses with bent ribs is not an adverse effect and has no toxicological significance. Therefore, the decreased mean litter incidence of bent ribs was considered to have arisen by chance and was not considered to be treatment related.
A statistically significantly decreased mean litter incidence of 14th rudimentary ribs was noted at 120 mg/kg compared to the control value (31.3% versus 61.0% per litter). At 30 and 60 mg/kg, 47.3% and 48.7% of fetuses per litter, respectively, had 14th rudimentary ribs. All values were within the historical control data range (17.9%-72.4% per litter) and because there was no dose-dependent relationship noted for other supernumerary ribs (14th full ribs, 7th cervical full ribs and 7th cervical rudimentary ribs (see table 1 and 2), the decrease of 14th rudimentary ribs at 120 mg/kg was not considered to be treatment related.
Remaining skeletal variations noted in this study were ossified cervical centrum no. 1, slightly to moderately malaligned sternebrae, supernumerary ossification site in sternum, branched sternebrae, caudal shift of pelvic girdle and reduced ossification of ribs. These variations were not considered to be treatment related, because they occurred at similar frequencies in the control group, occurred infrequently, occurred without dose-relationship and/or occurred at frequencies within the historical control range.
Key result
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Sex:
not specified
Basis for effect level:
fetal/pup body weight changes
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
In conclusion, based on the results in this prenatal developmental toxicity study the maternal NOAEL for C16-18, C18-unsaturated-alkyl dipropylene triamine was established as being 30 mg/kg. Based on the observation of pale adrenals in 7 fetuses, and signs of retarded skeletal ossification seen at 60 and 120 mg/kg, a developmental NOAEL of 30 mg/kg was selected.
Executive summary:

Mated female Wistar Han rats were assigned to four dose groups, each containing twenty-two animals. The test item was administered once daily by gavage from Day 6 to 19 post-coitum at doses of 30, 60 and 120 mg/kg (Groups 2, 3 and 4 respectively). The rats of the control group received the vehicle, propylene glycol, alone. Accuracy, homogeneity and stability of formulations were demonstrated by analyses.

 

Maternal findings

Maternal toxicity was evident in both the 60 and 120 mg/kg groups and included mortality, adverse clinical signs (hunched posture, rales, piloerection, salivation and lean appearance, among others), reduced food consumption, and substantially lower body weights and body weight loss compared to controls: 7% lower for 60 mg/kg and 25% lower BW for 120 mg/kg treated animals. Treatment related macroscopic findings (emaciated appearance, enlarged adrenal glands, small and large intestines thickened and distended with gas, among others) were also seen for mid- and high-dose animals as well.

No toxicologically relevant maternal findings were noted with treatment up to 30 mg/kg.

 

Developmental findings

No significant differences were observed between control and treated groups regarding the number of corpora lutea, implantation sites, viable or dead fetuses, early or late resorptions, or pre- and postimplantation loss. The percentage of early resorptions, total resorptions and post-implantation loss was relatively higher (not statistically significant) for females at 120 mg/kg than controls. This was attributable to two females who both had 100% post-implantation loss, consisting of early resorptions.

No effects were observed on litter size and sex-ratio.

Fetal body weights were significantly lower at 120 mg/kg (2.8 g) compared to controls (3.4 g), which was secondary to the lower gains/maternal weight loss at this dose level.

 

External malformations and variations:

No external malformations and developmental variations were observed in any of the fetuses.

 

Visceral malformations and variations:

Visceral malformations were observed in 4(3), 0(0), 4(4) and 1(1) fetuses (litters) in the control, 30, 60 and 120 mg/kg groups, respectively. None of the malformations noted at 60 and 120 were considered to be treatment related.

Visceral variations included a dose related increase of yellow-white discolored (anaemic?) adrenal glands in the 60 and 120 mg/kg groups in 7(2) and 48(5) fetuses (litters), whereas none occurred in the control and 30 mg/kg groups. The physiological effects of this variation are unknown, but the discoloration could not be ignored and was considered to be treatment related.

Other variations noted in test substance treated groups were small supernumerary liver lobes, liver appendix, partially undescended thymus horns, convoluted ureter, dilated ureter and right subclavian originating from the aortic arch. These variations were not considered to be treatment related, because they occurred at similar frequencies in the control group, occurred infrequently, occurred without dose-relationship and/or occurred at frequencies within the historical control range.

 

Skeletal Malformations and Variations:

Skeletal evaluations included the observation of effects that that were interpreted as polydactyly and malpositioned metatarsals. This finding is further discussed below.

The incidences of bent limb bones in the 30 and 60 mg/kg groups (2.9% and 3.0% per litter, respectively) were higher than the historical control data range (0.0%-1.6% per litter) and concurrent control value (1.5% per litter), but as no cases occurred in the high dose group, a relation to treatment could not be established.

Remaining skeletal malformations observed in fetuses of test substance treated groups were sternoschisis (nos. A045-03 and -07) and vertebral anomaly with or without associated rib anomaly (no. A065-06). Because these findings occurred at a low incidence, were seen in historical controls and, in the case of vertebral anomaly with or without associated rib anomaly, occurred in a concurrent control fetus (no. A018-01) they were not considered to be treatment related. The only other skeletal malformation in this study was a vertebral centra anomaly, noted in a control fetus.

 

Skeletal variations were observed in 87.7%, 84.1%, 83.3% and 80.1% of fetuses per litter in the control, 30, 60 and 120 mg/kg groups, respectively.

Retarded skeletal ossification was evidenced at 120 mg/kg by the variations of unossified sternebrae nos. 5 and/or 6, unossified sternebrae nos. 1, 2, 3 and/or 4, entire sternum unossified, reduced ossification of sternebrae, unossified vertebral centra, reduced ossification of vertebral centra, reduced ossification of vertebral arches, unossified hyoid, unossified metacarpals and/or metatarsals, unossified or reduced ossification of pubis and unossified or reduced ossification of ischium. This delayed skeletal ossification was in line with the reduced fetal weights at 120 mg/kg; these were all secondary to the maternal toxicity at this dose level.

Signs of retarded skeletal ossification were also present at 60 mg/kg, and were demonstrated by higher incidences of unossified sternebrae nos. 1, 2, 3 and/or 4, reduced ossification of sternebrae, reduced ossification of vertebral arches, unossified or reduced ossification of pubis and unossified or reduced ossification of ischium.

 

 

Discussion reported skeletal malformations

Malformations

fetuses

Dose level

control

30 mg/kg

60 mg/kg

120 mg/kg

Number examined skeletally

270

259

236

217

Polydactyly

no. of fetuses (litters)

0(0)

5(3)

1(1)

2(1)

% per litter

0

1.7

0.4

0.7

fetuses affected

-

A023-09,12
A032-11,12
A044-09

A057-12

A080-01,09

Metatarsal(s)- Malpositioned

no. of fetuses (litters)

1(1)

2(1)

4(3)

1(1)

% per litter

0.4

0.7

1,5

0,3

fetuses affected

A001-10

A023-10,12

A046-09
A048-09,12
A053-03

A080-11

For polydactyly of a total number of 712 fetuses from treated groups there are 10 (1.40%) affected.

Historical Control: (2008-2012; Crl:WI(Han; outbred, SPF-Quality)

23 studies; fetuses/litters examined externally 4557 / 384, skeletal 3122 / 376

 Polydactyly (external examination): 1 fetus

 Metatarsal(s)- Malpositioned (skeletal examination): 3 fetuses

 

No obvious dose relation could be established for these separate malformations, but they were considered to be related findings because both malformations were localized in the same region and result from patterning errors during limb development. These incidences of hindpaw malformations show an increase at 30, 60 and 120 mg/kg when compared to the control group and therefore were considered to be a result of treatment.

As polydactyly generally has a genetic background, the parentage of the affected fetuses and their mothers were checked. Affected litters are not all derived from the same fathers, and the supplied females were not siblings.

 

At the request of the Sponsor, an independent external consultant examined the affected fetuses. This expert concluded that these findings were not true malformations but were attributable to tissue mechanical damage or processing artefacts and subsequent displacement of the digits, which could give the appearance of extra hind paw structures. One case of polydactyly could not be discounted, though for the other fetuses no agreement could be established. (See attached review and discussion documents).

 

Based on the skeletal examinations in the main study, skeletal exams of the paws were performed for all fetuses (all dose groups). At 150 mg/kg, an extra metatarsal on the hind paw was seen for a single fetus. (See attached RF-extra metatarsal.pdf) However, the description “extra metatarsal” is rather dubious. The photograph shows no clear evidence of a supernumerary structure. The small brown area could be anything; the colouration certainly does not indicate ossified bone. Such areas can be often seen if the soft tissue clearing is not good. When ossification is less than optimal, it can sometimes appear to be fragmented. A reason for the unclear picture could be that long alcohol fixation of almost a year adversely affects the staining process. After about three months, the results of the staining procedure are usually less than optimal. The soft tissue clearing tends to be poor.

 

Overall, the polydactyly findings are not considered a genuine, test substance related finding. Supportive arguments for artefact are:

  • The consulted expert indicated to have seen such artefacts in the past.
  • No actual superfluous bone structures were observed.
  • External evaluation of fetuses did not result to one observation of polydactyly at all. Polydactyly should be visible upon external evaluations by experienced examinators.
  • Only seen in hind legs, and only unilateral. This is very uncommon. In literature, in cases for which no genetic cause could be found, and therefore a possible teratogenic cause was suspected, the polydactyly was bilateral.
    (In fowl, when polydactylous strains are cross-bred with normal toed strains, there is indeed the possibility of single sided polydactyly (heterodactylism). However, the incidence is only a small percentage of the overall polydactyly incidence. Furthermore, heterodactyly only occurred as a rare exception in animals which are homozygous for polydactyly. So this means it is actually only observed because of the absence of a digit that should normally be there, rather than as a single sided additional toe.)
  • No dose-response relation. (Although a biphasic response is possible, this is generally the case when increased effects rather lead to death of fetus rather than increase of incidence. In this study there is no such indication.)
  • Not seen in any other related substance in the category although all other properties and effects in repeated dose studies are the same.
  • Mechanistically difficult to understand: these substances have a MOA of general cytotoxicity at contact. They are not well absorbed and do not pass membranes easy (placenta?). A specific (cytotoxic?) action at a certain place and time in de fetal development is difficult to accept without being further effects. And a specific effect in the development of digits, it would be more likely to have missing structures. The signalling to induce specific cell development and growth would be easier inhibited then partially stimulated. Also the most common cause for polydactyly is genetically. The occurrence of random aneugenicity can be expected to lead to more effects (syndromes) rather that a very specific single sided occurrence of polydactyly. Besides, the concentrations at which polyploidy started to occur in vitro are not conceivable in vivo, and polyploidy is not the same aneugenicity. ‘Model’ compounds for aneugenicity as Colchicine and vinblastine indeed show a great spectrum of teratogenic effects, including polydactyly. If the mechanism for the triamine was based on aneugenicity, more developmental defects should be found, also, and maybe especially, in the affected foetuses.

In conclusion, based on the results in this prenatal developmental toxicity study the maternal NOAEL for C16-18, C18-unsaturated-alkyl dipropylene triamine was established as being 30 mg/kg. Based on the observation of pale adrenals in 7 fetuses, and signs of retarded skeletal ossification seen at 60 and 120 mg/kg, a developmental NOAEL of 30 mg/kg was selected.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
30 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Consistent results from all studies within the whole group of Polyamines, indicating a no concerns for reproduction toxicity. No developmental toxicity was observed in an OECD 422 screening study.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Quality of whole database:
Physical-chemical properties of Tallow tripropylenetetraamine indicate a low likelihood for exposure via inhalation. The paste has a melting point of 31 °C, a boiling point > 300 °C and a low vapour pressure (4.7 x 10-5 Pa at 20°C for the coco dipropylene triamine, with the shortest average alkyl chain length representing the highest vapour pressure for the group of polyamines). Its use is limited to industrial and professional users and does not involve the forming of aerosols, particles or droplets of an inhalable size. So exposure to humans via the inhalation route will be unlikely to occur. Furthermore, as the substance is classified as corrosive, such testing should normally not be conducted.
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Quality of whole database:
Manufacture and use are highly controlled. Its use is limited to industrial and professional users where following its severe corrosive properties will provide for sufficient protection measures to prevent exposure.
Additional information

No developmental toxicity was observed in an OECD 422 screening study with Tallow tripropylenetetraamine.

 

Cross-reading and category approach:

Within the category of the Polyamine there are further studies available that clearly demonstrate the same toxicological profile for the various substances over this category, and a clear lack for concern for developmental (Foetal toxicity and teratogenicity) toxicity from the range of available relevant studies.

(See the document for the category justification for the polyamines that is attached to this dossier).

 

A similar OECD 422 study on Coco dipropylenetrimine, and a two generation study C12 -Y-triamine are available. These reproduction studies have also shown no indication of concern for developmental toxicity:

                                                                                                                                  

To strengthen the cross-reading approach with this category of Polyamines, an additional OECD 414 study was performed on Tallow dipropylenetriamine. This substance contains the same alkyl chain length as Tallow tripropylenetetraamine, but contains one diaminepropane (DP) less. Results from Tallow dipropylenetriamine can be considered a worst case situation for Tallow tripropylenetetraamine:

Tallow dipropylenetriamine was administered once daily by gavage from day 6 to 19 post-coitum at doses of 30, 60 and 120 mg/kg. Maternal toxicity was evident in both 60 and 120 mg/kg groups and included mortality, adverse clinical signs (hunched posture, rales, piloerection, salivation and lean appearance, among others), reduced food consumption, and substantially lower body weights and body weight loss compared to controls: 7% lower for 60 mg/kg and 25% lower BW for 120 mg/kg treated animals. Treatment related macroscopic findings (emaciated appearance, enlarged adrenal glands, small and large intestines thickened and distended with gas, among others) were also seen for mid- and high-dose animals as well. No toxicologically relevant maternal findings were noted with treatment up to 30 mg/kg. No significant differences were observed between control and treated groups regarding the number of corpora lutea, implantation sites, viable or dead foetuses, early or late resorptions, or pre- and post implantation loss. The percentage of early resorptions, total resorptions and post-implantation loss was relatively higher (not statistically significant) for females at 120 mg/kg than controls. This was attributable to two females who both had 100% post-implantation loss, consisting of early resorptions. No effects were observed on litter size and sex-ratio. Foetal body weights were significantly lower at 120 mg/kg (2.8 g) compared to controls (3.4 g), which was secondary to the lower gains/maternal weight loss at this dose level. Also signs of retarded skeletal ossification were observed at 60 and 120 mg/kg. Visceral variations included a dose related increase of yellow-white discoloured (anaemic?) adrenal glands in the 60 and 120 mg/kg groups in 7(2) and 48(5) foetuses (litters), whereas none occurred in the control and 30 mg/kg groups. Based on these effects a developmental NOAEL of 30 mg/kg was selected.

 

Additionally, there is a low likelihood of exposure to these substances as they are only applied in professional or industrial setting in asphalt applications. Usage results to the inclusion into or onto a matrix. Consumers/general population will not be exposed. Because of corrosive properties adequate use of protective gloves and other equipment, such as face shields, aprons and good work practices are mandatory. Likelihood of exposures via inhalation is also low considering the high boiling point (> 300 °C) and very low vapour pressure (< 4.7 x 10-5 Pa at 20°C).

Toxicity to reproduction: other studies

Description of key information

No further studies available.

Justification for classification or non-classification

Alll available data from studies involving the evaluation of reproduction and developmental parameters have not shown any indication of reproductive or developmental effects. Therefore no classification is required for this endpoint.

Additional information